Mycology Final Exam- Chapters (Modules) 1-7

CHAPTER 1: BASICS OF MYCOLOGY Thallic Condidiogenesis (type of asexual reproduction)

-A septum forms near the end of a parent, and the growing point ahead of it becomes the daughter conidium. The daughter develops AFTER it is separated, although still attached to the parent. Thallic development may be holothallic or arthric.

CHAPTER 2: LABORATORY PROCEDURES FOR FUNGAL CULTURE AND ISOLATION PRIMARY FUNGAL MEDIA SELECTIVE MEDIA WITH ANTIBIOTICS 2.) Brain heart infusion agar with blood, gentamicin, and chloramphenicol Give characteristics of this selective media

-BHIB with gentamicin and chloramphenicol is commercially available BLOOD IN BHIB WITH GENTAMICIN AND CHLORAMPHENICOL: -blood makes it especially nutritious GENTAMICIN AND CHLORAMPHENICOL IN BHIB: -these antibiotics inhibit bacteria -chloramphenicol also inhibits funguslike bacteria, Nocardia asteroides (partially inhibited), Nocardia brasiliensis, and yeast phases of dimorphic fungi (yeast phases are inhibited only at 37 degrees celcius, not 25 degrees celcius) THE ANTIBIOTICS IN BHIB INHIBIT WHAT TYPES OF FUNGI AND WILL NOT GROW? 1.) Nocardia 2.) Other aerobic actinomycetes WHAT TYPES OF FUNGUS GROW WELL ON THIS SELECTIVE MEDIA? 1.) Fungal pathogens 2.) Fastidious fungal pathogens (example: H. capsulatum and B. dermatitidis.

CHAPTER 2: LABORATORY PROCEDURES FOR FUNGAL CULTURE AND ISOLATION PRIMARY FUNGAL MEDIA SELECTIVE MEDIA WITH ANTIBIOTICS 3.) Brain heart infusion agar with blood, gentamicin, chloramphenicol, and cycloheximide Give characteristics of this selective media

-BHIB with gentamicin, chloramphenicol, and cycloheximide is commercially available WHAT IS BHIB WITH GENTAMICIN, CHLORAMPHENICOL, AND CYCLOHEXIMIDE BEST USED FOR? -it's best used for fastidious fungal pathogens WHAT WILL BE INHIBITED IN BHIB WITH GENTAMICIN, CHLORAMPHENICOL, AND CYCLOHEXIMIDE? 1.) Bacteria 2.) Aerobic actinomycetes 3.) Fungal opportunists

CHAPTER 1: BASICS OF MYCOLOGY Phialoconidium (Plural is Phialoconidia) (asexual reproduction)

-Conidium arising from a phialide. The first develops holoblastically, but the succeeding conidia are enteroblastically derived. -Phialide is a tube-shaped or vase-shaped conidiogenous cell. The first phialoconidium is holoblastic and the rest are eneteroblastic, arising from a fixed locus in the phialide. The phialide may be ringed at the top by a cup-shaped collarette (terminal cup-shaped remnant of material, at the tip of a phialide or at the base of a columnella). -Penicillium and Aspergillus may have these phialide structures. These phialides were formerly called sterigmata; this old word is now reserved for organisms in the taxonomic phylum Basidiomycota. -the supporting conidiophore in a phialoconidium may be simple or it may be elaborately branched. -See figures 1-17 and 1-18 on page 13 for images.

CHAPTER 1: BASICS OF MYCOLOGY Annelloconidium (plural is annelloconidia) (asexual reproduction)

-Conidium arising from an annellide. The first is holoblastic, but the rest develop enteroblastically. -annelloconidia are grown from inside a vase-shaped conidiogenous annellide. -The first annelloconidium is holoblastic, while the rest are enteroblastic. -As each conidium is released, a ring of parent outer cell wall material remains behind, giving a distinct saw-toothed appearance to the sides of the annellide. -The established supporting structure is termed an annellophore, and it may be simple or it may be elaborately branched. -see figure 1-19 on page 13 for images. -Annelloconidia are observed in Scopulariopsis. -

CHAPTER 2: LABORATORY PROCEDURES FOR FUNGAL CULTURE AND ISOLATION PRIMARY FUNGAL MEDIA SELECTIVE MEDIA WITH ANTIBIOTICS 4.) Dermatophyte test medium (DTM) Give characteristics of this selective media

-DTM is commercially available WHERE IS DTM FREQUENTLY USED? -in dermatology offices DTM CONSISTS OF WHAT TWO THINGS? 1.) Antibiotics AND 2.) Phenol red indicator WHY DOES DTM HAVE PHENOL RED INDICATOR? -when dermatophytes grow, they change the color from yellow to red, although few fungi other than dermatophytes can accomplish this also.

CHAPTER 3: COMMON FUNGAL OPPORTUNISTS CHART 3-4: Some organisms that cause opportunistic mycoses

-GO STUDY ON PAGE 106, IS A GOOD SUMMARY FOR THE CHAPTER BASICALLY

CHAPTER 2: LABORATORY PROCEDURES FOR FUNGAL CULTURE AND ISOLATION CULTURING THE SPECIMEN PRIMARY FUNGAL MEDIA SELECTIVE MEDIA WITH ANTIBIOTICS 1.) Inhibitory mold agar (IMA) Give characteristics of this selective media

-IMA is commercially available WHICH ANTIBIOTIC DOES IMA CONTAIN? -it contains gentamicin WHAT DOES IMA (DUE TO GENTAMICIN) INHIBIT? -IMA inhibits bacteria WHAT DOES IMA ALLOW TO GROW? IMA allows the growth of: 1.) Yeasts 2.) Fungal opportunists 3.) Pathogenic fungi, including dermatophytes and some H. capsulatum

CHAPTER 1: BASICS OF MYCOLOGY Aerial Hyphae

-In a cross-section of a mold culture, aerial hyphae extend above the agar surface and may support reproductive structures of mold (reproductive structures of mold are commonly called conidia) at their tips. -see page 6, figure 1-3 for a cross view of vegetative and aerial hyphae -aerial hyphae are considered as the colony FRONT of an agar plate. -see page 235, colony plate #1 for an image of aerial hyphae

CHAPTER 1: BASICS OF MYCOLOGY Vegetative Hyphae

-In a cross-section of a mold culture, the vegetative, or food-absorbing, portion of the mycelium is under the surface of the agar. -Vegetative hyphae are considered as being the colony REVERSE of an agar plate -see page 6, figure 1-3 for a cross view of vegetative and aerial hyphae -see page 235, colony plate #2 for an image of vegetative hyphae

CHAPTER 1: BASICS OF MYCOLOGY Dimorphism Explanation

-Most fungi exist only as molds or as yeasts. -Some fungi, however, may exhibit dimorphism, meaning that they possess two distinct phases. -these two distinct phases are mold and yeast, and each phase is dependent on temperature.

CHAPTER 1: BASICS OF MYCOLOGY Septate Hyphae

-Septate hyphae have cell cross walls that are very evident, as in the phyla Ascomycota, Basidiomycota, and Deuteromycota. -septate hyphae are 3-4 micrometers in diameter -most hyphae are septate -see page 5, figure 1-2 to see what it looks like

CHAPTER 1: BASICS OF MYCOLOGY What is an annellide? (asexual reproduction)

-Tube- or vase-shaped conidiogenous cell. -The first anneloconidium arises holoblastically, while the rest develop enteroblastically, from a tip which exhibits a new ring of material as each annelloconidium passes through. -The outer rings provide a saw-toothed appearance at the annellide tip.

CHAPTER 1: BASICS OF MYCOLOGY Basidiospores (sexual reproduction)

-a binucleate mycelium is formed by fusion of two compatible hyphae or yeast cells, usually with the aid of CLAMP CONNECTIONS -the terminal cell of the resulting mycelium enlarges into a club-shaped structure called a basidium. -the two nuclei within the basidium fuse to form a zygote, then undergo meiosis to produce four haploid nuclei -then four little protrusions (basidiospores) extend out from the end of the basidium, and each haploid nucleus travels into a basidiospore -some basidiomycetes, such as mushrooms, produce a protective basidiocarp to lodge the basidia and basidiospores -the only really important basidiomycete in medical mycology is filobasidiella neoformans, the sexual stage of crytococcus neoformans -basidiospores are formed by the phylum basidiomycota

CHAPTER 1: BASICS OF MYCOLOGY superficial mycoses

-affect only the outermost layers of skin and hair -usually are of little or no pathology -patients are mainly worried about cosmetic effects EXAMPLES OF SUPERFICIAL MYCOSES: 1.) otomycosis 2.) black and white piedra 3.) pityriasis versicolor 4.) tinea nigra

CHAPTER 1: BASICS OF MYCOLOGY Sporangiosphores

-are only formed in aseptate fungi -sporangiosphores are formed in internal cleavage of the contents of a sac called a sporangium (plural is sporangia) -the sporangium is supported on a base called a columnella, and it in turn is supported by a stalk-like sporangiophore -new sporangia may be empty, while older ones are filled with spores -when the sporangium dissolves and disperses its contents, the columnella remains -sporangiosphores are only observed in the taxonomic phylum Zygomycota (are aseptate hyphae)

CHAPTER 1: BASICS OF MYCOLOGY Macroconidia (asexual reproduction)

-arises by conversion of an ENTIRE HYPHAL element into a multicelled conidium -macroconidia may be: 1.) thick or thin-walled 2.) spiny or smooth 3.) club-shaped or oval 4.) sessile (arising on the sides of the hyphae) 5.) supported by conidiophores 6.) observed individually 7.) observed in clusters

CHAPTER 1: BASICS OF MYCOLOGY Microconidia (asexual reproduction)

-arises by conversion of an ENTIRE HYPHAL element into a single-celled conidium, but is different from macroconidia in that it remains aseptate -microconidia are usually one-celled and round, oval, or club-shaped -they may be sessile (arising on the sides of the hyphae), supported alone, or in clusters by a conidiophore

CHAPTER 1: BASICS OF MYCOLOGY Blastoconidium (plural is blastoconidia)

-asexual reproduction of fungi -holoblastic conidium produced by budding -yeasts are typical examples of blastoconidia, although some molds also demonstrate them -blastoconidia, produced by budding, may be seen in molds, such as Cladosporium, or in yeasts, such as candida. -In some yeast species, these blastoconidia elongate to form pseudohyphae, which often align end to end. -see figures 1-14 and 1-15 on page 12 to see images of pseudohyphae and blastoconidia

CHAPTER 1: BASICS OF MYCOLOGY colonial color

-be as specific about the colony colors as possible -EXAMPLE: instead of describing a culture as brown, use words such as beige, tan, khaki, or mahogany. -if there are concentric rings of different colors, observe each one -be sure to characterize color of both the front and reserve sides of the culture

CHAPTER 1: BASICS OF MYCOLOGY what is colonial topography?

-colonial topography describes the various designs of hills and valleys seen on fungal cultures -topography is often masked (hidden) by the aerial hyphae (front colony); therefore this characteristic is better observed on the REVERSE side of the colony (vegetative side) -a colony may posses no topography; that is, it is flat

CHAPTER 1: BASICS OF MYCOLOGY What is an annellophore? (asexual reproduction)

-conidiophore, or stalk, which supports annellides.

CHAPTER 1: BASICS OF MYCOLOGY What is the primary aid in identifying dimorphic fungi?

-conversion from one form to the other (conversion between mold form and yeast form).

CHAPTER 1: BASICS OF MYCOLOGY colonial cottony or woolly texture

-cottony or woolly colonies produce a very high, dense aerial mycelium (see color plate 3 in back of book)

CHAPTER 1: BASICS OF MYCOLOGY Pseudohyphae

-elongated blastoconidium made by some yeast species -cells of pseudohyphae are constricted at their points of attachment, while those of true hyphae are not. -see figures 1-14 and 1-15 on page 12 to see images of pseudohyphae and blastoconidia

CHAPTER 2: LABORATORY PROCEDURES FOR FUNGAL CULTURE AND ISOLATION GENERAL RULES FOR GOOD FUNGAL SPECIMEN COLLECTION 5.) The specimen must be adequately labelled, including the possible disease the physician suspects

-for certain fungal diseases, a special protocol is followed. FOR EXAMPLE: -if actinomycosis is suspected, the specimen must be maintained, processed, and cultured under anaerobic conditions, or the causative (etiologic) organism will die. -the final lab results are only as good as the specimen that the lab has to work with.

CHAPTER 2: LABORATORY PROCEDURES FOR FUNGAL CULTURE AND ISOLATION EXAMINING THE CULTURE What is the final identification of molds based on?

-for molds, the final identification is basically dependent on MICROSCOPIC morphology, although there are a few useful biochemical tests.

CHAPTER 2: LABORATORY PROCEDURES FOR FUNGAL CULTURE AND ISOLATION EXAMINING THE CULTURE What is the final identification of yeasts and funguslike bacteria based on?

-for yeasts and funguslike bacteria, BIOCHEMICAL TESTS are the primary bases for identification, with LESS emphasis on microscopic and macroscopic appearance

CHAPTER 1: BASICS OF MYCOLOGY Arthroconidia (arthrospores) (asexual reproduction)

-fragment from the hyphae through the septation points -they separate within the hyphal strand before dispersing -arthroconidia may form: 1.) adjacent to each other within the hyphae 2.) may be separated by alternating empty spaces, disjunctor cells, giving a checkered appearance -it is IMPORTANT to determine if the arthroconidia are adjacent or alternate, as coccidoides immitis is partly identified by the presence of disjunctor cells. -arthroconidia mature to be: 1.) thick-walled AND 2.) barrel-shaped or rectangular

CHAPTER 2: LABORATORY PROCEDURES FOR FUNGAL CULTURE AND ISOLATION GENERAL RULES FOR GOOD FUNGAL SPECIMEN COLLECTION 2.) Use sterile technique in collecting the specimen

-fungus in specimen may contaminate your hands and could possibly cause an infection -use the following tools for sterile technique: 1.) Use only flamed and cooled forceps (an instrument shaped like a pair of large spoons or salad tongs) 2.) Use only sterile swabs 3.) Use only sterile specimen containers

CHAPTER 1: BASICS OF MYCOLOGY colonial glabrous or waxy texture

-glabrous or waxy colonies have a smooth surface because they produce no aerial mycelium (they do not have a front colony) -usually YEASTS form a glabrous macroscopic appearance -see color plate 6

CHAPTER 3: COMMON FUNGAL OPPORTUNISTS ASEPTATE OPPORTUNISTS CHART 3-1: Key to identification of clinically important zygomycota

-go see pages 80-81 for a summary of the aseptate hyphae opportunistic pathogens

CHAPTER 2: LABORATORY PROCEDURES FOR FUNGAL CULTURE AND ISOLATION Chart 2-4: Growth Rates of Various Fungi

-go study the chart and write it down or type it out, it's on page 56

CHAPTER 3: COMMON FUNGAL OPPORTUNISTS SEPATATE OPPORTUNISTS HYALINE SEPTATE OPPORTUNISTS CHART 3-3: Identification of the most common species of Aspergillus

-go to page 99 RIGHT NOW AND LEARN ALL OF THEM

CHAPTER 1: BASICS OF MYCOLOGY colonial granular or powdery texture

-granular or powdery colonies are flat and crumbly because of the dense production of conidia -the granular texture is rougher; like granulated sugar -the powdery texture is like flour (see color plate 5 for powdery texture) -these two terms (granular, powdery) are often used interchangeably.

CHAPTER 1: BASICS OF MYCOLOGY Poroconidia (asexual reproduction)

-holoblastic conidium produced through a pore in the parent cell wall. -poroconidia asexual reproduction is found in bipolaris. -poroconidia are formed by the daughter pushing through a minute pore in the parent cell. -the parent may be in a long stalk (conidiophore) or it may be a specialized conidiogenous cell -see figure 1-16 on page 12 for poroconidium

CHAPTER 1: BASICS OF MYCOLOGY Aseptate Hyphae

-hyphae may be considered aseptate, meaning they have no cross walls, as in the taxonomic phylum Zygomycota. -aseptate hyphae are wide and ribbonlike, measuring 6-10 micrometers in diameter. -Zygomycetous organisms are USUALLY aseptate, but cross walls (septate) may be evident at damaged areas and near reproductive structures. -some hyphae are aseptate, but most hyphae are septate. -see figure 1-1 on page 5 of textbook to see what it looks like

CHAPTER 1: BASICS OF MYCOLOGY Racquet Hyphae

-hyphae resembles tennis racquets placed end to end -see figures 1-8 and 1-9 on page 7 to see what racquet hyphae look like

CHAPTER 2: LABORATORY PROCEDURES FOR FUNGAL CULTURE AND ISOLATION GENERAL RULES FOR GOOD FUNGAL SPECIMEN COLLECTION 3.) The specimen must be adequate

-if several cultures are requested, ask for ADDITIONAL swab specimens for use in mycology, because: -often the physician will send down one swab to be used on bacteriology, tuberculosis, and fungal media -by the time the swab is rolled over multiple media, any organisms present might be removed and the fungal culture could be falsely negative

CHAPTER 4: SUPERFICIAL AND DERMATOPHYTIC FUNGI Systemic pathogens and cutaneous infections

-if the skin is abraded, systemic pathogens, for example, Coccidioides immitis, also may rarely produce cutaneous infections without involving the rest of the body. SECONDARY CUTANEOUS INFECTIONS AND SYSTEMIC PATHOGENS -systemic organisms may exhibit secondary cutaneous manifestations as part of the disseminated disease process. -secondary infections must be carefully differentiated from primary ones, as the prognosis and treatment are quite different.

CHAPTER 1: BASICS OF MYCOLOGY cutaneous mycoses

-involve the destruction of the keratin of skin, hair, and nails -there is rarely invasion of deeper body tissues -cutaneous mycoses are caused primarily by the DERMATOPHYTES and by CANDIDA. -dermatophytes include: 1.) Trichophyton 2.) Microsporum 3.) Epidermophyton

CHAPTER 1: BASICS OF MYCOLOGY Sexual reproduction of fungi

-involves fusion of two nuclei into a zygote

CHAPTER 1: BASICS OF MYCOLOGY systemic mycoses

-involves the deep tissues and organs of the body -in disseminated (spread throughout body) forms of systemic mycoses, subcutaneous and cutaneous areas may also be invaded -EXAMPLES of systemic mycoses include: 1.) blastomycosis 2.) paracoccidioidomycosis 3.) histoplasmosis 4.) coccidioidomycosis

CHAPTER 1: BASICS OF MYCOLOGY subcutaneous mycoses

-involves the following immediately below the skin: 1.) skin 2.) muscle 3.) connective tissue -deeper tissue involvement is RARE -EXAMPLES of subcutaneous mycoses include: 1.) chromoblastomycosis 2.) mycetoma 3.) sporotrichosis

CHAPTER 1: BASICS OF MYCOLOGY Arthric conidiogenesis (asexual reproduction)

-is a subheading under thallic; arthric daughter cells fragment within the hyphal strand before dispersing.

CHAPTER 2: LABORATORY PROCEDURES FOR FUNGAL CULTURE AND ISOLATION Subculturing: Potato Dextrose Agar (PDA)

-is used for the subculturing of molds -see box on the bottom of page 56 and study it for the medium preparation and procedure

CHAPTER 2: LABORATORY PROCEDURES FOR FUNGAL CULTURE AND ISOLATION Subculturing: Neutral Sabouraud Dextrose Agar (Emmon's Modification)

-is used for the subculturing of yeast -see box on the bottom of page 55 and study it for the medium preparation and procedure

CHAPTER 2: LABORATORY PROCEDURES FOR FUNGAL CULTURE AND ISOLATION FUNGAL SOURCES 4.) Respiratory: Bronchial Washings, Sputa, Throats, Transtracheal Aspirates If the respiratory specimen is tenacious or also to be used for TB culture, what should be done? also just include information from page 35 for this section, idk how to organize it tbh it makes no sense

-it is treated with an equal quantity of plain N-acetyl-L-cysteine (NALC) solution and vortexed to liquefy the mucus and equally distribute the organisms -do NOT use specimens that have been treated with sodium hydroxide, which is routinely used for processing sputa for mycobacteria, as this chemical destroys many fungi -the NALC treated specimen is poured into a sterile 50 mL centrifuge tube, brought up to the 50 mL mark with 0.07 M phosphate buffer (pH 6.8-7.1), and mixed. IF SPECIMEN IS TO BE USED FOR TB CULTURE: -divide the NALC treated specimen that has the phosphate buffer into two tubes -centrifuge both tubes at 2100 rpm for 15 minutes, and decant the supernatants -place some of the sediment from the mycology tube onto appropriate media MYCOBACTERIOLOGY TUBE -for the mycobateriology tube, do the following: 1.) add 2 mL of 1.0% NaOH to the sediment 2.) incubate 15 minutes 3.) bring the volume up to the 50 mL mark with buffer 4.) centrifuge at 2100 rpm for 15 minutes? 5.) decant the supernatant 6.) inoculate the sediment to the appropriate media MICROSCOPIC EXAMINATION OF FUNGUS 1.) a drop of concentrated sediment for fungus is put on a slide 2.) coverslip added 3.) specimen is examined under the microscope for budding yeasts, hyphae, spherules, or funguslike bacteria -also various stains can be used as described in the section on direct examination of the specimen TWO THROAT SWABS: 1.) roll one across a slide, stain, and examine for fungal elements 2.) roll second swab across appropriate fungal media

CHAPTER 2: LABORATORY PROCEDURES FOR FUNGAL CULTURE AND ISOLATION Direct examination of the specimen overview

-it's most important that each fungal specimen be examined microscopically before, or concurrently with culturing -a direct examination allows you to send out an immediate preliminary report to the physician POSITIVE DIRECT EXAMINATION RESULT -with a positive direct examination result, you may also inoculate special media to quickly isolate and specifically identify the organism BIGGEST ADVANTAGE OF A DIRECT EXAMINATION RESULT IS HELPING A PATIENT WITH AN ACUTE DISEASE -this can possible save a patient's life -if a fungus is observed, the physician can IMMEDIATELY begin treatment -some pathogenic fungi require a long incubation period (3-4 weeks) for growth. -by the time the patient may have died, and isolation and identification of the causative agent becomes academic IF A FUNGUS IS NOT OBSERVED ON A DIRECT MOUNT -the physician can avoid using antifungal drugs, which are generally toxic and require a long period of treatment; also, the physician may start thinking about other possible disease agents BE CAREFUL AND VERY THOROUGH WITH THE DIRECT EXAMINATION -many times this may require performing more than one type of direct mount

CHAPTER 1: BASICS OF MYCOLOGY Colonial morphology overview for diagnosis

-many fungi do not have a colonial characteristic enough to allow identification without additional criteria -although the above is true, some have very distinct colonial characteristics -there are certain general terms used to describe fungal colonies

CHAPTER 1: BASICS OF MYCOLOGY Spiral Hyphae

-may be flat or may turn like a corkscrew -they are commonly observed in older cultures -see figures 1-10 and 1-11 on page 8 to see an example of spiral hyphae.

CHAPTER 2: LABORATORY PROCEDURES FOR FUNGAL CULTURE AND ISOLATION DIRECT EXAMINATION Basically, what is direct examination in mycology?

-microscopic examination (under a microscope), BEFORE CULTURING OR WHILE THE ORGANISM IS BEING PUT ON PRIMARY CULTURE

CHAPTER 2: LABORATORY PROCEDURES FOR FUNGAL CULTURE AND ISOLATION FUNGAL SOURCES 1.) Blood and Bone Marrow Most blood culture media contain what? What does this do?

-most blood culture media contains SPS SPS does the following: 1.) inhibits complement activity 2.) inhibits lysozyme activity 3.) interferes with phagocytosis 4.) inactivates clinically achievable concentrations of aminoglycosides

CHAPTER 1: BASICS OF MYCOLOGY What are mycoses?

-mycoses are fungal diseases

CHAPTER 2: LABORATORY PROCEDURES FOR FUNGAL CULTURE AND ISOLATION FUNGAL SOURCES 3.) Cutaneous: Hair, Nails, and Skin explain nail specimen preparation and examination

-nails are cleaned with 70% alcohol -with a STERILE blade, scrape away and then dispose of the outer layers of the nail. -scrape bits of the INNER infected nail into a sterile Petri dish SO THE NAIL SPECIMEN OF CHOICE IS THE INNER INFECTED NAIL

CHAPTER 1: BASICS OF MYCOLOGY Nodular Organs

-nodular organs are knots of twisted hyphae -see figures 1-6 and 1-7 on page 7 to see what a nodular organ looks like

CHAPTER 2: LABORATORY PROCEDURES FOR FUNGAL CULTURE AND ISOLATION EXAMINING THE CULTURE Once a mature colony has formed, what must you do?

-once a mature colony has formed, observe it for macroscopic appearance -Colony appearance may help to identify a fungus. However, this morphology may vary greatly between fungal stains, times and temperatures of incubation, and culture media.

CHAPTER 1: BASICS OF MYCOLOGY Taxonomy overview

-organisms are classified by their sexual (telemorph) stage. -organisms with no known sexual stage are classifies in the phylum Deuteromycota -fungi in parenthesis represent the asexual (anamorph) name -fungi without parenthesis indicate the sexual (telemorph) name. -some asexual fungi may exhibit two or three different sexual appearances and thus may possess two or three different sexual names. ^^^- the reverse in also true; some sexual organisms may possess two or three different asexual names

CHAPTER 1: BASICS OF MYCOLOGY Fungi definition/characteristics

-organisms that contain true nuclei, making them eukaryotic -fungi do NOT have chlorophyll -fungi absorb all nutrients from the environment, especially from decaying organic matter

CHAPTER 2: LABORATORY PROCEDURES FOR FUNGAL CULTURE AND ISOLATION STUDY QUESTIONS 1-8, PAGES 36-37 3.) Why is a urine specimen obtained upon rising in the morning?

-overnight incubation and multiplication of fungi in the bladder will increase the chance of isolating them on culture

CHAPTER 1: BASICS OF MYCOLOGY Favic Chandeliers (hyphae characteristics-mold)

-resemble the antlers of a buck deer -ends of the hyphae blunt and branched -Favic chandeliers are diagnostic for identifying cultures of Trichophytom achoenleinii, an organism that causes dermatophytosis (ringworm of skin, hair, or nail). -see page 6, figure 1-4 and 1-5 to see what favic chandeliers look like

CHAPTER 1: BASICS OF MYCOLOGY colonial rugose topography

-rugose colonies have deep furrows irregularly radiating from the center of the culture on the reverse colony -see color plate 7

CHAPTER 1: BASICS OF MYCOLOGY Overview of asexual reproduction of conidium formation of fungi (conidiogenesis)

-see page 11 and study entire figure

CHAPTER 2: LABORATORY PROCEDURES FOR FUNGAL CULTURE AND ISOLATION FUNGAL SOURCES 3.) Cutaneous: Hair, Nails, and Skin If Malassezia furfur is a consideration of cutaneous specimen collection, what should be done?

-set up a SABHI tube layered with olive oil

CHAPTER 2: LABORATORY PROCEDURES FOR FUNGAL CULTURE AND ISOLATION FUNGAL SOURCES 1.) Blood and Bone Marrow when using blood cultures or bone marrow to identify Histoplasma, what should be done?

-since histoplasma can be fastidious (complex or particular nutrient requirements), the following should be done: 1.) nutrient-fortified media should be used 2.) hold cultures for 3 weeks before discarding as negative

CHAPTER 1: BASICS OF MYCOLOGY Why are sporangiospores and conidia put in different groups if they are both a part of asexual reproduction?

-sporangiospores are formed only in aseptate fungi -conidia are formed only in septate fungi

CHAPTER 1: BASICS OF MYCOLOGY Arthric Conidiogenesis (type of asexual reproduction)

-thallic development whereby the daughter cells fragment within the hyphal strand BEFORE dispersing (separation). They may be holoarthric or enteroarthric.

CHAPTER 1: BASICS OF MYCOLOGY Ascospores (sexual reproduction)

-the nucleus from a male cell (known as antheridium in ascomycota) passes through a bridge into the female cell (known as ascogonium in ascomycota) -the male and the female cells may be from the same, self-compatible colony or from two colonies of opposite mating types -once the male and female nuclei fuse to form a zygote, the cell becomes an ascus (plural is asci) -the diploid zygote nucleus then divides by meiosis to form four haploid nuclei, which in turn divide by mitosis to form eight nuclei -then, each new nucleus walls off inside the ascus to form an ascospore -some ascomycetous fungi exhibit: 1.) un-protected asci (saccharomyces cerevisiae 2.) asci within a somatic saclike structure called an ascocarp (ex: pseudallescheria boydii) -the ascocarp observed in medically important fungi is completely enclosed and is termed as cleistothecium -ascospores are formed by the phylum ascomycota

CHAPTER 1: BASICS OF MYCOLOGY Prefix holo in asexual reproduction

-the prefix holo in front of the asexual reproduction words indicates that ALL wall layers of the parent cell are involved in a daughter conidium development.

CHAPTER 1: BASICS OF MYCOLOGY Prefix entero in asexual reproduction

-the prefix in front of the asexual reproduction words indicates that only the INNER CELL WALL LAYERS are included in daughter conidium development.

CHAPTER 1: BASICS OF MYCOLOGY Mycology definition

-the study of fungi

CHAPTER 1: BASICS OF MYCOLOGY Asexual reproduction of fungi

-there is only nuclear and cytoplasmic division, also known as mitosis.

CHAPTER 1: BASICS OF MYCOLOGY What are the fungi whose sexual stage does not exist or has not been discovered?

-these fungi are in the taxonomic phylum Deuteromycota and they are known as the fungi imperfecti

CHAPTER 1: BASICS OF MYCOLOGY What are the fungi that have a sexual (teleomorph) stage called?

-they are called the perfect fungi

CHAPTER 3: COMMON FUNGAL OPPORTUNISTS SEPATATE OPPORTUNISTS DERMATIACEOUS SEPTATE OPPORTUNISTS Opportunists with dark-colored hyphae may cause what?

-they may cause phaeohyphomycosis (infection caused by dermatiaceous fungi)

CHAPTER 1: BASICS OF MYCOLOGY chlamydoconidia (asexual reproduction)

-thick-walled survival conidia formed during unpleasant environmental conditions -chlamydoconidia will germinate and produce conidia when a better climade occurs -may be observed at: 1.) the hyphal tip (terminal) 2.) on the sides (sessile) 3.) within the hyphal strand (intercalary)

CHAPTER 1: BASICS OF MYCOLOGY Zygospores (sexual reproduction)

-two compatible hyphae each form an arm (zygophore) extending toward the other. -the hyphae may be from a self-compatible colony or from two colonies of opposite mating types. -when the zygophores meet, they fuse to form a thick-walled, protective zygosporangium within which a zygospore develops. -zygospores are formed by the taxonomic phylum zygomycota

CHAPTER 1: BASICS OF MYCOLOGY colonial umbonate topography

-umbonate colonies possess a buttonlike central evaluation -they may be accompanied by rugose furrows around the button, as seen in color plate 8

CHAPTER 1: BASICS OF MYCOLOGY the terms macroconidia and microconidia are NOT used unless what?

-unless both types are apparent in the same culture, when there is a need to differentiate them

CHAPTER 4: SUPERFICIAL AND DERMATOPHYTIC FUNGI The dermatophytes (Microsporum, Epidermophyton, Trichophyton) -what is the term that used to be used for diseases elicited by these three organisms?

-used be to termed as tinea, or ringworm -tinea has been replaced by the term dermatophytosis, followed by the body area affected EXAMPLE: tinea capitis should be replaced by dermatophytosis of the scalp.

CHAPTER 2: LABORATORY PROCEDURES FOR FUNGAL CULTURE AND ISOLATION FUNGAL SOURCES 4.) Respiratory: Bronchial Washings, Sputa, Throats, Transtracheal Aspirates overview of respiratory specimens

-usually the respiratory tract becomes infected with inhaled organisms -many mycoses exhibit a pulmonary origin and then spread to other parts of the body

CHAPTER 2: LABORATORY PROCEDURES FOR FUNGAL CULTURE AND ISOLATION FUNGAL SOURCES 7.) Vaginal, Uterine Cervix characteristics of these specimens. ^^^^^ (vaginal, uterine cervix)

-vaginal and uterine cervix sites normally may contain yeasts. -usually these specimens are obtained to determine if their yeast flora (the normal yeast in these sites), in particular candida albicans, have overgrown their normal quantities to produce an infection COLLECTION: 1.) the specimen is collected with two sterile swabs 2.) then the swabs are put into transport media (EXAMPLES INCLUDE: Stuart's or Amie's) and are refrigerated until they are processed in the laboratory -refrigeration slows multiplication of yeasts and bacterial normal flora EXAMINATION IN THE LABORATORY OF THE TWO SWABS: 1.) one swab is used to make a smear and stained for fungal elements 2.) the other swab is rolled over fungal media -any fungi that grow are semiquantitated (many, moderate, few) and identified.

CHAPTER 1: BASICS OF MYCOLOGY Nonreproductive Vegetative Hyphae

-vegetative hyphae may possess some very distinct non-reproductive characteristics, which may aid in speciation.

CHAPTER 1: BASICS OF MYCOLOGY colonial velvety texture

-velvety colonies produce a low aerial (front) mycelium which resembles the fabric velvet (see color plate 4 in the back of book)

CHAPTER 1: BASICS OF MYCOLOGY colonial verrucose topography

-verrucose colonies exhibit a winkled, convoluted (folded, twisted, coiled) surface -see color plate 9

CHAPTER 2: LABORATORY PROCEDURES FOR FUNGAL CULTURE AND ISOLATION FUNGAL SOURCES 8.) Wounds, Subcutaneous Lesions, Mucocutaneous Lesions Characteristics of these specimens ^^^^ (wounds, subcutaneous lesions, mucocutaneous lesions)

-wounds, subcutaneous lesions, and mucocutaneous lesions may be brought out by fungi COLLECTION: 1.) scrapings of the crusted portions and from the deep center and edge of active lesions are taken with a sterile scalpel 2.) remove the scrapings to 1 mL of sterile saline for transport 3.) in addition to scrapings, material may be aspirated by needle and syringe from deep cysts or abscesses in the tissue VARIOUSLY COLORED GRANULES OR TINY BLACK DOTS FOUND IN SPECIMEN, HOW DO U COLLECT? if there are any variously colored granules or tiny black dots, these are collected by laying sterile saline-wetted gauze on top of the lesions and pulling off the gauze -the granules should be trapped in the gauze mesh PROCESSING: -all specimens are kept in ANAEROBIC containers in case Actinomyces is the causative agent PREPARATION AND EXAMINATION: 1.) In the lab, a potassium hydroxide preparation is made with some of the scrapings or aspirate, and the mount is searched for fungal elements 2.) the color of the granules is noted, since this may provide a clue as to the causative organism 3.) some of the granules are gently teased apart in a drop of sterile water or stained -they are observed microscopically for: 1-bacteria 2-funguslike bacteria 3-hyphae interspersed with swollen chlamydospore-like cells -some granules may contain a cementlike matrix or center of necrotic debris 4.) A potassium hydroxide preparation is also made with some of the black dots if present and are observed for: thick-walled, dark-brown bodies (also known as sclerotic bodies), which may be divided into two or four cells 5.) At the same time as the direct mounts are being done, the rest of the skin scrapings, aspirated material, or black dots are inoculated to aerobic and anaerobic fungal media -BE SURE TO FIRST PLATE THE ANAEROBIC AGAR AND IMMEDIATELY PLACE IT UNDER ANAEROBIC CONDITIONS TO ENSURE CULTURE INTEGRITY GRANULE PREPARATION -the granules are washed several times in sterile saline to remove most contamination before culturing, then crushed and placed on media.

CHAPTER 2: LABORATORY PROCEDURES FOR FUNGAL CULTURE AND ISOLATION FUNGAL SOURCES 1.) Blood and Bone Marrow If a nonautomated system (specifically Septi-check) is used (one that does not rely on carbon dioxide or gas pressure), what can be added? which nonautomated system can not use this technique? what does the added chemical do?

1 mL of 3% hydrogen peroxide can be added to each 50 mL of blood culture broth media in Septi-check -ISOLATOR CANNOT USE THIS METHOD, even though it is nonautomated, because it doesn't use broth media, but Septi-check does -the released catalase from breakdown of blood by hydrogen peroxide enhances yeast growth

CHAPTER 1: BASICS OF MYCOLOGY CHAPTER 1 FINAL EXAM QUESTIONS, PAGES 24-26 5.) In each blank, write the letter of the correct answer below: The taxonomic phylum Zygomycota is distinct in that the hyphae are usually ______________. The typical asexual reproductive structures are ______________, and the sexual stage is characterized by the formation of ______________.

1- D. aseptate 2- G. sporangiospores (asexual reproductive structures only found in aseptate hyphae) 3- C. zygospores

CHAPTER 1: BASICS OF MYCOLOGY CHAPTER 1 FINAL EXAM QUESTIONS, PAGES 24-26 4.) Choose the correct answer out of the two options in each parenthesis based on figure 1-40 on page 26: This fungus produces (phialoconidia/annelloconidia). It has no known sexual stage; thus this figure must represent a or an (anamorph/teleomorph) and is classified in the phylym (Basidiomycota/Deuteromycota)

1- phialoconidia 2- anamorph 3- Deuteromycota

CHAPTER 3: COMMON FUNGAL OPPORTUNISTS How are the common fungal opportunists categorized in this chapter?

1.) ASEPTATE OPPORTUNISTS 2.) SEPTATE OPPORTUNISTS -2A: Dematiaceous septate opportunists -2B: Hyaline septate opportunists

CHAPTER 3: COMMON FUNGAL OPPORTUNISTS ASEPTATE OPPORTUNISTS What are the zygomycota aseptate opportunists that are discussed in this chapter?

1.) Absidia species (terminal vesicle ABSENT) 2.) Apophysomyces species (terminal vesicle ABSENT) 3.) Mucor species (terminal vesicle ABSENT) 4.) Rhizopus species (terminal vesicle ABSENT) 5.) Saksenaea species (terminal vesicle ABSENT) 6.) Cunninghamella species (terminal vesicle present) 7.) Syncephalastrum species (terminal vesicle present)

CHAPTER 3: COMMON FUNGAL OPPORTUNISTS SEPATATE OPPORTUNISTS HYALINE SEPTATE OPPORTUNISTS What are the 9 hyaline septate opportunists?

1.) Acremonium (Cephalosporium) species 2.) Aspergillus species 3.) Chrysosporium species 4.) Fusarium species 5.) Gliocladium species 6.) Paecilomyces species 7.) Penicillium species 8.) Scopulariopsis species 9.) Sepedonium species

CHAPTER 3: COMMON FUNGAL OPPORTUNISTS SEPATATE OPPORTUNISTS DERMATIACEOUS SEPTATE OPPORTUNISTS What are the 8 dermatiaceous septate opportunists?

1.) Alternaria species 2.) Aureobasidium species 3.) Bipolaris species 4.) Cladosporium (Hormondendrum) species 5.) Curvularia species 6.) Exserohilum species 7.) Epicoccum species 8.) Nigrospora species

CHAPTER 1: BASICS OF MYCOLOGY What are the 3 commonly observed sexual mechanisms in medically important fungi?

1.) Ascospores 2.) Basidiospores 3.) Zygospores

CHAPTER 3: COMMON FUNGAL OPPORTUNISTS STUDY QUESTIONS 1-4 ON PAGE 105 2.) Aspergillus and Synchephalastrum resemblke each other microscopically. Put an A in front of each characteristic that pertains to Aspergillus sp., a B in front of those items that apply to Synchephalastrum sp. and a C where neither fungus is characterized. 1.) Aseptate 2.) Vesicles 3.) Blastoconidia 4.) Merosporangia 5.) Foot cells 6.) Phialides

1.) Aseptate B 2.) Vesicles A, B 3.) Blastoconidia C 4.) Merosporangia B 5.) Foot cells A 6.) Phialides A

CHAPTER 1: BASICS OF MYCOLOGY What are the different methods of reproduction for fungi?

1.) Asexually 2.) Sexually 3.) Depending on the circumstances, fungi may use more than one of these methods to multiply

CHAPTER 1: BASICS OF MYCOLOGY What are the two ways that a conidia may originate? (Asexual reproduction)

1.) BLASTICALLY -the conidiogenous (parent) cell enlarges, then a septum separates the enlarged portion into a daughter cell. 2.) THALLICALLY -the septum forms first, and the growing point ahead of it becomes the daughter

CHAPTER 3: COMMON FUNGAL OPPORTUNISTS STUDY QUESTIONS 1-3 ON PAGE 96 3.) Place the letter of the answer from Column B in front of the corresponding words in Column A. An answer may be used more than once. 1.) Black yeast 2.) Shield cells 3.) Enlarged central cell 4.) Arthrooconidia 5.) Single, one-celled, black conidia

1.) Black yeast C. Aureobasidium sp. 2.) Shield cells E. Cladosporium sp. 3.) Enlarged central cell F. Curvularia sp. 4.) Arthroconidia C. Aureobasidium sp. 5.) Single, one-celled, black conidia A. Nigrospora sp.

CHAPTER 2: LABORATORY PROCEDURES FOR FUNGAL CULTURE AND ISOLATION What are the different specific fungal sources?

1.) Blood and Bone Marrow 2.) Cerebrospinal fluid 3.) Cutaneous: hair, nails, and skin 4.) Respiratory: bronchial washings, sputa, throats, transtracheal aspirates 5.) Tissue, Biopsies 6.) Urine 7.) Vaginal, Uterine Cervix 8.) Wounds, Subcutaneous Lesions, Mucocutaneous Lesions

CHAPTER 4: SUPERFICIAL AND DERMATOPHYTIC FUNGI What are primary cutaneous mycoses caused by?

1.) Candida sp. 2.) The dermatophytes 1- Microsporum 2- Epidermophyton 3- Trichophyton

CHAPTER 3: COMMON FUNGAL OPPORTUNISTS STUDY QUESTIONS 1-4 ON PAGE 105 4.) The fungi listed in column A bellow all possess single, one-celled, terminal conidia. The items in column B may be used to differentiate them. Place the letter of the characteristic from Column B in front of the corresponding organism in Column A. More than one characteristic may be placed in front of each fungus. 1.) Chrysosporium sp. 2.) Sepedonium sp. 3.) Scedosporium apiospermum 4.) Blastomyces dermatitidis 5.) Histoplasma capsulatum

1.) Chrysosporium sp. B. rapid growing 2.) Sepedonium sp. B. rapid growing C. may possess microconidia 3.) Scedosporium apiospermum D. cleistothecia common E. annellidic 4.) Blastomyces dermatitidis A. dimorphic 5.) Histoplasma capsulatum A. dimorphic C. may possess microconidia

CHAPTER 1: BASICS OF MYCOLOGY Even though Blastoconidia are typically done by yeasts, which molds also demonstrate them?

1.) Cladosporium 2.) Aureobasidium 3.) Nigrospora

CHAPTER 1: BASICS OF MYCOLOGY What are the four main categories to describe fungal colonial texture?

1.) Cottony (or woolly texture) 2.) Velvety 3.) Granular (or powdery texture) 4.) Glabrous (or waxy) -see figure 1-38 on page 20 to see what they each look like

CHAPTER 2: LABORATORY PROCEDURES FOR FUNGAL CULTURE AND ISOLATION STUDY QUESTIONS 1-7 ON PAGES 71-72 5.) Give four methods for fungal stock culture preservation

1.) Distilled water 2.) Freezing 3.) Lyophilization 4.) Oil overlay

CHAPTER 2: LABORATORY PROCEDURES FOR FUNGAL CULTURE AND ISOLATION FUNGAL SOURCES 1.) Blood and Bone Marrow What are the commercial blood culture systems currently in use that contain aerobic and anaerobic blood culture bottles, which support growth of bacteria and yeasts?

1.) ESP blood culture system (Difco) -monitors cultures every 12 to 24 minutes by measuring headspace gas pressure 2.) The BacT/Alert (Organon Teknika) -measures carbon dioxide production every 10 minutes 3.) BACTEC 9000 system (beckton-dickinson diagnostic instrument systems) -measures carbon dioxide production every 10 minutes (old BACTEC would measure carbon dioxide production twice a day for 2 days, then once a day thereafter) -has an additional fungal medium, which enhances recovery of yeasts that might be overgrown by bacteria in mixed cultures

CHAPTER 4: SUPERFICIAL AND DERMATOPHYTIC FUNGI What are the 4 different types of superficial organisms that cause superficial mycoses?

1.) Exophiala (Phaeoannellomyces, Cladosporium) werneckii 2.) Malassezia furfur (Pityrosporum ovale, Pityrosporum orbiculare) 3.) Piedraia hortai 4.) Trichosporon beigeli (Cutaneum)

CHAPTER 2: LABORATORY PROCEDURES FOR FUNGAL CULTURE AND ISOLATION DIRECT MOUNT EXAMINATION 8.) Other stains Stains for fungal detection may be performed in laboratory sections other than microbiology. List the additional stains that technologists in histology may perform and explain each one.

1.) GOMORI METHENAMINE SILVER (GMS) STAIN -fungi and actinomycetes appear black against a green background 2.) ACID-SCHIFF (PAS) STAIN -fungal elements (but NOT actinomycetes) look magenta against a light pink or green background 3.) MAYER'S MUCICARMINE STAIN -may be performed when Cryptococcus is suspected -the capsules of Cryptococcus appear red against a yellow background, while other similar-appearing fungi do NOT take up the red stain. 4.) HEMATOXYLIN AND EOSIN (H&E) STAIN -is the "workhorse" of pathologists -the background is pink, with tissue nuclei and fungal elements staining purple -an added advantage to the H&E preparation is that stained sections may be placed directly under a fluorescent microscope (340 nm wavelength) where fungi , such as: 1-blastomyces 2-cryptococcus 3-candida 4-aspergillus 5-coccidioides 6-paracoccidioides 7-occasionally histoplasmsa -these will brightly autofluoresce while tissue and background material remain dark 5.) WRIGHT STAIN -performed on blood and bone marrow smears to look for purple pseudoencapsulated yeast forms of Histoplasma capsulatum inside polymorphonuclear cells and monocytes 6.) FONTANA-MASSON STAIN -may be used to differentiate dematiaceous fungi from hyaline fungi in tissue -the melaninlike pigment in dematiaceous fungi stains positive, whereas hyaline fungi are negative

CHAPTER 2: LABORATORY PROCEDURES FOR FUNGAL CULTURE AND ISOLATION CULTURING THE SPECIMEN PRIMARY FUNGAL MEDIA SELECTIVE MEDIA WITH ANTIBIOTICS What are the 4 common types of selective media with antibiotics in mycology?

1.) Inhibitory mold agar (IMA) 2.) Brain heart infusion agar with blood, gentamicin, and chloramphenicol 3.) Brain heart infusion agar with blood, gentamicin, chloramphenicol, and cycloheximide 4.) Dermatophyte test medium (DTM)

CHAPTER 2: LABORATORY PROCEDURES FOR FUNGAL CULTURE AND ISOLATION What are the 5 general rules for good fungal specimen collection?

1.) Make sure the specimen is collected from the area most likely to be affected 2.) Use sterile technique in collecting the specimen 3.) The specimen must be adequate 4.) The specimen must be promptly delivered to the laboratory and the laboratory must quickly process the specimen 5.) The specimen must be adequately labelled, including the possible disease the physician suspects

CHAPTER 4: SUPERFICIAL AND DERMATOPHYTIC FUNGI DERMATOPHYTES List the different dermatophytes and the body sites they infect

1.) Microsporum 1- skin 2- ectothrix hair 2.) Epidermophyton 1- skin 2- nails 3.) Trichophyton mentagrophytes, rubrum, and verrucosum 1- skin 2- nails 3- ectothrix hair 4.) Trichophyton tonsurans, schoenleinii, and violaceum 1- skin 2- nails 3- endothrix hair

CHAPTER 2: LABORATORY PROCEDURES FOR FUNGAL CULTURE AND ISOLATION What are the 5 different categories of therapeutic agents for mycoses?

1.) Polyene macrolides 2.) Pyrimidine analogues 3.) Azole antifungals 4.) Griseofulvin 5.) Allylamines

CHAPTER 2: LABORATORY PROCEDURES FOR FUNGAL CULTURE AND ISOLATION STUDY QUESTIONS 1-7 ON PAGES 71-72 6.) List the five basic classes of antifungal agents and give one example of each. Discuss which mycoses (systemic, dermatophytic, yeast, subcutaneous) each class usually treats

1.) Polyene macrolides -AMP B (systemic) -NYSTATIN (yeast) 2.) Pyrimidine analogues -5PC (yeast) 3.) Azoles -CLOTRIMAZOLE -MICRONAZOLE -KETOCONAZOLE -ITRACONAZOLE -FLUCONAZOLE -collectively will treat systemic, yeast, and dermatophytes 4.) Griseofulvin -treats dermatophytes 5.) Allylamines -NAFTIFINE -TERBINAFINE -both will treat dermatophytes

CHAPTER 3: COMMON FUNGAL OPPORTUNISTS FINAL EXAM QUESTIONS 1-14 ON PAGES 110-113 3.) In front of numbers 1 to 3, write the letter(s) of the fungus that represents each characteristic 1.) Produces rhizoids 2.) Branching sporangiophores 3.) Internodal sporangiophores

1.) Produces rhizoids A. Absidia sp. C. Rhizopus sp. 2.) Branching sporangiophores A. Absidia sp. B. Mucor sp. 3.) Internodal sporangiophores A. Absidia sp.

CHAPTER 1: BASICS OF MYCOLOGY What are the 3 different types of colonial topography?

1.) Rugose topography 2.) Umbonate topography 3.) Verrucose topography see figure 1-39 on page 21 to see each

CHAPTER 2: LABORATORY PROCEDURES FOR FUNGAL CULTURE AND ISOLATION CULTURING THE SPECIMEN PRIMARY FUNGAL MEDIA NONSELECTIVE MEDIA What are the 3 types of nonselective media for fungi?

1.) Sabouraud Brain Heart Infusion Agar (SABHI) 2.) Brain Heart Infusion Agar with Blood (BHIB) 3.) Subouraud Dextrose Agar (SDA)

CHAPTER 2: LABORATORY PROCEDURES FOR FUNGAL CULTURE AND ISOLATION What are the different types of direct mounts?

1.) Saline wet mount 2.) Lactophenol cotton blue (LPCD) wet mount 3.) Potassium hydroxide (KOH) preparation 4.) Gram stain 5.) Acid-fast stain 6.) India ink preparation 7.) Calcofluor white stain 8.) Other stains

CHAPTER 2: LABORATORY PROCEDURES FOR FUNGAL CULTURE AND ISOLATION FUNGAL SOURCES 1.) Blood and Bone Marrow What are the two non-automated commercial systems for blood specimens?

1.) Septi-Check (Becton-Dickinson) -is a biphasic system in which inoculated broth is periodically inverted over three agars -the bottle should be vented and agitated constantly for the first 24-48 hours to improve yield. 2.) Isolator (Wampole Labs) -is a centrifugation-lysis system in which blood is collected into a special tube, the blood is lysed, the tube is centrifuged, and the pellet is inoculated to fungal agar media.

CHAPTER 1: BASICS OF MYCOLOGY What are the different types of mycoses based on body site?

1.) Superficial mycoses 2.) Cutaneous mycoses 3.) Subcutaneous mycoses 4.) Systemic mycoses

CHAPTER 2: LABORATORY PROCEDURES FOR FUNGAL CULTURE AND ISOLATION EXAMINING THE CULTURE What are the 5 frequently used procedures to microscopically examine a CULTURE?

1.) Tease mount method 2.) Cellophane tape method 3.) Slide culture method 4.) Modified slide culture method 5.) Coverslip sandwich technique

CHAPTER 4: SUPERFICIAL AND DERMATOPHYTIC FUNGI STUDY QUESTION ON PAGE 122 Place the letter(s) of the words in Column B in front of the corresponding words in Column A. 1.) Trichosporon beigelii 2.) Spaghetti & meatballs 3.) Black patches on skin 4.) Black piedra

1.) Trichosporon beigelii E. Light brown hair nodules (white piedra) 2.) Spaghetti & meatballs A. Multicolored patches on skin D. Malassezia furfur 3.) Black patches on skin B. Tinea nigra F. Black mold mistaken for Candida or Auerobasidium 4.) Black piedra C. Hard, black hair nodules

CHAPTER 1: BASICS OF MYCOLOGY What are the 2 ways that fungi are observed?

1.) Under the microscope (microscopically) 2.) As a colony on an agar plate (macroscopically)

CHAPTER 3: COMMON FUNGAL OPPORTUNISTS OPPORTUNISTIC MYCOSES What are the 6 opportunistic mycoses described in this chapter?

1.) Zygomycosis (already described previously during aseptate opportunist section- the first section of the chapter) 2.) Aspergillosis 3.) Penicilliosis 4.) Keratomycosis 5.) Otomycosis 6.) Sinusitis

CHAPTER 2: LABORATORY PROCEDURES FOR FUNGAL CULTURE AND ISOLATION STUDY QUESTIONS ON PAGE 49, QUESTIONS 1-10 Questions 2-7 Match the direct mount stain with the proper characteristic from column B on page 49, matching question. 2.) ______ KOH preparation 3.) ______ Acid-fast stain 4.) ______ India ink preparation 5.) ______ Wright stain 6.) ______ Gram stain 7.) ______ Calcofluor white preparation

2.) D. Hair 3.) A. Nocardia 4.) F. Cryptococcus neoformans 5.) B. Histoplasma capsulatum 6.) G. Fungi stain blue 7.) E. Fungi fluoresce

CHAPTER 2: LABORATORY PROCEDURES FOR FUNGAL CULTURE AND ISOLATION FINAL EXAM QUESTIONS 1-7 ON PAGES 66-67 1.) Why should you not accept 24-hour collections on respiratory specimens?

A 24-hour collection becomes easily overgrown with bacterial contaminants and fungal opportunists

CHAPTER 2: LABORATORY PROCEDURES FOR FUNGAL CULTURE AND ISOLATION STUDY QUESTIONS 1-7 ON PAGES 71-72 4.) What is the principle behind the DNA probe procedure?

A known luminescent labelled single-stranded DNA probe to the organism in questioned is allowed to hybridize with ribosomal RNA from a sonicated fungal culture. If the fungus in question is present, the fungal rRNA will combine with the probe DNA and luminesce

CHAPTER 2: LABORATORY PROCEDURES FOR FUNGAL CULTURE AND ISOLATION STUDY QUESTIONS 1-6 ON PAGE 57 3.) The best medium to isolate yeasts from a vaginal specimen is:

A. Brain heart infusion agar with blood, gentamicin, and chloramphenicol EXPLANATION: -While SABHI will also grow yeasts, bacteria will overrun the culture; therefore use gentamicin and chloramphenicol. -Do NOT use cycloheximide as it will inhibit yeasts

CHAPTER 3: COMMON FUNGAL OPPORTUNISTS FINAL EXAM QUESTIONS 1-14 ON PAGES 110-113 7.) In these blanks, write the genus identified from each drawing. A. B. C. D. E. F. G. H. I. J. K. L.

A. Curvularia sp. B. Syncephalastrum sp. C. Penicillium sp. D. Mucor sp. E. Scopulariopsis sp. F. Acremonium sp. G. Aspergillus sp. H. Cladosporium sp. I. Alternaria sp. J. Paecilomyces sp. K. Rhizopus sp. L. Auerobasidium sp.

CHAPTER 2: LABORATORY PROCEDURES FOR FUNGAL CULTURE AND ISOLATION STUDY QUESTIONS 1-7 ON PAGES 71-72 2.) State if the following serological methods detect fungal antigen, fungal antibody, or both. A. IMMUNODIFFUSION B. LATEX AGGLUTINATION C. DIRECT FLUORESCENT ANTIBODY D. ELISA

A. IMMUNODIFFUSION -both B. LATEX AGGLUTINATION -both C. DIRECT FLUORESCENT ANTIBODY -antigen D. ELISA -both

CHAPTER 1: BASICS OF MYCOLOGY PAGE 17-18, STUDY QUESTIONS: 1.) See figure 1-29. What type of reproductive structure is observed?

A. Macroconidium

CHAPTER 1: BASICS OF MYCOLOGY PAGE 17-18, STUDY QUESTIONS: 6.) From figure 1-31, fill in the blanks: A. _________ B. _________ C. _________

A. Sporangiosphore B. Columnella C. Sporangiophore

CHAPTER 3: COMMON FUNGAL OPPORTUNISTS STUDY QUESTIONS 1-2 ON PAGE 87 From figure 3-16, fill in the blanks: A. _______ B. _______ C. _______

A. Sporangium B. Rhizoid C. Stolon

CHAPTER 2: LABORATORY PROCEDURES FOR FUNGAL CULTURE AND ISOLATION FINAL EXAM QUESTIONS 1-7 ON PAGES 66-67 6.) List the advantages and disadvantages of the following: A. Tease Mount B. Slide Culture/Modified Slide Culture C. Coverslip Sandwich

A. Tease Mount ADVANTAGES -quick to make; no incubation period DISADVANTAGES -may destroy conidial juxtaposition B. Slide Culture/Modified Slide Culture ADVANTAGES -beautiful conidial juxtaposition; 2 mounts from one slide culture DISADVANTAGES -takes time to set up; incubation period required C. Coverslip Sandwich ADVANTAGES -beautiful conidial juxtaposition; several mounts from one plate DISADVANTAGES -incubation period required

CHAPTER 1: BASICS OF MYCOLOGY CHAPTER 1 FINAL EXAM QUESTIONS, PAGES 24-26 1.) From drawings A through L, fill in the blanks: A. B. C. D. E. F. G. H. I. J. K. L.

A. Zygosporangium (or zygospore inside zygosporangium) B. Blastoconidia C. Racquet hyphae D. Macroconidium E. Verrucose topagraphy F. Ascospore G. Reverse colony; vegetative hyphae H. Sessile chlamydoconidium I. Granular or powdery texture J. Dimorphism K. Basidiospore L. Arthroconidia

CHAPTER 3: COMMON FUNGAL OPPORTUNISTS FINAL EXAM QUESTIONS 1-14 ON PAGES 110-113 9.) Otomycosis involves:

A. external ear infections.

CHAPTER 2: LABORATORY PROCEDURES FOR FUNGAL CULTURE AND ISOLATION EXAMINING THE CULTURE Explain the coverslip sandwich technique

ADVANTAGES OF THE COVERSLIP SANDWICH TECHNIQUE: 1.) more than two preparations may be made 2.) each preparation can be removed from the agar at different intervals 3.) the set up is easier than for the slide culture 4.) the juxtaposition of reproductive structures to the hyphae is almost as good as the slide culture method DISADVANTAGES OF THE COVERSLIP SANDWICH TECHNIQUE: 1.) the coverslip easily breaks while being inserted into the afar or when taken out 2.) cottony fungi grow past the edges of the coverslip before forming reproductive structures 3.) there is a waiting period for incubation on the potato dextrose agar plate in addition to incubation on the primary isolation medium ONLY AERIAL STRUCTURES SHOULD BE OBSERVED FOR MYCOLOGIC IDENTIFICATION -be aware that fungal elements formed on the coverslip beneath the agar are vegetative; only the aerial structures should be observed for mycologic identification. SEE BOX ON PAGE 64 FOR THE MEDIUM PREPARATION AND PROCEDURE FOR THE COVERSLIP SANDWICH TECHNIQUE -seriously, go study it

CHAPTER 1: BASICS OF MYCOLOGY PAGE 9- STUDY QUESTIONS True or false: fungi resemble plants in that both contain chlorophyll

ANSWER: False -plants contain chlorophyll; fungi do not

CHAPTER 1: BASICS OF MYCOLOGY PAGE 9- STUDY QUESTIONS A patient's sputum specimen contains a dimorphic fungus. If the sputum is put on a slide and a coverslip added, what would you expect to observe- hyphae or yeasts?

ANSWER: Yeasts -the sputum came from body temperature (37 degrees celcius) -at 37 degrees celcius, a dimorphic organism is in a yeast phase

CHAPTER 1: BASICS OF MYCOLOGY PAGE 9- STUDY QUESTIONS Figure 1-13 on page 9 is a drawing of: A. racquet hyphae B. nodular organs C. yeasts D. favic chandeliers E. aerial hyphae

ANSWER: D. Favic chandeliers

CHAPTER 1: BASICS OF MYCOLOGY PAGE 9- STUDY QUESTIONS Are knots of twisted hyphae called cinidia or nodular organs?

ANSWER: Nodular organs

CHAPTER 3: COMMON FUNGAL OPPORTUNISTS STUDY QUESTIONS 1-2 ON PAGE 87 1.) In your own words, describe at least 5 common properties that fungal opportunists possess

ANY 5 OF THE FOLLOWING WILL WORK FOR COMMON PROPERTIES THAT FUNGAL OPPORTUNISTS POSSESS 1.) most fungal opportunists form mature colonies in 4-5 days 2.) they live in soil 3.) they become airborne occasionally 4.) they are normally inhaled 5.) respiratory specimens may normally yield a few colonies of fungal opportunists 6.) fungal opportunists may be normal skin flora 7.) they may contaminate laboratory cultures 8.) opportunists are usually nonpathogenic 9.) they are opportunistic pathogens in debilitated patients 10.) antibiotics in fungal media inhibit fungal opportunists 11.) fungal opportunists must be repeatedly isolated in large numbers from cultured patient specimens to be considered the causative agent of a disease

CHAPTER 3: COMMON FUNGAL OPPORTUNISTS STUDY QUESTIONS 1-4 ON PAGE 105 1.) Penicillium, Gliocladium, Paecilomyces, and Scopulariopsis all possess a penicillus. Describe three ways they may be distinguished from each other.

ANY THREE OF THE FOLLOWING WILL SUFFICE: 1.) Gliocladium forms balls of conidia; the rest produce chains 2.) Paecilomyces exhibits tapering (elongated) conidiophores; the rest do not 3.) Scopulariopsis conidia are lemon shaped; the rest are round 4.) Scopulariopsis colonies are tan; the rest are usually shades of green 5.) All except Gliocladium have been numerously reported as human pathogens (Gliocladium is not known to be a human pathogen)

CHAPTER 3: COMMON FUNGAL OPPORTUNISTS FINAL EXAM QUESTIONS 1-14 ON PAGES 110-113 4.) Epicoccum sp., Ulocladium sp., and Stemphylium sp. all demonstrate similar conidia. Describe at least two ways to differentiate them

ANY TWO OF THE FOLLOWING: 1.) Epicoccum has sporodchia, while Ulocladium possesses a bent-knee conidiophore, and Stemphylium has a straight conidiophore. 2.) Epicoccum produces orange colonies; the others produce black ones. 3.) Stemphylium conidial cross septa are constricted; those in the other two fungi are not

CHAPTER 3: COMMON FUNGAL OPPORTUNISTS FINAL EXAM QUESTIONS 1-14 ON PAGES 110-113 A 10-year-old boy was admitted to the hospital in diabetic ketoacidosis. Three days later, he complained of nasal sinus blockage. Stained histologic preparations of material removed from his sinuses showed the wide, ribbonlike hyphae seen in Figure 3-58. On SABHI agar, a white cottony mold rapidly grew. 14.) Since this is an opportunistic infection, should the physician wait for repeated cultures before initiating treatment? Why or why not?

Absolutely not. This disease rapidly erodes into surrounding blood vessels, with systemic spread and death within 10 days after the initial sinus/eye symptoms. Treatment should begin as soon as the disease is suspected.

CHAPTER 2: LABORATORY PROCEDURES FOR FUNGAL CULTURE AND ISOLATION STUDY QUESTIONS 1-8, PAGES 36-37 1.) List three general rules for good collection of fungal specimens

Any of the following three would be correct: 1.) Make sure the specimen is collected from the area that is most likely affected 2.) Use sterile technique in collected the specimen 3.) The specimen must be adequate 4.) The specimen must be delivered promptly to the laboratory 5.) The specimen must be adequately labeled

CHAPTER 2: LABORATORY PROCEDURES FOR FUNGAL CULTURE AND ISOLATION STUDY QUESTIONS ON PAGE 49, QUESTIONS 1-10 1.) Why is it important to perform a direct mount examination on every specimen? Give two reasons.

Any two of the following answers are adequate: 1.) A direct exam allows you to send out an immediate preliminary report to the physician, so he or she may initiate treatment or look for other diagnoses. 2.) With the direct exam results, you will know whether to inoculate any special media. 3.) The direct exam allows you to observe the yeast phase of dimorphic organisms. 4.) The direct exam may provide a clue as to the identity of the causative agent, without having to wait for the fungus to incubate

CHAPTER 2: LABORATORY PROCEDURES FOR FUNGAL CULTURE AND ISOLATION FINAL EXAM QUESTIONS 1-7 ON PAGES 66-67 2.) Fill in the blanks: Two fungi that may be found in cerebrospinal fluid are ___________ and ___________.

Any two of the following: 1.) Coccidioides immitis 2.) Histoplasma capsulatum 3.) Actinomyces 4.) Nocardia 5.) Candida 6.) Cryptococcus neoformans -opportunists repeatedly isolated

CHAPTER 2: LABORATORY PROCEDURES FOR FUNGAL CULTURE AND ISOLATION STUDY QUESTIONS 1-6 ON PAGE 57 1.) The best medium to isolate fungal opportunists from a nonsterile site is:

B. Inhibitory mold agar (IMA) EXPLANATION: -DTM and cycloheximide inhibit fungal opportunists. -BHIB (without antibiotics) will be overgrown by bacteria, making isolation of opportunists difficult

CHAPTER 2: LABORATORY PROCEDURES FOR FUNGAL CULTURE AND ISOLATION STUDY QUESTIONS 1-7 ON PAGES 71-72 1.) All of the following are true concerning skin tests for blastomycosis, histoplasmosis, and coccidioidomycosis except:

B. Perform the skin tests before taking blood for serology

CHAPTER 3: COMMON FUNGAL OPPORTUNISTS STUDY QUESTIONS 1-3 ON PAGE 96 2.) Which of the following possess conidia with vertical and horizontal cross-walls?

B. Ulocladium sp. D. Epicoccum sp. F. Alternaria sp. G. Stemphylium sp.

CHAPTER 2: LABORATORY PROCEDURES FOR FUNGAL CULTURE AND ISOLATION CULTURING THE SPECIMEN PRIMARY FUNGAL MEDIA NONSELECTIVE MEDIA 2.) Brain Heart Infusion Agar with Blood (BHIB) Give characteristics of BHIB -see page 53, first box for BHIB medium preparation and procedure

BHIB VS. SABHI: -BHIB is a general medium that is nutritionally richer than SABHI. BHIB SHOULD ONLY BE USED IN THE FOLLOWING SITUATIONS: -since BHIB is nutritionally richer than SABHI, it should be reserved for specimens.....: 1.) From normally a sterile site OR FOR 2.) Anaerobic actinomycetes BHIB MEDIUM PREPARATION AND PROCEDURE: -go to page 53 and type out or write out the whole box (first box), thoroughly study it

CHAPTER 2: LABORATORY PROCEDURES FOR FUNGAL CULTURE AND ISOLATION FUNGAL SOURCES 4.) Respiratory: Bronchial Washings, Sputa, Throats, Transtracheal Aspirates If actinomycosis is suspected, what should be done?

BRONCHIAL WASHINGS OR TRANSTRACHEAL ASPIRATES: -specimens are sent as soon as possible for processing and anaerobic culturing, with EXPLICIT instructions to culture for Actinomyces RESPIRATORY SWAB SPECIMENS: -are immediately placed in an anaerobic transport container USING A SYRING TO OBTAIN THE SPECIMEN: -if a syringe is used to obtain the specimen, be sure to seal the needle with a cork; utilize safety precautions when corking the needle

CHAPTER 2: LABORATORY PROCEDURES FOR FUNGAL CULTURE AND ISOLATION STUDY QUESTIONS 1-7 ON PAGES 71-72 7.) Discuss problems with successful antifungal treatment of immunocompromised patients

Because the immune system isn't there to help infection, and also patients are frequently granulocytopenic, antifungal agents alone only temporarily work, with frequent relapses and eventually antifungal drug resistance.

CHAPTER 1: BASICS OF MYCOLOGY What is the temperature for the YEAST phase (also known as TISSUE PHASE) of a dimorphic fungi?

Body temperature (37 degrees celcius) or incubator temperature (35 degrees celcius)

CHAPTER 3: COMMON FUNGAL OPPORTUNISTS FINAL EXAM QUESTIONS 1-14 ON PAGES 110-113 A 10-year-old boy was admitted to the hospital in diabetic ketoacidosis. Three days later, he complained of nasal sinus blockage. Stained histologic preparations of material removed from his sinuses showed the wide, ribbonlike hyphae seen in Figure 3-58. On SABHI agar, a white cottony mold rapidly grew. 12.) Three agents of this mycosis are:

C. Absidia, Mucor, and Rhizopus

CHAPTER 1: BASICS OF MYCOLOGY CHAPTER 1 FINAL EXAM QUESTIONS, PAGES 24-26 3.) A patient is seen by his physician for a hard, nonmoving nodule below the skin on his right index finger. There are no other symptoms. The infection that this patient is most likely presenting is:

C. subcutaneous

CHAPTER 2: LABORATORY PROCEDURES FOR FUNGAL CULTURE AND ISOLATION STUDY QUESTIONS 1-7 ON PAGES 71-72 3.) On cryptococcal antigen testing list three causes of false-positive results, and one way to decrease them

CAUSES OF FALSE POSITIVE, ANY THREE OF THE FOLLOWING: 1.) Trichosporon infection 2.) collagen vascular disease 3.) malignancy 4.) rheumatoid arthritis 5.) syneresis fluid 6.) complement WAYS TO DECREASE FALSE-POSITIVE RESULTS, ANY OF THE FOLLOWING: 1.) heat inactivation of nonspecific interference and complement 2.) pronase pretreatment 3.) 2-beta-mercaptoethanol pretreatment 4.) removal of syneresis fluid from agar plates before taking colonies for testing

CHAPTER 1: BASICS OF MYCOLOGY What is the difference between chlamydospores and chlamydoconidia?

CHLAMYDOSPORES: -thick-walled yeast vesicles (seen in candida albicans) that are NOT conidia, since they neither germinate nor produce conidia when mature CHLAMYDOCONIDIA: -applies to the germinating structures

CHAPTER 2: LABORATORY PROCEDURES FOR FUNGAL CULTURE AND ISOLATION FUNGAL SOURCES 3.) Cutaneous: Hair, Nails, and Skin explain hair specimen preparation and examination

COLLECTION OF HAIR: -hair is plucked out by the roots with sterile forceps CHOOSING PROPER HAIR SPECIMEN: -many fungi that infect hair will fluoresce with a Wood's lamp (366 nm) -hairs that fluoresce should be chosen for the specimen -if none of the hairs fluoresce, choose ones that are broken and scaly PLACING HAIR SPECIMEN FOR PROCESSING: -place the hair specimen in a sterile Petri dish for processing

CHAPTER 2: LABORATORY PROCEDURES FOR FUNGAL CULTURE AND ISOLATION FUNGAL SOURCES 2.) Cerebrospinal fluid cerebrospinal fluid (CSF) specimen characteristics

COLLECTION: -CSF is aseptically (free from contamination caused by harmful bacteria, viruses, or other microorganisms) collected as for bacteriology. PREPARATION AND EXAMINATION: 1.) the specimen is centrifuged 2.) one drop of sediment is placed on a slide, coverslip is added, and the mount is microscopically examined for fungal elements 3.) the remaining sediment is inoculated to AEROBIC fungal media -make sure to also include ANAEROBIC media if actinomyces is suspected INDICATION FOR MORE DIRECT MOUNTS -the following direct mounts may be indicated depending on the suspected agent: 1.) India ink preparation 2.) Wright stain

CHAPTER 2: LABORATORY PROCEDURES FOR FUNGAL CULTURE AND ISOLATION FUNGAL SOURCES 4.) Respiratory: Bronchial Washings, Sputa, Throats, Transtracheal Aspirates Bronchial washing characteristics

COLLECTION: -bronchial washings are obtained by threading a tube down the patient's throat to their bronchi, injecting a little sterile saline to pick up organisms in the lungs, and aspirating up the material -bronchial washings should be collected upon rising in the morning -overnight incubation and multiplication of fungi in the lungs will increase the chance of isolating fungi on culture -a 24-hour collection is DISCOURAGED (DON'T DO IT) because it becomes easily overgrown with bacterial and fungal contaminants CONTAMINATION ISSUES: -since the tube passes down the throat, bronchial washings are usually contaminated with throat flora

CHAPTER 2: LABORATORY PROCEDURES FOR FUNGAL CULTURE AND ISOLATION FUNGAL SOURCES 4.) Respiratory: Bronchial Washings, Sputa, Throats, Transtracheal Aspirates Sputa characteristics

COLLECTION: -sputa should be collected upon rising in the morning -overnight incubation and multiplication of fungi in the lungs will increase the chance of isolating fungi on culture -a 24-hour collection is DISCOURAGED (DON'T DO IT) because it becomes easily overgrown with bacterial and fungal contaminants PATIENT PREPARATION: 1.) the patient should NOTTTTT eat before donating the specimen, as food left in the mouth may contain fungi that will grow on the patient's culture 2.) the patient should brush his/her teeth, rinse with water, then produce material from a DEEP COUGH SENDING SPUTA: -the sputa specimen is put in a sterile container and sent IMMEDIATELY to the laboratory CONTAMINATION: -since sputa is coughed up through the throat, it WILL BE contaminated with normal flora

CHAPTER 2: LABORATORY PROCEDURES FOR FUNGAL CULTURE AND ISOLATION FUNGAL SOURCES 6.) Urine Urine specimen characteristics

COLLECTION: -urine should be collected after overnight incubation in the bladder, so it should be a FIRST MORNING SPECIMEN -a clean catch or catheterized urine specimen collection method should be used, because it minimizes the presence of superficial genital flora PROSTATIC MASSAGE: -prostatic massage before urination may pick up: 1.) C. neoformans 2.) B. dermatitidis 3.) H. capsulatum 4.) C. immitis -these fungi can cause prostatitis, especially in acquire immunodeficiency syndrome (AIDS) patients. METHODS THAT ARE DISCOURAGED (DO NOT DO): 24-hour urine specimens are discouraged because of overgrowth with opportunists -urine should NOT be collected from a bedpan, as the pan may also contain opportunists PROCESSING: -the urine specimen is placed in a sterile container and sent IMMEDIATELY to the laboratory EXAMINATION AND PREPARATION: -in the lab, some of the urine is centrifuged and a direct mount for fungal exam is made with ONE DROP of the sediment -stains may also be performed as indicated IF FUNGI ARE OBSERVED IN URINE: -if fungi are observed, a preliminary report is sent to the physician FUNGAL IN URINE SIGNIFICANCE- HOW IT HAS CHANGED -usually urine is inoculated for bacteriology (calibrated loop, uncentrifuged urine, blood agar plate) and if fungi grow, they are quantitated and identified. -BUT, the efficacy of quantitating fungi has been questioned -clinically, for a clean catch urine, over 100,000 fungal colonies of one kind per milliliter of urine were significant of infection; 10,000 to 100,000 were suspect; and under 10,000 or three different fungal/bacterial organisms present on the culture were representative of probably genital contamination -NOW, significance of fungus in urine is based more on clinical findings, and identification is at the discretion of the individual physician COLLECTION OF URINE FROM A SUPRAPUBIC PUNCTURE AND FUNGUS SIGNIFICANCE -ANY fungal colonies obtained from this method are significant and should be identified IF AN UNCOMMON ORGANISM IS SUSPECTED, HOW IS THE CHANCE OF RECOVERY INCREASED? -chance of recovery is increased if the urine is centrifuged and the sediment inoculated to fungal media -the only downside of this ^^^ is that this procedure can NOT be used. to quantitate organisms, just any fungi that are grown are identified

CHAPTER 2: LABORATORY PROCEDURES FOR FUNGAL CULTURE AND ISOLATION FUNGAL SOURCES 4.) Respiratory: Bronchial Washings, Sputa, Throats, Transtracheal Aspirates throat specimen characteristics

COLLECTION: -throat specimens are obtained by rolling TWO sterile swabs INDIVIDUALLY over the affected area -collection should be performed PRIOR to eating -the swabs are kept moist with 0.5 mL sterile saline until they are processed in the laboratory IF CULTURING CANDIDA: -candida organisms do NOT stick well to the swab -therefore, scrape off material with a sterile tongue depressor, put the depressor in a sterile tube, and send IMMEDIATELY to the laboratory

CHAPTER 2: LABORATORY PROCEDURES FOR FUNGAL CULTURE AND ISOLATION FUNGAL SOURCES 5.) Tissue, Biopsies overview of tissue, biopsy specimens

COLLECTION: -tissue and biopsy specimens are obtained by the PHYSICIAN -specimens should include: 1.) normal tissue 2.) the center of the lesion (injury) 3.) the edge of the lesion (injury) STORAGE/PRESERVATION FOR PROCESSING -specimens are kept moist with sterile saline until ready for processing EXAMINATION OF TISSUES: -tissues are aseptically teased apart in a sterile Petri dish and examined for: 1.) granules 2.) pus 3.) necrosis (death of tissue) -use these areas (granules, pus, necrosis) of tissue for culture IF GRANULES ARE PRESENT IN TISSUE -if granules are present in tissue, proceed as described under wounds, subcutaneous lesions and mucocutaneous lesions (see page 36, last heading for specimen collection) IF AREAS OF PUS OR NECROSIS ARE EVIDENT IN TISSUE -if areas of pus or necrosis are evident, inoculate these directly onto appropriate aerobic and anaerobic fungal media, smearing some on slides for: 1.) potassium hydroxide preparation 2.) stained preparations IF THERE ARE NO AREAS OF PUS, NECROSIS, OR GRANULES IN TISSUE -if there are no areas of pus, necrosis, or granules in tissue, then mince the rest of the tissue with a sterile scalpel and grind it in a tissue homogenizer. -inoculate the homogenized material onto aerobic and anaerobic fungal media, smearing some slides for: 1.) potassium hydroxide preparation 2.) stained preparations -and also make direct smears IF ACTINOMYCES IS SUSPECTED -is actinomyces is suspected, the specimen must be processed under anaerobic conditions

CHAPTER 2: LABORATORY PROCEDURES FOR FUNGAL CULTURE AND ISOLATION FUNGAL SOURCES 4.) Respiratory: Bronchial Washings, Sputa, Throats, Transtracheal Aspirates transtracheal aspirate characteristics

COLLECTION: -transtracheal aspirates are obtained by inserting a tube, through a surgically made slit in the trachea, down to the bronchi, aspirating lung secretions -this procedure should be done in the MORNING, when lung secretions have accumulated -this procedure bypasses the throat (does not come into contact with the throat), so NO throat flora contamination should be seen

CHAPTER 2: LABORATORY PROCEDURES FOR FUNGAL CULTURE AND ISOLATION CULTURING THE SPECIMEN PRIMARY FUNGAL MEDIA SELECTIVE MEDIA WITH ANTIBIOTICS What are the 3 antibiotics that are frequently added in different combinations to media to select for various fungi? What do these specific antibiotics inhibit?

COMMON ANTIBIOTICS ADDED TO SELECT FOR VARIOUS FUNGI: 1.) Gentamicin -inhibits bacteria 2.) Chloramphenicol -inhibits funguslike bacteria and yeast phases of dimorphic fungi 3.) Cycloheximide -inhibits funguslike bacteria, many yeasts, and many fungal opportunists

CHAPTER 3: COMMON FUNGAL OPPORTUNISTS COMMON PROPERTIES OF FUNGAL OPPORTUNISTS There are a number of generalization that can be made regarding fungal opportunists, explain them all

COMMON PROPERTIES OF FUNGAL OPPORTUNISTS: 1.) RAPID GROWING -most are rapid growers, forming mature colonies in 4 or 5 days 2.) SAPROBIC AND AIRBORNE -they are saprobic, meaning that they live on decaying organic matter in the soil, and sometimes become airborne 3.) NORMALLY INHALED -since we constantly inhale the conidia of fungal opportunists, routine cultures of sputum and other respiratory secretions may normally yield a few colonies of these organisms -also, since the conidia are in the air, they may contaminate the skin as well as laboratory cultures 4.) OPPORTUNISTIC PATHOGENS -Usually these organisms are nonpathogenic. However, they act as opportunistic pathogens -when the patient is debilitated in some way, as from illness or especially from immunosuppressive drugs, these common organisms may multiply and cause disease, often with fatal consequences 5.) LABORATORY DIAGNOSIS -in order to isolate an opportunist, be sure to inoculate media free of antibiotics, since the drugs inhibit these fungi. -because they are so common in the environment, opportunistic pathogens must be repeatedly isolated in large numbers from cultures specimens of the patient, or from normally sterile sites, to be considered the causative agent of the disease -since fungal opportunists are differentiated on the bases of their microscopic morphology, isolates should be transferred to media such as potato dextrose or corn meal agar to enhance formation of characteristic conidia.

CHAPTER 3: COMMON FUNGAL OPPORTUNISTS OPPORTUNISTIC MYCOSES Keratomycosis characteristics

CORNEA OF THE EYE AND INFECTION -the cornea of the eye is resistant to infection -however, with predisposing factors, fungi can invade the cornea and cause keratomycosis FACTORS THAT CAN LEAD TO KETAOMYCOSIS INFECTION: 1.) trauma to the eye 2.) the use of corticosteroids (antiinflammatory medications used in conjunction with antibiotics for supposed bacterial infection of the eye) 3.) glaucoma WHAT HAPPENS AFTER TRAUMA TO THE EYE ALLOWS INFECTION? -a white or cream-colored infiltrate develops after the conidia germinate, and hyphal growth forms in the area of trauma. -ulceration occurs in the cornea, eventually causing scarring and subsequent blindness. FUNGAL LESION PAIN: -fungal lesions range from extremely painful to the unnoticed-unnoticed possibly because of decreased sensation of the eye which follows nerve damage or long-continued wearing of contact lenses -the lesions may elicit an inflammatory response (pus cell) response as described above, or none at all. WHAT SHOULD BE TAKEN FOR DIRECT MOUNTS AND CULTURE? -corneal scrapings WHAT MUST BE DONE FOR CONFIRMATION? -since the causative organisms are mostly opportunists, a fungus must be repeatedly isolated to be considered etiologically significant -there are over 80 fungi that may cause keratomycosis TREATMENT FOR KERATOMYCOSIS: -the drug of choice is natamycin, a polyene antifungal agent which, because of its wide spectrum activity, is effective against yeasts as well as hyaline and dematiaceous molds. -natamycin is administered in the form of a 5% suspension which must be applied to the infected eye as often as every hour for the first few days. -rapid improvement of the infection occurs with this regimen

CHAPTER 3: COMMON FUNGAL OPPORTUNISTS SEPATATE OPPORTUNISTS HYALINE SEPTATE OPPORTUNISTS Aspergillus species characteristics

CULTURE: -SABHI agar -room temperature -rapid-growing colony -colony is rugose and velvety. -various colors of the colony are due to dense production of conidia: blue, green, yellow, black, and white MICROSCOPIC: -the mycelium is septate -unbranched, rough or smooth conidiophores with a FOOT CELL at their base support a large VESICLE at their tip -the vesicle in turn supports short, flask-shaped PHIALIDES in a SINGLE or DOUBLE row, which produce CHAINS of smooth or rough PHIALOCONIDIA. RESEMBLANCE: -Syncephalastrum sp. must not be confused with Aspergillus (Syncephalastrum sp. is a similar-appearing zygomycete). -Syncephalastrum is different from Aspergillus in that it is aseptate, possesses no phialides, and exhibits chains of spores in tubes (merosporangia) off the vesicle OTHER COMMENTS: -if possible, this genus should be speciated, especially A. fumigatus -A. fumigatus grows at 45 degrees celcius, while most other Aspergillus species do not. PATHOGENICITY: -A. fumigatus is the most common opportunistic pathogen of the genus -Aspergillus species cause: 1.) disseminated aspergillosis 2.) pulmonary disease 3.) allergic bronchopulmonary disease (farmer's lung) 4.) keratomycosis 5.) otomycosis 6.) infection of the nasal sinuses

CHAPTER 3: COMMON FUNGAL OPPORTUNISTS SEPATATE OPPORTUNISTS DERMATIACEOUS SEPTATE OPPORTUNISTS Curvularia species characteristics

CULTURE: -SABHI agar -room temperature -colony is moderately rapid-growing, cottony, and white, light pink, orange, or green, with a brown reverse MICROSCOPIC: -septate mycelium is DARK -large, four- to five-celled, dark POROCONIDIA are borne on a BENT-KNEE shaped CONIDIOPHORE. -the poroconidia are centrally distended owing to an OVER-ENLARGED CENTRAL CELL, and the ends are lighter than the middle PATHOGENICITY: -Curvularia causes the following: 1- keratomycosis (usually causes this) 2- mycetoma (draining subcutaneous lesions on the extremities) 3- endocarditis 4- pulmonary infection 5- allergies 6- infection of the nasal septum

CHAPTER 4: SUPERFICIAL AND DERMATOPHYTIC FUNGI SUPERFICIAL MYCOSES Trichosporon beigeli (Cutaneum) characteristics

CULTURE: -SABHI agar -room temperature -forms colony within 5 days MICROSCOPIC: -on corn meal-tween 80 agar -hyaline hyphae with blastoconidia and arthroconidia are observed -biochemical tests can be performed to identify this fungus PATHOGENICITY: 1.) Disease that trichosporon beigeli causes: -white piedra 2.) White piedra explanation -consists of light brown, soft nodules around beard and mustache hairs. -the nodules from white piedra are less firmly attached than those of black piedra 3.) White piedra and immunocompromised patients (disseminated white piedra) -in immunocompromised patients, invasion of the blood, kidneys, lungs, and skin may occur. -the disseminated form of disease usually manifests as fever, pulmonary infiltrates, and cutaneous lesions. 4.) Treatment for white piedra -treatment of white piedra consists of cutting or shaving all the hairs in the affected area. -in addition, topical application of clotrimazole cream, amphotericin B lotion, or 5% ammoniated mercury ointment is recommended to prevent recurrence of the infection 5.) T. beigelii resistance in disseminated disease -when causing disseminated disease, T. beigelii is resistant to amphotericin B

CHAPTER 4: SUPERFICIAL AND DERMATOPHYTIC FUNGI SUPERFICIAL MYCOSES Exophiala (Phaeoannellomyces, Cladosporium) werneckii characteristics

CULTURE: -SABHI agar -room temperature -slow-developing -black, yeasty colony which may later be covered with short olive-gray mycelium MICROSCOPIC: -the yeast portion of the colony contains only dark, one- or two-celled blastoconidia -the older mold portion, hyphae develop with one- or two-celled dark blastoconidia in large clusters, which resemble Candida, and blastoconidia in small clusters, which resemble the opportunist Aureobasidium -very old colonies produce annellides with clusters or chains of one- or two-celled dark annelloconidia RESEMBLANCE: 1.) Candida -the older mold portion, hyphae develop with one- or two-celled dark blastoconidia in large clusters, which resemble Candida 2.) Aureobasidium -in the older mold portion, the blastoconidia in small clusters resemble the opportunist Aureobasidium 3.) E. jeanselmie or Wangiella dermatitidis -E. werneckii may be easily mistaken for these two fungi; however, these organisms may be differentiated on the basis of nitrate utilization and maximum growth temperature OTHER COMMENTS: -E. wernickii may be classified in the genus Phaeoannelomyces based on the production of budding cells with annellides when grown on potato dextrose agar at 25 degrees celcius, but different culture and media conditions may have other morphological forms that predominate, so this classification is not accepted by some mycologists PATHOGENICITY: 1.) Condition that Exophilia wernickii causes -tinea nigra 2.) Explanation of tinea nigra -brown to black, nonscaly patches form primarily ont he palms of the hands and enlarge over a period of months to years 3.) Where tinea nigra is typically found -most cases in the United States occur in Southeastern states such as Louisiana, Alabama, and North Carolina 4.) Treatment for tinea nigra -with daily use of topical antifungal agents the patcvhes usually dissapear in 2-4 weeks -topical antifungal agents include: 1- undecylenic acid OR 2- imidazole

CHAPTER 4: SUPERFICIAL AND DERMATOPHYTIC FUNGI SUPERFICIAL MYCOSES Piedraia hortai characteristics

CULTURE: -SABHI agar -room temperature -slowly-forming -compact, greenish black, heaped, glabrous colony MICROSCOPIC: -produces dark, thick-walled hyphae with swellings. -ASCI (ascus) containing ascospores may be present PATOGENICITY: 1.) Condition that P. hortai causes: -black piedra 2.) Explanation of black piedra -consists of firmly attached, hard black nodules around the outside of scalp hairs 3.) Treatment of black piedra -treatment consists of cutting or shaving all the hairs in the affected area.

CHAPTER 3: COMMON FUNGAL OPPORTUNISTS SEPATATE OPPORTUNISTS DERMATIACEOUS SEPTATE OPPORTUNISTS Give characteristics of Alternaria species

CULTURE: -on SABHI agar -room temperature -light gray, woolly colony -rapidly matures to dark greenish black or brown, with a black reverse MICROSCOPIC: -reproductive structures and hyphae are DARK -the CHAINED POROCONIDIA, which contain HORIZONTAL and VERTICAL SEPTA, have club-shaped bases with tapered apices. -if the poroconidia are not in chains, they may be mistaken for the opportunist STEMPHYLIUM PATHOGENICITY: -Alternaria has been reported in: 1- keratomycosis 2- skin infections 3- osteomyelitis 4- pulmonary disease 5- nasal septum infection

CHAPTER 4: SUPERFICIAL AND DERMATOPHYTIC FUNGI SUPERFICIAL MYCOSES Malassezia furfur (Pityrosporum ovale, Pityrosporum orbiculare) characteristics

CULTURE: -M. furfur will NOT grow on routine culture media unless a lipid such as olive oil is layered over it and the culture is incubated at 37 degrees celsius -the Dupont Isolator system is recommended for recovery of M. furfur from blood -alternatively, addition of 3% palmitic acid to the BACTEC Peds Plus blood culture bottles allows detection of M. furfur within 24-48 hours with the BACTEC NR 660 system LABORATORY DIAGNOSIS CONFIRMATION -laboratory diagnosis is confirmed by observing growth of yeast on medium (SABHI or blood agar) overlaid with olive oil or on Faergemann agar (Remel), versus absence of growth on medium without olive oil MICROSCOPIC: -in KOH or stained preparations from skin disease -M. furfur appears as thick, round to oval cells in clusters, accompanied by short, angular hyphae- a SPAGHETTI AND MEATBALLS semblance. -in patients with sepsis, the organism can rarely be seen on smears of peripheral blood. -when recovered in laboratory culture, budding yeast cells are observed. -buds form repeatedly from the same pole of the mother cell (UNIPOLAR), resulting in a "collarette" formation PATHOGENICITY: 1.) Diseases caused by M. furfur 1- pityriasis versicolor 2- nasal sinusitis 3- cases of folliculitis (particularly an antibiotic-resistant form occurring on the trunk and legs of AIDS patients) 4- intravenous line sepsis in patients receiving intravenous lipid infusions (especially premature infants of very low birth weight) 2.) Explanation of pityriasis versicolor -An asymptomatic skin infection characterized by scaly patches of different colors: reddish brown, brown, and white. The patches fluoresce under a Wood's lamp. 3.) Treatment for M. furfur conditions 1- Treatment for pityriasis versicolor -topical application of selenium sulfide or a cream of clotrimazole or miconazole is usually effective therapy. 2- Treatment for systemic disease -M. furfur is susceptible to miconazole and ketaconazole and moderately susceptible to amphotericin B. -Flucytosine will NOT work as treatment, because M. furfur is resistant to this treatment

CHAPTER 3: COMMON FUNGAL OPPORTUNISTS SEPATATE OPPORTUNISTS DERMATIACEOUS SEPTATE OPPORTUNISTS Epicoccum species characteristics

CULTURE: -SABHI agar -room temperature -colonial rings of yellow, orange, and brown are seen, and pigments of the same color may diffuse into the agar MICROSCOPIC: -thick clusters of SPORODCHIA (short conidiophores) support terminal DARK round conidia with unconstricted HORIZONTAL and VERTICAL SEPTA -with age, the conidia become rough-walled PATHOGENICITY: -has been associated with allergies RESEMBLANCE: -Epicoccum species resembles the opportunists: 1.) Ulocladium 1- dark round to oval poroconidia 2- unconstricted 3- no sporodchia: bent-knee shaped conidiophore 4- gray to black colony, reverse black 2.) Stemphylium 1- dark oval poroconidia 2- constricted 3- no sporodchia: new poroconidium produced through old scar 4- gray to black colony, reverse light gray -----Epicoccum have the following that differs from the other species: 1- dark round conidia 2- unconstricted horizontal and vertical septa in conidia 3- sporodchia 4- orange colony, reverse brown

CHAPTER 3: COMMON FUNGAL OPPORTUNISTS SEPATATE OPPORTUNISTS DERMATIACEOUS SEPTATE OPPORTUNISTS Aureobasidium species characteristics

CULTURE: -SABHI agar -room temperature -colonies are initially shiny white and yeastlike -with age, colonies become shiny black and leathery with a white fringe (BLACK YEAST) MICROSCOPIC: -light to DARK brown conidiophores are not differentiated from the hyphae -short DENTICLES support HYALINE solitary or clustered CONIDIA, from which secondary blastoconidia may arise -with AGE, conidiophores become dark, one- to two- celled ARTHROCONIDIA PATHOGENICITY: -Aureobasidium species have been reported in the following: 1- foot and leg lesions 2- in allergies 3- in a case of cheloid blastomycosis

CHAPTER 3: COMMON FUNGAL OPPORTUNISTS SEPATATE OPPORTUNISTS HYALINE SEPTATE OPPORTUNISTS Penicillium species characteristics

CULTURE: -SABHI agar -room temperature -the rapid-growing colony is initially velvety and white, later becoming powdery and BLUE GREEN with a white periphery and colorless reverse MICROSCOPIC: -mycelium is septate -PENICILLI bear flask-shaped PHIALIDES, which in turn support CHAINS or round PHIALOCONIDIA -conidiophores and phialoconidia may be hyaline to pigmented, and smooth to rough, depending on the species PATHOGENICITY: Penicillium causes the following: 1.) keratomycosis 2.) penicilliosis 3.) otomycosis 4.) onychomycosis 5.) rarely causes deep infection PENICILLIUM MARNEFFEI CULTURE: -SABHI agar -room temperature -the rapid-growing, grayish fluccose colonies have a finely wrinkled surface and a DARK RED pigment that diffuses throughout the agar -at 37 degrees celsius, on blood agar, yeastlike colonies may appear in 3-4 days and are grayish white, waxy, and attached to the agar surface PENICILLIUM MARNEFFEI MICROSCOPIC: -at room temperature, the septate mycelium is typical of Penicillium with conidiophores bearing up to five short broad METULAE (branched conidiophores). -the WIDE PHIALIDES, which taper to narrow apices, are borne in groups of four to six on the metulae -smooth, lemon-shaped phialoconidia occur in chains -growth at 37 degrees celsius on blood agar is yeastlike; filamentous forms and yeast forms may be seen PENICILLIUM MARNEFFEI PATHOGENICITY: -Penicillium marneffei causes a disseminated form of penicilliosis -Penicillium marneffei is a dimorphic fungus.

CHAPTER 3: COMMON FUNGAL OPPORTUNISTS SEPATATE OPPORTUNISTS DERMATIACEOUS SEPTATE OPPORTUNISTS Bipolaris species characteristics

CULTURE: -SABHI agar -room temperature -the rapid-growing colony is velvety or woolly, at first appearing grayish-brown; later the center is matted and black, and the reverse is light or dark MICROSCOPIC: -the septate hyphae are DARK -numerous dark CYLINDRICAL, four- or five- celled POROCONIDIA with TRUNCATE HILA (points of attachment) are usually present in clusters along a BENT-KNEE shaped CONIDIOPHORE. -when a poroconidium germinates, germ tubes may arise from one or both POLES (ends) and grow along the axis of the conidium. -Bipolaris may be confused with the rarely pathogenic Drechslera species, but the poroconidia of Drechslera species have rounded, nonprotruding hila and germ tubes that arise perpendicular to the axis of the conidium PATHOGENICITY: -Bipolaris causes: 1- keratomycosis (frequently) 2- fungal sinusitis (frequently) 3- subcutaneous abscesses (occasionally) 4- phaeohyphomycosis (occasionally) 5- meningitis 6- allergies 7- peritonitis -long-term therapy with amphotericin B may be effective in some, but not all, cases.

CHAPTER 3: COMMON FUNGAL OPPORTUNISTS SEPATATE OPPORTUNISTS HYALINE SEPTATE OPPORTUNISTS Sepedonium species characteristics

CULTURE: -SABHI agar -room temperature -the waxy and white colony rapidly becomes velvety and lemon-colored with a peripheral fringe, and white reverse MICROSCOPIC: -single or clustered, thick-walled, smooth to rough (MACRO) conidia form at the ends of simple or branched conidiophores which are not well differentiated from the vegetative mycelium. -formerly, it was thought that single, smooth, and thin-walled, elliptical (micro) conidia were also produced: it is now thought that these only represent young macroconidia RESEMBLANCE: -Sepedonium greatly imitates the systemic dimorph Histoplasma capsulatum which possesses macro- and microconidia, but Sepedonium does NOT convert to a yeast phase at 37 degrees celsius, but H. capsulatum does PATHOGENICITY: -Sepedonium is not known to be a human pathogen

CHAPTER 3: COMMON FUNGAL OPPORTUNISTS SEPATATE OPPORTUNISTS DERMATIACEOUS SEPTATE OPPORTUNISTS Nigrospora species characteristics

CULTURE: -SABHI agar -room temperature -white, woolly colony with a black reverse rapidly fills the plate -with age, the aerial mycelium turns gray MICROSCOPIC: -the hyphae are dark -short, fat conidiophores support SINGLE, oval, smooth-walled, BLACK conidia at the tips PATHOGENICITY: -Nigrospora has been reported as a causative agent of keratomycosis

CHAPTER 3: COMMON FUNGAL OPPORTUNISTS SEPATATE OPPORTUNISTS DERMATIACEOUS SEPTATE OPPORTUNISTS Cladosporium (Hormodendrum) species characteristics

CULTURE: -SABHI agar -room temperature -the colony is moderately slow-growing for an opportunist, requiring 7 days -colony is powdery or velvety, heaped and folded, and dark gray-green with the reverse black MICROSCOPIC: -the septate hyphae are DARK -short CHAINS of dark one- to four-celled BLASTOCONIDIA with a distinct SCAR at each point of attachment are borne from REPEATEDLY FORKING SHIELD CELLS (shield-shaped conidiogenous cells) OTHER COMMENTS: -in the past opportunistic cladosporium species were differentiated from pathogenic ones by the opportunist's ability to hydrolyze nutrient gelatin but this test is NOT reliable. -differentiation should be done by growth rate, microscopic morphology, and clinical picture. PATHOGENICITY: -may cause: 1.) keratomycosis 2.) allergies

CHAPTER 3: COMMON FUNGAL OPPORTUNISTS SEPATATE OPPORTUNISTS HYALINE SEPTATE OPPORTUNISTS Chrysosporium species characteristics

CULTURE: -SABHI agar -room temperature -a heaped, velvety, buff-colored colony rapidly forms, with a white, yellow, or reddish brown reverse MICROSCOPIC: -SINGLE, round to club-shaped, smooth or rough CONIDIA perch on top of short conidiophores, which are poorly differentiated from the vegetative mycelium. -conidia also develop directly off the hyphae, and swollen arthroconidia may be present RESEMBLANCE: 1.) Chrysosporium colonially and microscopically resembles the subcutaneous pathogen Scedosporium apiospermum -Chrysosporium is a rapid-grower, while S. apiospermum is an intermediate-grower -Scedosporium has annellides and the common presence of sexual stage cleistothecia, while Chrysosporium does not. 2.) Chrysosporium resembles the mold phase of the systemic dimorphs, Blastomyces dermatitidis and Histoplasma capsulatum -Chrysosporium is a rapid-grower, while the systemic dimorphs are slow-growers -Chrysosporium cannot be converted to a yeast phase at 37 degrees celsius, while the systemic dimorphs can. PATHOGENICITY: -Chrysosporium has rarely been reported as a pathogen.

CHAPTER 3: COMMON FUNGAL OPPORTUNISTS SEPATATE OPPORTUNISTS HYALINE SEPTATE OPPORTUNISTS Gliocladium species characteristics

CULTURE: -SABHI agar -room temperature -colony is initially white but rapidly fills the plate with a green, furry growth mimicking a GREEN LAWN. -some strains may remain white or turn rose colored -the reverse is white MICROSCOPIC: -PENICILLI (brushlike conidiophores) bear flask-shaped PHIALIDES, which in turn produce terminal masses of hyaline to green PHIALOCONIDIA, held together in a LARGE BALL by a gelatinous matrix RESEMBLANCE: 1.) the opportunist Trichoderma is also initially white and later green; however, the color is usually more yellowish green and the growth is cottony 2.) young penicilli with only one or two branches may be mistaken for Trichoderma and Verticillium, which exhibit single or whorled phialides with terminal balls of phialoconidia. -trichoderma structure are smaller and more delicate than Gliocladium; also, with careful searching, no penicilli should be found with either Trichoderma or Verticillium PATHOGENICITY: -Gliocladium is not known to be a human pathogen

CHAPTER 3: COMMON FUNGAL OPPORTUNISTS SEPATATE OPPORTUNISTS HYALINE SEPTATE OPPORTUNISTS Acremonium (Cephalosporium) species characteristics

CULTURE: -SABHI agar -room temperature -rapid-growing -colony is wrinkled, membranous, and white, gray, or rose, later becoming covered with loose aerial mycelium -the colony reverse is colorless, pale yellow, or pinkish MICROSCOPIC: -the mycelium is septate -unbranched tapering conidiophored support closely packed balls of sickle- or elliptical-shaped conidia. RESEMBLANCE: -if the conidiophores are in whorls around the hyphae, Acremonium may resemble the opportunist Verticillium; however, Veticillium does not have tapering conidiophores. -if the conidia are more loosely packed, Acremonium may resemble Sporothrix schenkii, but Sporothrix forms a yeast at 37 degrees celcius, while Acremonium does not PATHOGENICITY: 1.) keratomycosis 2.) mycetoma (rarely) 3.) lesions of hard palate 4.) meningitis 5.) arthritis 6.) systemic disease

CHAPTER 3: COMMON FUNGAL OPPORTUNISTS SEPATATE OPPORTUNISTS DERMATIACEOUS SEPTATE OPPORTUNISTS Exserohilum species characteristics

CULTURE: -SABHI agar -room temperature -the colony is rapid-growing with a light grayish woolly appearance at first, which becomes dark gray to black with a black reverse. MICROSCOPIC: -the septate hyphae are DARK -long, dark, CYLINDRICAL POROCONIDIA with 6-14 or more cells, and distinct, protruding, TRUNCATE HILA occur in clusters RESEMBLANCE: 1.) Exserohilum species resembles the nonpathogenic Helminthosporium sp., but Exserohilum has a bent-knee shaped conidiophore, while Helminthosporium sp. do not. 2.) Exserohilum species resembles Bipolaris and Drechslera, but Exserohilum has more cells in the conidia. PATHOGENICITY: 1.) phaeohyphomycosis (most often cause) 2.) fungal sinusitis (most often cause) Less Common: 3.) infected heart valve prosthesis 4.) aortic embolus 5.) corneal ulcer 6.) meningitis -infections may require long-term therapy with amphotericin B

CHAPTER 3: COMMON FUNGAL OPPORTUNISTS SEPATATE OPPORTUNISTS HYALINE SEPTATE OPPORTUNISTS Paecilomyces species characteristics

CULTURE: -SABHI agar -room temperature -the powdery, velvety, or cottony mycelium very rapidly matures to an OLIVE TAN, although shades of violet or brown may be seen. MICROSCOPIC: -single, whorled, or penicillus-type ELONGATED phialides bear chains of smooth or rough, hyaline to pigmented oval CONIDIA. RESEMBLANCE: -microscopically, Paecilomyces maintains a close resemblance to the opportunists Penicillium and Verticillium, but these opportunists do NOT exhibit elongated phialides PATHOGENICITY : Paecilomyceshas been implicated in cases of: 1.) penicilliosis 2.) endophthalmitis 3.) endocarditis 4.) pleural effusion 5.) skin lesions

CHAPTER 3: COMMON FUNGAL OPPORTUNISTS SEPATATE OPPORTUNISTS HYALINE SEPTATE OPPORTUNISTS Scopulariopsis species characteristics

CULTURE: -SABHI agar -room temperature -the rapid-growing colony is velvety, rugose, and white, later becoming light tan or brown, with a tan reverse MICROSCOPIC: -mycelium is septate -single, unbranching, or penicillus-type anellophores bear flask-shaped annellides, which in turn support large lemon-shaped annelloconidia in chains. -with age, the conidia become echinulate, or spiny PATHOGENICITY: Scopulariopsis causes: 1.) keratomycosis 2.) rarely bronchopulmonary disease (penicilliosis) 3.) otomycosis 4.) onychomycosis 5.) has been reported in an infection of the ankle and as the cause of an inguinal ulcer

CHAPTER 3: COMMON FUNGAL OPPORTUNISTS SEPATATE OPPORTUNISTS HYALINE SEPTATE OPPORTUNISTS Fusarium species characteristics

CULTURE: -SABHI agar -room temperature -the rapid-growing colony is white at first and woolly or cottony -later the colony becomes LAVENDER, or sometimes yellow or orange, with a light reverse MICROSCOPIC: -the mycelium is septate -conidiophores are single or branching, occasionally producing whorls, and they terminate in tapering phialides -microphialoconidia are one-celled and occur in balls -macrophialoconidia are two- to five-celled, BANANA- or CYLINDRICAL-SHAPED, with a distinctive GOOT CELL at the point of attachment. -chlamydoconidia are common in Fusarium RESEMBLANCE: 1.) if macrophialoconidia are not exhibited, Fusarium may be mistaken for Acremonium 2.) the opportunist Cylindrocarpon produces similar-appearing macrophialoconidia; however, the ends are rounded, there are no foot cells, and one to ten septa may occur. PATHOGENICITY: Fusarium is found in the following: 1.) Fusarium is the MOST COMMON cause of keratomycosis 2.) skin lesions on burn patients 3.) In onychomycosis (nail infection) 4.) otomycosis (fungus infection in the outer ear) 5.) varicose ulcers 6.) mycetoma 7.) osteomyelitis following trauma 8.) disseminated infection

CHAPTER 2: LABORATORY PROCEDURES FOR FUNGAL CULTURE AND ISOLATION FUNGAL SOURCES 1.) Blood and Bone Marrow what is the most common fungal organism isolated from blood culture?

Candida species

CHAPTER 1: BASICS OF MYCOLOGY Which organism is shown that is a spiral hyphae on page 8, figure 1-10?

Chrysosporium sp.

CHAPTER 1: BASICS OF MYCOLOGY Aerial Hyphae VS. Vegetative Hyphae

Color plates 1 and 2 on page 235 show the distinct color differences sometimes observed between the aerial hyphae (on the front side of the colony) and the vegetative hyphae (on the back side of the colony)

CHAPTER 2: LABORATORY PROCEDURES FOR FUNGAL CULTURE AND ISOLATION STUDY QUESTIONS 1-6 ON PAGE 57 4.) The best medium to isolate Crytococcus neoformans in bronchial washings from an AIDS patient is:

D. Birdseed agar EXPLANATION: -SABHI, IMA, and birdseed agar will all grow C. neoformans. However, bacteria and other yeasts may obscure its growth. -Birdseed agar will show dark colonies of C. neoformans that stand out.

CHAPTER 2: LABORATORY PROCEDURES FOR FUNGAL CULTURE AND ISOLATION STUDY QUESTIONS 1-6 ON PAGE 57 2.) The best medium to isolate fastidious Histoplasma capsulatum from sputum is:

D. Brain heart infusion agar with blood, gentamicin, chloramphenicol, and cycloheximide EXPLANATION: -While all these media may grow Histoplasma, fastidious strains require media with blood as extra nutrition. -Additionally, because sputum has many bacteria and fungal opportunists that would overrun the slower growing Histoplasma, the medium should contain inhibitory antibiotics

CHAPTER 2: LABORATORY PROCEDURES FOR FUNGAL CULTURE AND ISOLATION STUDY QUESTIONS 1-8, PAGES 36-37 4.) The fungal organism most commonly found in vaginal infections is

D. Candida albicans

CHAPTER 3: COMMON FUNGAL OPPORTUNISTS FINAL EXAM QUESTIONS 1-14 ON PAGES 110-113 1.) Banana-shaped macrophialoconidia with foot cells are diagnostic of

D. Fusarium sp.

CHAPTER 2: LABORATORY PROCEDURES FOR FUNGAL CULTURE AND ISOLATION STUDY QUESTIONS ON PAGE 49, QUESTIONS 1-10 9.) Which is the most useful mounting fluid for nail scrapings?

D. Potassium hydroxide

CHAPTER 1: BASICS OF MYCOLOGY PAGE 17-18, STUDY QUESTIONS: 4.) The conidiogenous cell in figure 1-30 is a(an):

D. annelide

CHAPTER 4: SUPERFICIAL AND DERMATOPHYTIC FUNGI DERMATOPHYTES Dermatophytes characteristics

DERMATOPHYTES BELONG TO THE FOLLOWING GENERA: 1.) Microsporum 2.) Epidermophyton 3.) Trichophyton WHAT ARE THE 3 TYPES OF SPECIES FOUND OF DERMATOPHYTES: 1.) Geophilic species -live in the soil 2.) Zoophilic species -live on animals 3.) Anthropophilic species -live on humans ARE ALL DERMATOPHYTES PATHOGENIC TO HUMANS? -no, not all dermatophytes are pathogenic to humans; nonpathogens may be found as contaminants on specimens WHAT ARE THE USUAL SPECIMENS FOR DERMATOPHYTE STUDY? 1.) Skin scrapings 2.) Nail scrapings 3.) Hair stubs (including the roots) STAINING SKIN WITH CALCOFLUOR WHITE TO THE KOH -this enhances visualization of hyphal fragments STAINING SKIN SCALE SPECIMENS WITH NEUTRAL RED -has been suggested to differentiate viable (positive or red staining) hyphal fragments from nonviable (negative or nonstaining) fragments HAIR SPECIMENS FOR DERMATOPHYTES -arthroconidia are sought in hairs, and it is noted whether they are outside the hair shaft (ectothrix invasion) or inside (endothrix invasion). -be careful in observing hairs, because ectothrix invasion begins within the shaft at the base and then moves further up the hair -ectothrix and endothrix characteristics are seen only in direct mounts, not in hair that have been cultured. DEMATOPHYTE TEST MEDIUM (DTM) -DTM is useful as a screening medium -after incubation at room temperature for 14 days, if the organism is a dermatophyte, it will change the yellow indicator to red. WHY CAN'T DTM BE USED AS DEFINITIVE IDENTIFICATION? -there are a few other fungi, for example, Cladosporium, that will also change the color of the medium; thus, slide cultures must be performed for definitive identification SABHI AGAR WITH CYCLOHEXIMIDE AND CHLORAMPHENICOL -is also another method for isolating dermatophytes

CHAPTER 1: BASICS OF MYCOLOGY PAGE 17-18, STUDY QUESTIONS: 5.) Blastoconidia that have elongated are termed

E. pseudohyphae

CHAPTER 2: LABORATORY PROCEDURES FOR FUNGAL CULTURE AND ISOLATION GENERAL RULES FOR GOOD FUNGAL SPECIMEN COLLECTION 1.) Make sure the specimen is collected from the area most likely to be affected

EXAMPLES: 1.) If hair infection is suspected, choose hair specimens that look broken and scaly (most likely will have organism) 2.) For a sputum specimen, instruct the patient to produce material from a DEEP COUGH, not just spit up saliva

CHAPTER 2: LABORATORY PROCEDURES FOR FUNGAL CULTURE AND ISOLATION DIRECT MOUNT EXAMINATION 1.) Saline wet mount What are the fungal elements that may be found? Explain the reagent preparation and procedure

FUNGAL ELEMENTS FOUND IN SALINE WET MOUNT: 1.) budding yeasts 2.) hyphae 3.) pseudohyphae 4.) conidia 5.) thin branching filaments resembling bacteria (funguslike bacteria) 6.) granules 7.) Coccidioides immitis spherules SALINE WET MOUNT REAGENT PREPARATION: Saline: Sodium chloride 0.85 g. Distilled water 100 mL 1.) Dissolve the sodium chloride in water and mix well SALINE WET MOUNT PROCEDURE: 1.) Place one drop of specimen on a glass slide and add one drop of saline 2.) Put on a coverslip and observe under low and high power under the microscope, using LOW light -organisms will appear refractile, or shiny, and slightly green -be sure to differentiate yeast cells from bubbles or red blood cells: yeasts will contain inclusions, while bubbles and red blood cells will not

CHAPTER 4: SUPERFICIAL AND DERMATOPHYTIC FUNGI DERMATOPHYTES Hair fluorescence

FUNGI INFECTING HAIRS MAY MAKE WHAT? -a metabolite, pteridine, which produced a bright greenish yellow fluorescence under a Wood's lamp (366 nm) WHICH ORGANISMS PRODUCE THIS FLUORESCENCE? 1.) Microsporum canis 2.) Microsporum audouinii 3.) Microsporum distortum 4.) Microsporum ferrugineum HOW DOES HAIR FLUORESCENCE HELP? 1.) Helps to tentatively identify the agent responsible fir the infection 2.) Fluorescence, if present, will help to choose infected hairs (those that fluoresce) from noninfected ones for culture NEGATIVE FLUORESCENCE OF ALL HAIRS -note that negative fluorescence of all hairs on the affected body site may still indicate a fungal infection, but by organisms other than those listed above.

CHAPTER 2: LABORATORY PROCEDURES FOR FUNGAL CULTURE AND ISOLATION PRIMARY FUNGAL MEDIA INCUBATION TIME

FUNGI MATURATION IS DIFFERENT AMONG FUNGI -some fungi mature within 3-4 days, while others may require 3-4 weeks GROWTH RATES VARY DEPENDING ON WHAT CONDITIONS? 1.) media used 2.) temperature of incubation 3.) inhibitors in the patient's specimen TO REDUCE THE RISK OF FALSE-NEGATIVE REPORTS, WHAT MUST BE DONE FOR ALL FUNGI CULTURES? -keep all cultures at least 1 month before reporting final results and discarding media -the only fungi that is different from this is H. capsulatum, see below HOW LONG SHOULD YOU INCUBATE A CULTURE SUSPECTED OF CONTAINING H. CAPSULATUM BEFORE DISCARDING? -incubate culture for 12 weeks before discarding as negative WHAT DOES MAINTAINING A MOIST ATMOSPHERE (40-50% HUMIDITY) HELP? -it helps keep inoculated media from drying out and enhances fungal growth WHY SHOULD YOU SEAL PLATES WITH AIR-PERMEABLE TAPE? -to prevent contamination and keep screw caps loose.

CHAPTER 2: LABORATORY PROCEDURES FOR FUNGAL CULTURE AND ISOLATION STUDY QUESTIONS 1-8, PAGES 36-37 6.) True or false: typically fungal infection is so overwhelming by the time it is isolated from blood or cerebrospinal fluid, there is no need to concentrate the specimen

False -many times even with concentration, the fungus won't grow

CHAPTER 2: LABORATORY PROCEDURES FOR FUNGAL CULTURE AND ISOLATION STUDY QUESTIONS ON PAGE 49, QUESTIONS 1-10 8.) True or false: With the India ink preparation, only encapsulated organisms are visible against the black background

False All bacteria and fungi will stand out against the black background. Look only for encapsulated organisms.

CHAPTER 3: COMMON FUNGAL OPPORTUNISTS STUDY QUESTIONS 1-4 ON PAGE 105 3.) True or false: Acremonium exhibits repeatedly forking conidiophores with terminal balls of conidia

False -Acremonium exhibits unbranching, gradually tapering conidiophores with balls of conidia.

CHAPTER 3: COMMON FUNGAL OPPORTUNISTS FINAL EXAM QUESTIONS 1-14 ON PAGES 110-113 5.) True or false: Isolates of Helminthosporium are distinguished from Bipolaris in that Helminthosporium possess conidiophores that grow in a bent-knee fashion, while Bipolaris conidiophores grow straight.

False -Bipolaris possesses the bent-knee conidiophore, while that of Helminthosporium is straight. Since the poroconidia are so similar, Bipolaris was often misidentified by helminthosporium in the past.

CHAPTER 3: COMMON FUNGAL OPPORTUNISTS FINAL EXAM QUESTIONS 1-14 ON PAGES 110-113 2.) True or false: Dematiaceous organisms exhibit light-colored hyphae and/or conidia

False -Dermatiaceous organisms exhibit dark-colored hyphae and/or conidia.

CHAPTER 3: COMMON FUNGAL OPPORTUNISTS FINAL EXAM QUESTIONS 1-14 ON PAGES 110-113 10.) True or false: The cornea of the eye is easily susceptible to infection.

False -The cornea is usually resistant to infection; keratomycosis primarily begins with trauma to the eye.

CHAPTER 2: LABORATORY PROCEDURES FOR FUNGAL CULTURE AND ISOLATION STUDY QUESTIONS 1-8, PAGES 36-37 8.) True or false: granules from subcutaneous lesions only represent necrotic material and should be disregarded

False -Granules are composed of organisms (bacteria, funguslike bacteria, or fungi) with or without a cementlike matrix or a center of necrotic material. They are the most likely source for isolating the etiologic agent of the infection

CHAPTER 2: LABORATORY PROCEDURES FOR FUNGAL CULTURE AND ISOLATION STUDY QUESTIONS 1-8, PAGES 36-37 5.) True or false: do not disinfect skin or nail areas with alcohol before taking scrapings, as the disinfectant will kill any pathogenic fungi you want to recover

False -alcohol removed surface contaminants; it does not affect fungal pathogens

CHAPTER 1: BASICS OF MYCOLOGY PAGE 17-18, STUDY QUESTIONS: 3.) True or false: Chalmydospores of Candida albicans will germinate to form new conidia when mature

False -candida albicans forms chlamydosphores, which are NON-germinating vesicles

CHAPTER 2: LABORATORY PROCEDURES FOR FUNGAL CULTURE AND ISOLATION FINAL EXAM QUESTIONS 1-7 ON PAGES 66-67 3.) True or false: Fungal growth can be easily detected in blood culture bottles because the medium quickly becomes turbid

False -unlike bacteria, fungi do NOT make the broth cloudy. This necessitates frequent gram staining

CHAPTER 2: LABORATORY PROCEDURES FOR FUNGAL CULTURE AND ISOLATION STUDY QUESTIONS ON PAGE 49, QUESTIONS 1-10 10.) True or false: With specimen lactophenol cotton blue mounts, which show fungi, the specimen may be removed from under the coverslip and cultured

False The phenol in LPCB kills any fungus that may be present; thus, it cannot be cultured.

CHAPTER 2: LABORATORY PROCEDURES FOR FUNGAL CULTURE AND ISOLATION PRIMARY FUNGAL MEDIA SELECTIVE MEDIA WITH ANTIBIOTICS Chart 2-3 Pathogenic fungi inhibited by cycloheximide and chloramphenicol, page 54

GO STUDY THAT CHART NOW AND TYPE IT OUT FULLY OR WRITE IT DOWN FULLY

CHAPTER 4: SUPERFICIAL AND DERMATOPHYTIC FUNGI DERMATOPHYTES Chart 4-3: Microscopic Characteristics of Dermatophyte Genera

GO STUDY THIS CHART ON PAGE 126 NOW

CHAPTER 2: LABORATORY PROCEDURES FOR FUNGAL CULTURE AND ISOLATION 1.) Blood and Bone Marrow FUNGAL SOURCES how long are blood cultures held when BACTERIAL (not fungus) pathogens are suspected? how long are blood cultures help when a slow-growing fungal organism is expected?

HOLDING FOR BACTERIAL PATHOGENS -typically held for 7 days HOLDING FOR SLOW-GROWING FUNGAL ORGANISMS -should be held for 3 WEEKS

CHAPTER 2: LABORATORY PROCEDURES FOR FUNGAL CULTURE AND ISOLATION THERAPEUTIC AGENTS FOR MYCOSES Griseofulvin

HOW DOES GRISEOFULVIN WORK? -this drug works by interaction with polymerized microtubules and disruption of the mitotic spindle IS GRISEOFULVIN FUNGISTATIC OR FUNGICIDAL? -it is fungicidal HOW IS GRISEOFULVIN TAKEN AND WHAT DOES IT TREAT? -griseofulvin is taken orally for the treatment of dermatophytosis (Trichophyton, Microsporum, and Epidermophyton), and must be continued until the infected hair, nail, or skin has completely grown out and been replaced.

CHAPTER 3: COMMON FUNGAL OPPORTUNISTS OPPORTUNISTIC MYCOSES Aspergillosis characteristics

HOW IS ASPERGILLOSIS USUALLY ACQUIRED? -through inhalation of the conidia HOW DOES ASPERGILLOSIS BEGIN AS? -it begins as a pulmonary disease, producing granulomatous (tumorous) lesions in the lungs or bronchi WHAT CAN THE PULMONARY DISEASE LEAD TO? -it may be spread from the lung tissue into surrounding blood vessels WHERE DOES THE DISEASE DISSEMINATE TO (IF IT EVEN GETS TO THAT POINT)? -the disease disseminates to the rest of the body, including the: 1.) Brain 2.) Gastrointestinal tract 3.) Kidneys THE INVASE, DISSEMINATED PULMONARY FORM OF ASPERGILLOSIS IS BEING INCREASING OBSERVED IN: -in debilitated patients receiving: 1.) Antibiotic 2.) Immunosuppressive 3.) Cancer therapy WHAT IS THE SEVERITY OF THE DISSEMINATED VERSION OF THIS DISEASE? -it is acute and fatal WHAT ARE THE TWO CHARACTERISTIC FORMS OF ASPERGILLOSIS THAT DO NOT HAVE TISSUE INVASION? 1.) Otomycosis (external ear infection 2.) Fungus ball (abscess) ASTHMA ATTACKS AND ASPERGILLOSIS -asthma attacks can be initiated in sensitized individuals by inhalation of Aspergillus conidia (allergic bronchopulmonary disease) WHAT ARE THE MISCALLANEOUS TYPES OF ASPERGILLOSIS? 1.) myocarditis 2.) meningitis 3.) osteomyelitis 4.) mycetoma 5.) burn infection 6.) invasion of the nasal sinuses 7.) onychomycosis 8.) keratomycosis 9.) toxin ingestion from eating contaminated foods DIRECT MOUNTS -direct mounts of specimens reveal septate hyphae that branch dichotomously (Y-shaped branching) -specimens from fungus balls may also show the typical Aspergillus fruiting heads CLASSICALLY, HOW IS ASPERGILLOSIS DIAGNOSED? -diagnosis is classically confirmed if repeated cultures of infected material grow large numbers of Aspergillus species. DIAGNOSIS IN CASES OF INVASIVE PULMONARY ASPERGILLOSIS -in cases of invasive pulmonary aspergillosis, only 25% of patients may produce positive cultures, and only half of those may possess more than one positive result -therefore, isolation of A. fumigatus or A. flavus should not be automatically considered as laboratory contamination but rather should be judged in the light of the patient's medical history. A. FUMIGATUS -is most commonly isolated from: 1.) Invase pulmonary aspergillosis 2.) Fungus ball 3.) Allergic bronchopulmonary disease A. FLAVUS -is seen in: 1.) Invasive pulmonary aspergillosis 2.) Allergic bronchopulmonary disease A. NIGER -produces: 1.) Fungus ball 2.) Otomycosis TREATMENT -aspergillosis usually responds to treatment with amphotericin B.

CHAPTER 3: COMMON FUNGAL OPPORTUNISTS SEPATATE OPPORTUNISTS Septate opportunists overview

HYPHAE OF MOST OPPORTUNISTS CONTAIN WHAT? -cross walls, also known as septate hyphae. -so, most opportunists are septate hyphae THE SEPTATE OPPORTUNISTS THAT ARE OF MEDICAL IMPORTANCE FALL UNDER WHICH PHYLUM? -the phylum Deuteromycota (also known as fungi imperfecti, as there is no known sexual phase) -WHAT ARE THE TWO CATEGORIES THAT SEPTATE OPPORTUNISTS ARE DIVIDED INTO? 1.) Dematiaceous septate opportunists -dark-colored hyphae and/or conidia AND 2.) Hyaline septate opportunists -light-colored hyphae and conidia COLONIAL COLOR AIDS IN THE -initial identification DEMATIACEOUS SEPTATE OPPORTUNIST CHARACTERISTICS -dark-colored hyphae and/or conidia -organisms with dark hyphae on tease mounts also have dark green to black colonies, especially on the colony reverse HYALINE SEPTATE OPPORTUNIST CHARACTERISTICS -light-colored hyphae and conidia -hyaline organisms exhibit light-colored colonial aerial hyphae, but they may be covered over with brightly colored conidia; thus, a tease mount is required.

CHAPTER 2: LABORATORY PROCEDURES FOR FUNGAL CULTURE AND ISOLATION PRIMARY FUNGAL MEDIA SPECIAL CONSIDERATIONS Actinomyces

IF ACTINOMYCES IS SUSPECTED, WHAT SHOULD BE DONE? 1.) Plate the specimen onto brain heart infusion agar with blood (NO antibiotics) as soon as possible after collection AND 2.) Immediately incubate under anaerobic conditions at 35-37 degrees celcius

CHAPTER 2: LABORATORY PROCEDURES FOR FUNGAL CULTURE AND ISOLATION PRIMARY FUNGAL MEDIA SPECIAL CONSIDERATIONS Nocardia

IF NOCARDIA IS SUSPECTED, WHAT CAN BE ADDED TO NONSELECTIVE FUNGAL MEDIA AND WHAT DOES IT DO? -paraffin may be added to nonselective fungal medium if Nocardia is suspected -paraffin does the following: 1.) Inhibits bacterial overgrowth AND 2.) Enhances growth of Nocardia NOCARDIA GROWS WELL ON WHAT OTHER TYPE OF MYCOBACTERIAL MEDIUM? -Nocardia grows well on 7H11 Mycobacterial medium WHAT TYPES OF MEDIA ARE USEFUL FOR PRIMARY MEDIA FOR ISOLATION OF NOCARDIA FROM CONTAMINATED RESPIRATORY SPECIMENS, SUCH AS SPUTUM? 1.) The Legionella nonselective buffered charcoal-yeast extract agar (BCYE) agar AND 2.) Other selective Legionella agars

CHAPTER 2: LABORATORY PROCEDURES FOR FUNGAL CULTURE AND ISOLATION Therapy for mycoses (fungal diseases)

IN THE PAST, TREATMENT OF SYSTEMIC FUNGAL INFECTIONS REQUIRED WHAT? -In the past, treatment of systemic fungal infections required amophotericin B, with its set of toxic renal effects. NEW FUNGICIDAL AGENTS -Now newer fungicidal agents are being evaluated in Europe, and many systemic fungistatic agents are available in the United States

CHAPTER 2: LABORATORY PROCEDURES FOR FUNGAL CULTURE AND ISOLATION STUDY QUESTIONS 1-6 ON PAGE 57 5.) According to this module, what is an intermediate growth rate?

Intermediate growth means that a mature colony forms in 6-10 days

CHAPTER 2: LABORATORY PROCEDURES FOR FUNGAL CULTURE AND ISOLATION DIRECT MOUNT EXAMINATION 3.) Potassium hydroxide (KOH) preparation What are the fungal elements that may be found? Explain the reagent preparation and procedure

KOH PREPARATION IS USED ON CUTANEOUS SKIN, HAIR, OR NAIL SPECIMENS DUE TO THE FOLLOWING REASONS: -in skin, hair, or nail specimens, the background cellular material may mask fungal elements -the potassium hydroxide in the KOH preparation will fix this by dissolving the keratin in these specimens, thus making any fungi more visible. KOH PREPARATION MAY ALSO BE USED TO PREPARE SPUTUM OR VAGINAL SECRETIONS IN THE FOLLOWING SITUATION: -if there is a lot of material in sputum or vaginal secretions, a KOH mount may be prepared to dissolve the background, thus making any yeasts more visible. WHY MUST A SALINE MOUNT BE DONE IN ADDITION TO THE KOH PREPARATION? -a saline mount must be additionally done to quantitate epithelial cells and white blood cells and to note the presence of Trichomonas KOH REAGENT PREPARATION: 10% potassium hydroxide: commercially available (Marion Scientific): Potassium hydroxide 10 g. Glycerol 20 mL Distilled water 80 mL 1.) Dissolve the potassium hydroxide in water; then add glycerol. -Glycerol prevents crystallization of the reagent and allows KOH preparations to be maintained for 2 days before drying up. KOH PROCEDURE: 1.) Thinly smear some of the specimen on a glass slide. 2.) Add one drop of 10% KOH, put on a coverslip, and gently heat by passing through a flame two to three times. Do NOT boil. 3.) When the specimen has cleared (about 20 minutes), observe it under low and high power for any mycologic elements: 1-hyphae 2-arthroconidia 3-yeasts -on hair specimens, determine if the fungus is growing outside the hair shaft (ectothrix invasion) or inside the hair shaft (endothrix invasion). -As with saline preparations, the organisms are NOT stained and thus they appear refractile. Low light is best for observing fungi.

CHAPTER 2: LABORATORY PROCEDURES FOR FUNGAL CULTURE AND ISOLATION FUNGAL SOURCES 1.) Blood and Bone Marrow What are the two ways that a blood specimen may be drawn? HINT: LABORATORY VS. BESIDE

LABORATORY -the blood specimen may be drawn into a transport Vacutainer tube with 0.35% polyantholsulfonate (SPS) for subsequent transfer into blood culture media once in the laboratory. BEDSIDE -blood may be drawn DIRECTLY into the blood culture media at the patient's bedside

CHAPTER 2: LABORATORY PROCEDURES FOR FUNGAL CULTURE AND ISOLATION FUNGAL SOURCES 3.) Cutaneous: Hair, Nails, and Skin IN THE LABORATORY: what should be done with large skin and nail scrapings? what should be done with small portions of soft skin or hair?

LARGE SKIN AND NAIL SCRAPINGS: -large skin and nail scrapings are cut into tiny sections SMALL PORTIONS OF SOFT SKIN OR HAIR: -a small portion of soft skin or hair is mixed with POTASSIUM HYDROXIDE, a coverslip is added, and the slide is gently warmed to aid clearing of tissue -the mount is examined microscopically for fungal elements -the rest of the specimen is inoculated onto fungal media ^^^ idk if the mounting and the inoculating is for all cutaneous specimens?

CHAPTER 4: SUPERFICIAL AND DERMATOPHYTIC FUNGI Superficial organisms overview

LOCATION: -superficial fungi are primarily observed in tropical climates -sometimes may be seen in the United States. SPECIMEN OF CHOICE: -skin scrapings and plucked hair are the specimens of choice

CHAPTER 1: BASICS OF MYCOLOGY In figure 1-6 on page 7, there is an image of _________________ with a nodular organ, which means that this is a ____ ______

Microscporum ferrugineum, twisted hyphae.

CHAPTER 1: BASICS OF MYCOLOGY Are most hyphae septate or aseptate?

Most hyphae are septate.

CHAPTER 2: LABORATORY PROCEDURES FOR FUNGAL CULTURE AND ISOLATION PRIMARY FUNGAL MEDIA SUBCULTURE MEDIA

ONCE FUNGI HAVE GROWN ON PRIMARY CULTURE, WHAT DO THEY FREQUENTLY NEED? -they frequently need subculturing for complete isolation -subculture media are used to promote identification WHAT ARE THE 2 SUBCULTURES FOR YEASTS? EXPLAIN IT. 1.) Neutral Sabouraud Dextrose Agar (Emmon's modification) -yeasts are subcultured to neutral sabouraud dextrose agar (Emmon's modification); is commercially available -this subculture for yeast has less glucose and a neutral pH compared to regular sabouraud dextrose agar, which allows for a better maintenance of yeasts 2.) Birdseed Agar -yeasts can also be subcultured to birdseed agar for quick identification of Cryptococcus neoformans. WHAT IS THE SUBCULTURE FOR MOLDS? EXPLAIN IT. -molds are transferred to potato dextrose agar -this agar promotes sporulation and pigmentation of colonies.

CHAPTER 2: LABORATORY PROCEDURES FOR FUNGAL CULTURE AND ISOLATION 1.) Blood and Bone Marrow FUNGAL SOURCES All of the blood culture systems (whether automated or nonautomated) adequately isolate yeasts, but only one system REPEATEDLY isolates Histoplasma capsulatum, which system does this? ALL the systems can NOT adequately isolate what type of fungi?

ONLY SYSTEM THAT REPEATEDLY ISOLATED HISTOPLASMA CAPSULATUM: Isolator ALL SYSTEMS CAN NOT ADEQUATELY ISOLATE: -filamentous fungi

CHAPTER 3: COMMON FUNGAL OPPORTUNISTS Introduction to common fungal opportunists

OPPORTUNISTS WILL BE SEEN VERY OFTEN IN ROUTINE FUNGAL CULTURES, AND IS THEREFORE IMPORTANT TO DO WHAT? -to be able to identify and differentiate them from the regularly pathogenic (disease producing) fungi, which are presented in modules 4-7

CHAPTER 2: LABORATORY PROCEDURES FOR FUNGAL CULTURE AND ISOLATION FUNGAL SOURCES 3.) Cutaneous: Hair, Nails, and Skin explain skin specimen preparation and examination

PREPARATION AND COLLECTION: -skin is first cleaned with 70% alcohol to remove surface bacteria and fungal opportunists IF DERMATOPHYTOSIS IS PRESENT: -if dermatophytosis is present, scrape the outer portions of the red ring with a sterile blade -if there is no ring, scape areas that look most infected -place the scrapings in a sterile Petri dish for processing

CHAPTER 1: BASICS OF MYCOLOGY How may pseudohyphae be differentiated from true hyphae?

Pseudohyphae may be differentiated from true hyphae in that the cells of pseudohyphae are constricted to their points of attachment, while true hyphae are not.

CHAPTER 3: COMMON FUNGAL OPPORTUNISTS ASEPTATE OPPORTUNISTS Aseptate opportunists overview

REMINDER OF ASEPTATE FUNGI -aseptate fungi are those with hyphae that usually do not contain cross-walls ASEPTATE FUNGI FALL UNDER WHICH TAXONOMIC PHYLUM? -Zygomycota

CHAPTER 3: COMMON FUNGAL OPPORTUNISTS FINAL EXAM QUESTIONS 1-14 ON PAGES 110-113 A 10-year-old boy was admitted to the hospital in diabetic ketoacidosis. Three days later, he complained of nasal sinus blockage. Stained histologic preparations of material removed from his sinuses showed the wide, ribbonlike hyphae seen in Figure 3-58. On SABHI agar, a white cottony mold rapidly grew. 13.) Slide cultures of the mold revealed unbranching sporangiophores at rhizoid nodes. Fill in the blank: The causative organism is _______.

Rhizopus sp.

CHAPTER 1: BASICS OF MYCOLOGY What is the temperature for the MOLD phase of a dimorphic fungi?

Room temperature (25 or 30 degrees celcius)

CHAPTER 2: LABORATORY PROCEDURES FOR FUNGAL CULTURE AND ISOLATION CULTURING THE SPECIMEN PRIMARY FUNGAL MEDIA NONSELECTIVE MEDIA 1.) Sabouraud Brain Heart Infusion Agar (SABHI) Give the characteristics of SABHI See the box on page 52 for the medium preparation and procedure for SABHI

SABHI VS. SABOURAUD DEXTROSE AGAR (SDA) -SABHI is becoming the medium of choice for general initial isolation, because the traditional SDA is not as rich nutritionally and thus will not support the growth of as many fungi. SABHI ALLOWS THE GROWTH OF THE FOLLOWING PATHOGENS: 1.) Bacteria 2.) Fungal opportunists 3.) Dermatophytes (cutaneous pathogens) 4.) Yeasts 5.) Subcutaneous pathogens 6.) Most systemic pathogens WHICH PATHOGENS MAY NOT PROSPER ON SABHI? -some fastidious strains of H. capsulatum and B. dermatitidis may not prosper. -SABHI is commercially available SABHI MEDIUM PREPARATION AND PROCEDURE -go to page 52 and type out or write out the whole box, thoroughly study it.

CHAPTER 2: LABORATORY PROCEDURES FOR FUNGAL CULTURE AND ISOLATION FINAL EXAM QUESTIONS 1-7 ON PAGES 66-67 7.) How do specimen site, colonial morphology, and general microscopic morphology (example: mold, yeast, dimorphic fungus, or funguslike bacteria) aid in identification?

SPECIMEN SITE -since you know that only certain organisms can be isolated from a particular specimen, many fungi are ruled out COLONIAL MORPHOLOGY -sometimes a colony has such a characteristic morphology, for example, Penicillium, that you suspect its identity even before observing the microscopic characteristics GENERAL MICROSCOPIC MORPHOLOGY -since you know that only certain organisms possess hyphae, yeast forms, filamentous bacterial characteristics, or dimorphic attributes, many fungi are ruled out.

CHAPTER 2: LABORATORY PROCEDURES FOR FUNGAL CULTURE AND ISOLATION PRIMARY FUNGAL MEDIA SPECIAL CONSIDERATIONS Crytococcus neoformans

SPUTUM FROM AIDS PATIENTS MAY BE PLATED ON WHAT TYPE OF AGAR TO SELECTIVELY ISOLATE CRYPTOCOCCUS NEOFORMANS? -birdseed agar (caffeic acid agar) WHAT DOES BIRDSEED AGAR (CAFFEIC ACID AGAR) DO FOR CRYTOCOCCUS? 1.) This medium prevents overgrowth of bacteria 2.) C. neoformans will grow dark brown while other yeasts will be white or beige -Birdseed agar (caffeic acid agar) is commercially available

CHAPTER 2: LABORATORY PROCEDURES FOR FUNGAL CULTURE AND ISOLATION FUNGAL SOURCES 1.) Blood and Bone Marrow The bone marrow is a sterile site, what advantage does this have?

Since the bone marrow is a sterile site, it can be inoculated to media WITHOUT antibiotics

CHAPTER 3: COMMON FUNGAL OPPORTUNISTS ASEPTATE OPPORTUNISTS Mucor species

TERMINAL VESICLE: -the terminal vesicle is absent, the sporangiophores of these organisms end in columellae around which sporangia form. CULTURE: -SABHI agar -room temperature -a white, fluffy mycelium quickly forms -colony becomes gray to brown with age MICROSCOPIC: -the mycelium is usually aseptate -single or BRANCHING sporangiophores support round, spore-filled sporangia. -sometimes empty sporangial sacs or bare columellae with collarettes may be seen. -the columellae is variable in shape and light to pigmented in color -there are NOOO, NONEEE, NOOO rhizoids or stolons present in Mucor species. PATHOGENICITY: -Mucor may cause: 1.) zygomycosis 2.) otomycosis (ear infection) 3.) allergies

CHAPTER 3: COMMON FUNGAL OPPORTUNISTS ASEPTATE OPPORTUNISTS Rhizopus species characteristics

TERMINAL VESICLE: -the terminal vesicle is absent, the sporangiophores of these organisms end in columellae around which sporangia form. CULTURE: -SABHI agar -room temperature -white, dense, cottony aerial hyphae rapidly form, which later become dotted with brown or black sporangia MICROSCOPIC: -the hyphae are usually aseptate -UNBRANCHED SPORANGIOPHORES arise OPPOSITE RHIZOIDS at the nodes -each sporangiophore supports a round spore-filled sporangium with a flattened base -sometimes the sporangia are completely black, or they may be empty -when the sporangial wall dissolves, a bare hemispherical columella WITHOUT a collarette is observed -STOLONS connect the groups of rhizoids with each other PATHOGENICITY: -Rhizopus causes: 1.) zygomycosis 2.) otomycosis (ear infection)

CHAPTER 3: COMMON FUNGAL OPPORTUNISTS ASEPTATE OPPORTUNISTS Saksenaea species characteristics

TERMINAL VESICLE: -the terminal vesicle is absent, the sporangiophores of these organisms end in columellae around which sporangia form. CULTURE: -SABHI agar -room temperature -rapidly growing woolly white colonies are seen MICROSCOPIC: -the broad hyphae are usually aseptate with sparsely septate and branched. -on blocks of SABHI agar growing in distilled water-yeast extract medium at 37 degrees celsius, typical FLASK-SHAPED SPORANGIA are observed that produce smooth, elongated sporangiospores. -RHIZOIDS are produce opposite the sporangiophores. PATHOGENICITY: Saksenaea may cause: 1.) zygomycosis 2.) osteomyelitis (inflammation or swelling that occurs in the bone)

CHAPTER 3: COMMON FUNGAL OPPORTUNISTS ASEPTATE OPPORTUNISTS Absidia species characteristics

TERMINAL VESICLE: -the terminal vesicle is absent, the sporangiophores of these organisms end in columellae around which sporangia form. CULTURE: -on SABHI agar -at room temperature -a woolly gray colony rapidly matures -the reverse side of the colony is colorless MICROSCOPIC -the mycelium is usually ASEPTATE, with BRANCHING SPORANGIOPHORES BETWEEN the RHIZOIDS (rootlike hyphae) on the STOLONS (interconnecting runners) -there is a slight swelling below the columella, and sporangia are PEAR-SHAPED -when the sporangial wall dissolves, a collarette remains at the base of the columella PATHOGENICITY: -absidia species may cause the following: 1.) Zygomycosis 2.) Keratomycosis

CHAPTER 3: COMMON FUNGAL OPPORTUNISTS ASEPTATE OPPORTUNISTS Apophysomyces species characteristics

TERMINAL VESICLE: -the terminal vesicle is absent, the sporangiophores of these organisms end in columellae around which sporangia form. CULTURE: -on SABHI agar -room temperature -cottony white colonies may mature rapidly, becoming off-white to yellow with age -is thermo-tolerant, growing at temperatures up to 42 degrees celsius MICROSCOPIC: -can be grown on the following: 1.) SABHI agar or even on nutritionally deficient media, such as: 2.) corn meal 3.) potato dextrose agar -hyphae usually FAIL to sporulate at room temperature -to induce sporulation, it is suggested to aseptically transfer blocks of SABHI afar inoculated with fungus to tubes of sterile distilled water- 10% yeast extract media (without hair) used for the in vitro hair preparation test (see page 134), and incubating at 37 degrees celsius for 10 to 14 days -under the special growth characteristics listed prior, the following can be seen: 1.) unbranched sporangiophores with FOOT CELLS (bases) AND 2.) funnel-shaped APOPHYSES (swollen columellae) 3.) thin-walled RHIZOIDS may form opposite the sporangiophores 4.) sporangia are pyriform and contain oblong (an object or flat figure in an elongated rectangle or oval shape) sporangiospores PATHOGENICITY: -apophysomyces is a cause of zygomycosis

CHAPTER 3: COMMON FUNGAL OPPORTUNISTS ASEPTATE OPPORTUNISTS Syncephalastrum species

TERMINAL VESICLE: -the terminal vesicle is present, meaning that sporangiophores of these organisms end in spherical vesicles from which spore-forming structures arise CULTURE: -SABHI agar -room temperature -a woolly dark colony rapidly matures MICROSCOPIC: -the mycelium is usually aseptate -BRANCHED sporangiophores terminate in VESICLES. -round sporangiospores form in a ROW within cylindrical MEROSPORANGIA (specialized sporangia) which radiate around the entire surface of the vesicle -RUDIMENTARY RHIZOIDS are seen -the microscopic morphology of Syncephalastrum species may be easily confused with ASPERGILLUS SPECIES; however, Aspergillus has SEPTATE hyphae, no rhizoids, and vesicles surrounded by phialides from which phialoconidia arise in chains PATHOGENICITY: Syncephalastrum species is NOT KNOWN to be a human pathogen

CHAPTER 3: COMMON FUNGAL OPPORTUNISTS ASEPTATE OPPORTUNISTS Cunninghamella species

TERMINAL VESICLE: -the terminal vesicle is present, meaning that sporangiophores of these organisms end in spherical vesicles from which spore-forming structures arise CULTURE: -SABHI agar -room temperature -white cottony colonies grow rapidly, turning gray with age -good growth is seen at 37 degrees celsius and some species grow at 42 degrees celsius MICROSCOPIC: -the hyphae are usually aseptate -BRANCHED sporangiophores terminate in VESICLES (swollen cells) on which one-celled round sporangiola form at the tips of swollen DENTICLES (small toothlike projections) PATHOGENICITY: -Cunninghamella is a cause of zygomycosis in NEUTROPENIC (A condition called neutropenia occurs when the number of neutrophils (a type of white blood cell that helps your body fight infection) in your bloodstream falls below normal, putting you at a high risk for getting an infection) patients

CHAPTER 2: LABORATORY PROCEDURES FOR FUNGAL CULTURE AND ISOLATION Seromycology supplemental rationale tests: -there are tests to detect fungal antibody from serum and there are tests to detect fungal antigen -Explain the tests to detect fungal antibody from serum

TESTS TO DETECT FUNGAL ANTIBODY FROM SERUM INCLUDE: 1.) immunodiffusion 2.) indirect latex agglutination 3.) complement fixation 4.) indirect immunofluorescence 5.) counterimmunoelectrophoresis 6.) enzyme-linked immunosorbent assay (ELISA) ISSUES WITH THE USEFULNESS OF TESTS TO DETECT FUNGAL ANTIBODY FROM SERUM: -a fourfold rise in titer of paired sera drawn 2-3 weeks apart increases sensitivity; however, the delay in diagnosis diminishes the usefulness of the procedure SOME OF THE NEWER TESTS TO DETECT FUNGAL ANTIBODY FROM SERUM INCLUDE: 1.) ELISA for H. capsulatum 2.) enzyme immunoassay for P. brasiliensis IgM and IgG 3.) premier enzyme immunoassay for C. immitis IgM and IgG THE BIOMERIEUX TEST FOR IGM AND IGG ANTIBODY IN DISSEMINATED CANDIDIASIS SENSITIVITY AND SPECIFICITY -is 100% sensitive and 100% specific

CHAPTER 2: LABORATORY PROCEDURES FOR FUNGAL CULTURE AND ISOLATION Seromycology supplemental rationale tests: -there are tests to detect fungal antibody from serum and there are tests to detect fungal antigen -Explain the tests to detect fungal antigen overview

TESTS TO DETECT FUNGAL ANTIGEN SPECIMEN MAY USE: 1.) specimen directly (example: cryptococcal antigen from serum or CSF) OR 2.) bits of fungal colony once isolated (example: exoantigen testing of systemic pathogens by immunodiffusion) TECHNIQUES TO DETECT FUNGAL ANTIGEN SPECIMEN: 1.) immunodiffusion/exoantigen 2.) indirect reverse latex agglutination 3.) direct fluorescent antibody 4.) enzyme immunoassay 5.) avidin-biotin ELISA EXOANTIGEN TESTS ARE 100% SPECIFIC FOR WHICH FUNGAL ANTIGENS WITHIN 48-72 HOURS? 1.) B. dermatitidis 2.) C. immitis 3.) P. brasiliensis TO KNOW THE REST ABOUT ANTIGEN TESTING START AT TOP OF PAGE 68 AND READ THE REST -seriously go read and study it NOW, type it out or write it down FULLY

CHAPTER 1: BASICS OF MYCOLOGY CHAPTER 1 FINAL EXAM QUESTIONS, PAGES 24-26 2.) See color plates 10 and 11, describe the following of this colony: TEXTURE: TOPOGRAPHY: FRONT COLOR: REVERSE COLOR:

TEXTURE: velvety to woolly (cottony) texture TOPOGRAPHY: rugose FRONT COLOR: -front color is white with a white peripheral ring REVERSE COLOR: (starting from the periphery and moving inward) -white, orange, yellow-orange, center orange-tan OR -white peripheral rings, with inner rings of varying shades of orange (yellow-orange, orange, and orange-tan)

CHAPTER 2: LABORATORY PROCEDURES FOR FUNGAL CULTURE AND ISOLATION GENERAL RULES FOR GOOD FUNGAL SPECIMEN COLLECTION 4.) The specimen must be promptly delivered to the laboratory and the laboratory must quickly process the specimen

THE ISSUE: -slow-growing, pathogenic fungi are rapidly overgrown by bacterial and fungal opportunists, making it hard to retrieve the fungus that is actually causing the infection. PRESERVATION METHODS: 1.) If the specimen must sit before processing, REFRIGERATE it. -keeps fungi alive and bacterial/fungal opportunist colony counts down -prevents yeasts like Candida albicans from multiplying and therefore increasing their significance in the specimen. 2.) If the specimen must be mailed, add the following two options, choose one: 1- add 50,000 units of penicillin and 100,000 micrograms of streptomycin 2- add 0.2 mg of chloramphenicol to each milliliter of material

CHAPTER 2: LABORATORY PROCEDURES FOR FUNGAL CULTURE AND ISOLATION EXAMINING THE CULTURE Explain the slide culture method

THE TWO ADVANTAGES OF THE SLIDE CULTURE METHOD 1.) fungal elements are grown and maintained in their original juxtaposition, thus making it easier to morphologically identify the organism 2.) two mounts may be obtained from one culture THE THREE DISADVANTAGES OF THE SLIDE CULTURE METHOD 1.) this method requires technical expertise to set up the slide culture 2.) there is a waiting period for incubation of the slide culture in addition to incubation on the primary isolation medium before identifying fungus 3.) cottony fungi, for example the phylum zygomycota, grow past the edges of the cover glass before forming reproductive structures SEE BOX ON PAGE 59 FOR THE PROCEDURE -seriously, go study it

CHAPTER 2: LABORATORY PROCEDURES FOR FUNGAL CULTURE AND ISOLATION FINAL EXAM QUESTIONS 1-7 ON PAGES 66-67 5.) One role of the mycology laboratory is to isolate fungi as quickly as possible, while keeping down costs. In your opinion, what is the best general incubation temperature for primary fungal cultures? Justify your answer.

THERE ARE 3 POSSIBLE ANSWERS FOR THIS QUESTION, DEPENDING ON THE INDIVIDUAL'S PHILOSOPHY AND THE LABORATORY'S ECONOMIC SITUATION. 1.) Some laboratories incubate all their cultures at room temperature, so a costly incubator is not required. Fungi will grow at this temperature, but it takes longer for them to mature and thus identify. 2.) If all cultures are put at 30 degrees celcius, they will grow faster, thus aiding quick results, but an incubator is necessary. -A 30 degrees celcius temperature is recommended in this module. 3.) Some laboratories incubate one set of cultures at 37 degrees celcius to isolate the yeast phase of dimorphic fungi, and a second set of cultures at room temperature (25 degrees celcius) or 30 degrees celcius for other organisms. This is probably NOT cost effective since so few dimorphic fungi are isolated. Also the yeast phase could be immediately observed in specimen direct mounts.

CHAPTER 1: BASICS OF MYCOLOGY Dimorphic organisms produce what types of diseases?

They typically produce serious and often fatal systemic diseases.

CHAPTER 1: BASICS OF MYCOLOGY What is the scientific name for ringworm (dermatophytosis) of skin, hair, or nail?

Trichophytom achoenleinii -see page 6, figure 1-4 to see an image of ringworm and the favic chandeliers it has.

CHAPTER 1: BASICS OF MYCOLOGY Which organism is shown in figure 1-8 on page 7 that is a racquet hyphae?

Trichophyton ajelloi

CHAPTER 1: BASICS OF MYCOLOGY PAGE 17-18, STUDY QUESTIONS: 2.) True or false: Arthroconidia reproduce by fragmentation

True

CHAPTER 2: LABORATORY PROCEDURES FOR FUNGAL CULTURE AND ISOLATION STUDY QUESTIONS 1-6 ON PAGE 57 6.) True or false: Potato dextrose agar promotes sporulation

True

CHAPTER 2: LABORATORY PROCEDURES FOR FUNGAL CULTURE AND ISOLATION STUDY QUESTIONS 1-8, PAGES 36-37 2.) True or false: with a sputum culture, one would expect to observe growth of throat organisms

True

CHAPTER 2: LABORATORY PROCEDURES FOR FUNGAL CULTURE AND ISOLATION STUDY QUESTIONS 1-8, PAGES 36-37 7.) True or false: sputum specimens may be placed in plan N-acetyl-L-cysteine solution for concurrent fungal and TB processing

True

CHAPTER 3: COMMON FUNGAL OPPORTUNISTS FINAL EXAM QUESTIONS 1-14 ON PAGES 110-113 6.) True or false: Aspergillus fumigatus produces phialides and phialoconidia only at the end of the vesicle

True

CHAPTER 3: COMMON FUNGAL OPPORTUNISTS FINAL EXAM QUESTIONS 1-14 ON PAGES 110-113 8.) True or false: In aspergillosis, the etiologic fungus may be isolated only once and still be considered significant

True

CHAPTER 2: LABORATORY PROCEDURES FOR FUNGAL CULTURE AND ISOLATION FUNGAL SOURCES 1.) Blood and Bone Marrow the isolator tube is drawn in a similar manner to......

Vacutainer SPS tube

CHAPTER 2: LABORATORY PROCEDURES FOR FUNGAL CULTURE AND ISOLATION Explain skin supplemental rationale tests

WHAT A SKIN TEST IS AND HOW IT WORKS -an extract of the antigen (known fungus) is injected into the patient's skin -after 24-48 hours, the site is observed for erythema (redness) and swelling, and measured for induration (hardness). -this reaction is caused by a delayed hypersensitivity in patients who have been previously exposed to the antigen. SKIN TESTS ARE USUALLY PERFORMED IN PATIENTS SUSPECTED OF HAVING WHAT? 1.) histoplasmosis 2.) blastomycosis OR 3.) coccidioidomycosis SINCE THERE IS A CROSS REACTIVITY BETWEEN THE THREE ANTIGENS FROM THE ABOVE SUSPECTED CONDITIONS, WHAT MUST BE DONE? -since there is a cross reactivity between the 3 antigens of histoplasmosis, blastomycosis, and coccidioidomycosis, all three are injected at the same time but at DIFFERENT sites WHAT IS THE CRITERIA IN DETERMINING THE ORGANISM THAT THE PATIENT WAS PREVIOUSLY EXPOSED TO? -more than one site from the three antigens may give hardness and redness; however, the one with induration (hardness) of 5 mm or more in diameter is the organism the patient was previously exposed to ARE SKIN TESTS GOOD FOR DETERMINING PRESENT ACTIVE INFECTION? EXPLAIN WHY. -NO, the skin test is of limited use in diagnosis of present infection because the skin test will be positive years after the patient came in contact with the antigen. -also, the skin test may be negative in the acute stage of infection (because in the acute stage there is no immunologic response synthesized yet), in patients who are immunosuppressed, or in patients who have overwhelming fungal infection WHY CAN'T YOU ADMINISTER A SKIN TEST BEFORE TAKING BLOOD FOR OTHER SEROLOGICAL STUDIES? -injected skin test antigen will stimulate antibody production, which may give a false-positive reaction in serologic procedures

CHAPTER 2: LABORATORY PROCEDURES FOR FUNGAL CULTURE AND ISOLATION Explain DNA testing supplemental rationale tests

WHAT ARE DNA PROBES EXTREMELY USEFUL FOR? -DNA probes are extremely useful for quickly identifying clinical isolates of slower growing fungi. A COMMERCIALLY AVAILABLE SYSTEM (ACCUPROBE, MADE BY GENPROBE) EXISTS FOR WHICH FUNGAL PATHOGENS? 1.) H. capsulatum 2.) B. dermatitidis 3.) C. immitis 4.) C. neoformans HOW DOES A DNA PROBE TEST WORK? -a known luminescent-labelled, single-stranded DNA probe to the organism in question is allowed to hybridize with unknown ribosomal RNA from a sonicated culture. -if the fungus in question is present, the fungal rRNA will combine with probe DNA and luminesce when placed in a luminometer SENSITIVITY AND SPECIFICITY OF DNA PROBE TESTING -this method is the most sensitive and specific of any on the market DNA PROBE SENSITIVTY AND SPECIFICITY FOR H. CAPSULATUM, C. NEOFORMANS, AND C. IMMITIS -is 100% sensitive and 100% specific DNA PROBE SENSITIVTY AND SPECIFICITY FOR B. DERMATITIDIS -the B. dermatitidis AcuProbe is 97.3% sensitive, but 10 of 17 P. brasiliensis isolates were identified as B. dermatitidis POLYMERASE CHAIN REACTION (PCR) -another technique which shows great promise is the polymerase chain reaction (PCR); however, no commercial kits are yet available PCR CAN BE PERFORMED ON -PCR can be performed on clinical specimens with suspected fungi, or on fungal isolates PCR CAN DETECT WHAT? WHAT OTHER METHOD WITH PCR ACTUALLY IDENTIFIES THE FUNGUS? -PCR can detect as little as one strand of fungal DNA in a specimen, as the technique amplifies DNA a millionfold. -another method such as restriction enzyme analysis then identifies the fungus. WHAT IS THE PROBLEM WITH PCR? -the problem is that these techniques may be actually too sensitive PCR AND ASPERGILLUS -PCR has been used to detect Aspergillus in respiratory specimens, but about 25% of negative controls give positive results, probably from Aspergillus colonization. PCR AND CANDIDA -candida has been isolated from blood specimens in a similar manner, but again there was a problem of contamination with Candida skin flora from when the blood was drawn. PCR STUDY -another study was able to break down unknown fungi into 5 groups: 1.) Candida 2.) Cryptococcus and Trichosporon 3.) Aspergillus 4.) Zygomycetes 5.) Dimorphs -with more research needed to specifically identify the organisms

CHAPTER 2: LABORATORY PROCEDURES FOR FUNGAL CULTURE AND ISOLATION THERAPEUTIC AGENTS FOR MYCOSES Polyene macrolides

WHAT ARE THE 2 AGENTS UNDER POLYENE MACROLIDES? 1.) Amp B 2.) Nystatin HOW DO POLYENE MACROLIDES WORK? -these agents are inserted into the fungal membrane phospholipid, creating pores that increase membrane permeability, allowing small molecules to leak out INTRAVENOUS AMP B REMAINS THE EMPIRIC (Empiric therapy or empirical therapy is medical treatment or therapy based on experience and, more specifically, therapy begun on the basis of a clinical "educated guess" in the absence of complete or perfect information.) THERAPY FOR WHAT -Intravenous Amp B remains the empiric therapy for suspected systemic fungemia in neurotropenic (Neutropenia is when a person has a low level of neutrophils) patients. INTRAVENOUS AMP B IS USED FOR WHICH MYCOSES? 1.) candidiasis 2.) crytococcus 3.) histoplasmosis 4.) blastomycosis 5.) paracoccioidomycosis 6.) coccidioidomycosis 7.) aspergillosis 8.) extracutaneous sporotrichosis 9.) zygomycosis WHAT ARE THE MOST COMMON SIDE EFFECTS OF INTRAVENOUS AMP B? 1.) renal failure 2.) hypokalemia 3.) hypomagnesemia 4.) anemia SIDE EFFECTS OF INTRAVENOUS AMP B ARE IMPROVED BY WHAT? 1.) heavy hydration with normal saline before and after the antibiotic AND 2.) potassium/magnesium supplements WHICH ORGANISMS ARE NOW BECOMING RESISTANT TO AMP B? 1.) T. beigelii 2.) C. tropicalis 3.) C. lusitaniae 4.) C. guillermondii 5.) C. parapsilosis WHAT IS NYSTATIN USED FOR? -Nystatin is used orally, vaginally, or topically for Candida. WHAT ARE OTHER NAMES FOR NYSTATIN? -mycostatin and nilstat SINCE NYSTATIN IS NOT GIVEN INTRAVENOUSLY, WHAT IS THE PROS OF THIS? -virtually there are no side effects WHAT IS THE DISADVANTAGE OF NYSTATIN? -it is not very potent; thus it must be given for a prolonged period of time to show efficacy

CHAPTER 2: LABORATORY PROCEDURES FOR FUNGAL CULTURE AND ISOLATION THERAPEUTIC AGENTS FOR MYCOSES Azole antifungals

WHAT ARE THE 5 DIFFERENT TYPES OF AZOLE ANTIFUNGALS? 1.) clotrimazole (lotrimin) 2.) miconazole (micatin, monistat) 3.) ketoconazole (nizoral) 4.) itraconazole (sporanox) 5.) fluconazole (diflucan) HOW CAN THE AZOLE ANTIFUNGALS BE GIVEN DEPENDING ON THE AZOLE DERIVATIVE? 1.) topically 2.) orally OR 3.) intravenously HOW DO AZOLE ANTIFUNGALS WORK? 1.) azoles bind to the heme portion of cytochrome P450 to interfere with mixed oxidase functions 2.) they also block formation of ergosterol in the fungal cell membrane, inhibiting growth. 3.) while azoles are usually fungistatic, some topical preparations can be fungicidal AZOLE ANTIFUNGALS ARE USEFUL AGAINST WHICH CONDITIONS? 1.) candidiasis 2.) cryptococcosis 3.) coccidioidomycosis 4.) blastomycosis 5.) histoplasmosis 6.) scedosporiosis 7.) pityriasis versicolor 8.) dermatophytosis (ringworm) 9.) aspergillosis 10.) sporotrichosis PROPHYLAXIS (action taken to prevent disease, especially by specified means or against a specified disease) FOR CANDIDIASIS IN IMMUNOSUPPRESSED TRANSPLANT PATIENTS -prophylaxis for candidiasis in immunosuppressed transplant patients is oral ketoconazole (or itraconazole , because of its less toxic side effects). -both ketoconazole and itraconazole cause increased cyclosporin levels (cyclosporin is used as an immunosuppressive to prevent graft rejection); thus a lesser dose of cyclosporin may successfully be used along with the azole to prevent both graft rejection and mycotic infection FLUCONAZOLE USE WITH AIDS PATIENTS -fluconazole does NOT raise cyclosporin levels -fluconazole is the agent of choice for oral or esophageal candidiasis in AIDS patients because it's more reliably absorbed despite AIDS gastropathy. WHAT IS THE UNFORTUNATE DISADVANTAGE OF FLUCONAZOLE? 1.) unfortunately, resistant strains are surfacing 2.) unfortunately, in immunocompromised or granulocytopenic patients, fluconazole has been shown to obscure (hide) the onset of aspergillosis WHAT CAN BE USED INSTEAD OF FLUCONAZOLE WITH THE FLUCONAZOLE-RESISTANT THRUTH IN AIDS PATIENTS? -use oral Amp B or itraconazole FLUCONAZOLE AND CRYPTOCOCCAL MENINGITIS IN AFRICAN AIDS PATIENTS -fluconazole has been used successfully to treat cryptococcal meningitis in African AIDS patients, with a clinical cure of 63% and negative culture after 60-90 days of therapy in 76% of patients PATIENTS WITH CHRONIC MUCOCUTANEOUS CANDIDIASIS HAVE HYPERKERATOTIC LESIONS THAT DO NOT RESPOND TO TOPICAL THERAPY, IN THESE CASES WHAT ARE THE AGENTS OF CHOICE? -oral ketoconazole or itraconazole are the agents of choice

CHAPTER 2: LABORATORY PROCEDURES FOR FUNGAL CULTURE AND ISOLATION THERAPEUTIC AGENTS FOR MYCOSES Allylamines

WHAT ARE THE TWO TYPES OF ALLYLAMINES AND HOW ARE THEY TAKEN? 1.) Naftifine (Naftin) -topical 2.) Terbinafine (Lamisil) -oral/topical HOW DO NAFTIFINE AND TERBINAFINE WORK? -they act by inhibiting squalene epoxidase, an essential step in ergosterol synthesis WHAT ARE NAFTIFINE AND TERBINAFINE USED FOR? -they are both used for dermatophytosis

CHAPTER 2: LABORATORY PROCEDURES FOR FUNGAL CULTURE AND ISOLATION EXAMINING THE CULTURE Explain the cellophane tape method

WHAT DOES THE CELLOPHANE TAPE METHOD WORK BEST WITH? -the cellophane tape method works best with MATURE colonies growing on PLATES, rather than tubes, of media. ADVANTAGE OF CELLOPHANE TAPE METHOD -the advantage of this method is that it doesn't disturb the position of structures; however, it cannot be sealed for permanent storage DISADVANTAGE OF CELLOPHANE TAPE METHOD -this method can not be sealed for permanent storage CELLOPHANE TAPE METHOD DESCRIBED: 1.) prepare a microscope slide with a drop of LPCB and set aside 2.) take a 2-inch piece of transparent cellophane tape, hold it between the fingers with the sticky side out, and touch the sticky side to the top of the fungal colony. -sporulation structures should stick to the tape. 3.) now place the tape, sticky side down, on the microscope slide over the LPCB while stretching the tape and pressing both ends to attach it firmly to the slide. -the tape acts as a coverglass 4.) examine under the microscope for fruiting structures AN IMPROVED METHOD OF THE CELLOPHANE TEST -this improved method can actually be sealed for permanent storage -The improved method uses a device which dispenses a thin layer of transparent adhesive to the surface of a cover glass. Touch the sticky side of the cover glass to the top of the fungal colony, then place the cover glass (sticky side down) over a drop of LPCB on a microscope slide. -this approved mount can be made permanent by sealing the edges with clear nail polish.

CHAPTER 2: LABORATORY PROCEDURES FOR FUNGAL CULTURE AND ISOLATION DIRECT MOUNT EXAMINATION 2.) Lactophenol cotton blue (LPCB) wet mount What are the fungal elements that may be found? Explain the reagent preparation and procedure

WHAT DOES THE PHENOL IN THE LPCB WET MOUNT DO? -kills any organisms WHAT DOES THE LACTIC ACID IN THE LPCB WET MOUNT DO? -preserves fungal structures WHAT DOES COTTON BLUE STAIN IN THE LPCB WET MOUNT? -it stains the chitin in the fungal well walls THE SAME STRUCTURES MAY BE SEEN IN THE LPCB WET MOUNT LIKE THE STRUCTURES FROM THE SALINE WET MOUNT, BUT WHAT MAKES THE LPCB PREPARATION DIFFERENT? -the LPCB preparation can be made PERMANENT -structures that are seen in both the saline wet mount and the LPCB wet mount include: 1.) budding yeasts 2.) hyphae 3.) pseudohyphae 4.) conidia 5.) thin branching filaments resembling bacteria (funguslike bacteria) 6.) granules 7.) Coccidioides immitis spherules LPCB WET MOUNT REAGENT PREPARATION: LPCB: commercially available (Marion Scientific): Phenol, concentrated 20 mL Lactic acid 20 mL Glycerol 40 mL Cotton blue (China blue) 0.05 g. Distilled water 20 mL 1.) Dissolve cotton blue in distilled water, then add the rest of the ingredients. Mix well. LPCB WET MOUNT PROCEDURE: 1.) Place one drop of specimen on a slide, add one drop of LPCB. -some specimens, for example, pleural fluid, will precipitate with this stain 2.) Mix well and add a coverslip. Observe under the microscope for fungal elements 3.) For a permanent preparation, rim the coverslip with clear nail polish or Permount

CHAPTER 3: COMMON FUNGAL OPPORTUNISTS OPPORTUNISTIC MYCOSES Penicilliosis characteristics

WHAT IS ANOTHER NAME FOR PENICILLIOSIS? -also known as hyalohyphomycosis caused by penicillus-producing fungi HOW IS PENICILLIOSIS ACQUIRED? -usually is acquired by inhaling the conidia of the opportunists: 1.) Penicillium 2.) Scopulariopsis 3.) Paecilomyces WHAT DOES PENICILLIOSIS START AS? -a pulmonary disease DISSEMINATED VERSION OF PENICILLIOSIS -it starts as a pulmonary disease but may spread into the adjacent blood vessels. -subsequently it disseminates to the rest of the body, including: 1.) Spinal fluid 2.) Kidneys (Penicillium) 3.) Endocardium (Paecilomyces and Penicillium) -total invasion occurs in DEBILITATED patients PENICILLIOSIS MAY ALSO INCLUDE THE FOLLOWING: 1.) Infections of the nails (Scopulariopsis) 2.) Otomycosis (Penicillium) 3.) Keratomycosis (Penicillium and Scopulariopsis) 4.) Inguinal ulcers (Scopulariopsis) 5.) Allergic bronchial asthma in sensitized people (Penicillium and Scopulariopsis) DIRECT MOUNT -observing hyphae and conidia in direct mounts in itself if not significant unless the organism is also repeatedly isolated in large numbers THE DISSEMINATED FORM OF PENICILLIOSIS CAUSED BY PENICILLIUM MARNEFFEI HAS THE FOLLOWING CLINICAL MANIFESTATIONS THAT ARE ACUTE ONSET: 1.) fever 2.) chills 3.) cough 4.) weakness 5.) loss of weight 6.) leukocytosis 7.) anemia 8.) enlarged lymph nodes 9.) enlarged sleen 10.) enlarged liver DISSEMINATED FORM OF PENICILLIOSIS CAUSED BY PENICILLIUM MARNEFFEI MAY HAVE THE FOLLOWING PRIMARY FORMS OF INFECTION: 1.) pulmonary (inhalation of the organism) 2.) intestinal (ingestion of the organism) WHERE DOES THE DISSEMINATED FORM OF PENICILLIOSIS CAUSED BY PENICILLIUM MARNEFFEI OCCUR? -it occurs in Southeast Asia, especially in the following areas: 1.) China 2.) Thailand 3.) Hong Kong 4.) Vietnam ALTHOUGH THE DISSEMINATED FORM OF PENICILLIOSIS CAUSED BY PENICILLIUM MARNEFFEI HAS OCCURRED IN IMMUNOCOMPETENT INDIVIDUALS, IT OCCURS MOST OFTEN IN WHICH TYPES OF PATIENTS? -the immunocompromised and AIDS patients, some of whom may be diagnosed in the United States or Europe following travel to Southeast Asia DISSEMINATED FORM OF PENICILLIOSIS CAUSED BY PENICILLIUM MARNEFFEI: WHAT DO HEMATOXYLIN AND EOSIN (H&E) STIANS OF AFFECTED TISSUE REVEAL? -yeast bodies occurring intracellularly within macrophages WHAT DO THE YEAST BODIES OCCURRING INTRACELLULARLY WITHIN MACROPHAGES ON THE H&E STAIND OF P. MARNEFFEI RESEMBLE? -they resemble the appearance of H. capsulatum but Histoplasma appears pseudoencapsulated. THE YEAST BODIES OF P. MARNEFFEI -the yeast bodies of P. marneffei divide by elongating into sausage forms, developing a septum across the width of the cell, and splitting at the septum into two cells TREATMENT OF THE DISSEMINATED FORM OF PENICILLIOSIS CAUSED BY PENICILLIUM MARNEFFEI -antifungal agents include: 1.) amphotericin B 2.) ketoconazole 3.) fluconazole 4.) 5-fluorocytosine -without treatment with these antifungal agents, the disease is usually fatal

CHAPTER 2: LABORATORY PROCEDURES FOR FUNGAL CULTURE AND ISOLATION DIRECT MOUNT EXAMINATION 7.) Calcofluor white stain give characteristics of the calcofluor white stain -calcofluor white stain reagent preparation and procedure can be seen on page 48 in the box.

WHAT IS CALCOFLUOR WHITE TYPICALLY USED FOR IN INDUSTRIES OTHER THAN THE MEDICAL FIELD? -calcofluor white is a bleaching agent that is used in the paper and textile industry WHY IS THE CALCOFLUOR WHITE STAIN NOT ABSORBED INTO HUMAN TISSUE? -calcofluor white is taken up into the chitin of the fungal cell wall; background human material does not possess chitin, thus the dye is not absorbed into human tissue THE CALCOFLUOR WHITE IN THE FUNGUS PRODUCES WHAT APPEARANCE UNDER AN ULTRAVIOLET MICROSCOPE? -the dye in the fungus produces a chalk-white or apple-green fluorescence depending upon excitation wavelength (usually 340 nm) when observed under an ultraviolet microscope. WHEN WILL CALCOFLUOR WHITE BE MIXED WITH 10% KOH JUST IMMEDIATELY BEFORE USE TO DISSOLVE BACKGROUND MATERIAL? -in a particularly hard specimen, such as a nail specimen. ISSUES WITH CALCOFLUOR WHITE STAIN -calcofluor will not fluoresce inside coccidioides spherules, and vaginal wet preparations are difficult to interpret CALCOFLUOR WHITE STAIN PREPARATION AND PROCEDURE -see the box on page 48, and GO STUDY IT FROM THERE, type everything or write everything out fully when studying

CHAPTER 3: COMMON FUNGAL OPPORTUNISTS OPPORTUNISTIC MYCOSES Otomycosis characteristics

WHAT IS OTOMYCOSIS? -it is a fungal infection of the external auditory opening or ear canal WHAT ARE THE SYMPTOMS OF OTOMYCOSIS? -inflammation, itching, scaling, and partial deafness due to the ear canal being filled with a hyphal plug TRAUMA OFTEN STARTS THE DISEASE, GIVE EXAMPLES OF HOW THIS CAN HAPPEN. 1.) Cleaning the ears with matches or similar objects causes abrasions that readily become infected 2.) Secondary to bacterial infection 3.) Previous use of antibiotics 4.) Too much heat and moisture WHAT ARE THE SPECIMENS OF CHOICE FOR DIRECT MOUNT AND CULTURE? -scrapings and purulent discharge are the specimens of choice WHAT MUST BE DONE TO CONFIRM SIGNIFICANCE OF OPPORTUNIST? -many of the fungal etiologic agents are opportunists and thus must be repeatedly isolated in large numbers before being considered significant WHICH FUNGI CAN CAUSE OTOMYCOSIS? 1.) Fungal opportunists 2.) Yeasts 3.) Dermatophytes

CHAPTER 3: COMMON FUNGAL OPPORTUNISTS OPPORTUNISTIC MYCOSES Sinusitis characteristics

WHAT IS SINUSITIS? -it is an infection of the nasal sinuses WHAT ARE THE SYMPTOMS OF SINUSITIS? -symptoms include sinus pain, nasal discharge, nasal obstruction, and inflammation -eosinophilia -patients present with thick nasal discharge that may occasionally be bloody and contain pieces of brownish tissue. SINUSITIS CAN BE CAUSED BY WHAT? -the syndrome may be caused by a number of bacterial and fungal species, and it can be either acute or chronic. WHAT ARE THE IMPORTANT AGENTS OF FUNGAL SINUSITIS? -certain species of dematiaceous fungi: 1.) Bipolaris 2.) Curvularia 3.) Alternaria -Aspergillus used to be considered the usual cause of fungal sinusitis SINUSITIS USUALLY OCCURS IN WHAT TYPES OF PATIENTS? -it usually occurs in immunocompetent patients with a history of allergy and nasal polyps. WHICH BLOOD CELL IS COMMONLY FOUND IN SINUSITIS -eosinophilia is common WHAT IS NECESSARY FOR EFFECTIVE TREATMENT OF CHRONIC SINUSITIS? -surgical debridement IF SINUSITIS IS LEFT UNTREATED, WHAT COULD HAPPEN? -the paranasal sinuses, orbital sinuses or central nervous system may become involved with potentially life-threatening consequences

CHAPTER 2: LABORATORY PROCEDURES FOR FUNGAL CULTURE AND ISOLATION EXAMINING THE CULTURE Explain the tease mount method

WHAT IS THE ADVANTAGE OF THE TEASE MOUNT METHOD? -the advantage of this method is that it can be performed and examined immediately after maturation of the fungal colony on the primary isolation plate. WHAT IS THE DISADVANTAGE OF THE TEASE MOUNT METHOD? -the disadvantage is that the rough action of teasing apart hyphae disturbs the position of conidia so that oftentimes the structural morphology of the organism cannot be discerned (recognized). SEE BOX AND FIGURE 2-6 ON PAGE 58 -study and look at both

CHAPTER 2: LABORATORY PROCEDURES FOR FUNGAL CULTURE AND ISOLATION Culture preservation

WHAT IS THE EASIEST WAY TO MAINTAIN STOCK FUNGAL CULTURES? -the distilled water method of Castellani EXPLAIN THE DISTILLED WATER METHOD OF CASTELLANI: 1.) after growth and sporulation of the fungus on solid medium, place 2-3 mL of sterile distilled water over the culture 2.) scrape the sporulating structures into the water with a sterile wire loop or pipette tip 3.) aspirate the water with a sterile pipette and transfer to a sterile screw-capped tube 4.) tighten the cap and store as room temperature or in the refrigerator WHAT IS THE OVERALL SURVIVAL FOR THE FIRST 5 YEARS WHEN USING THE DISTILLED WATER METHOD OF CASTELLANI FOR FUNGUS PRESERVATION? -it is 97% for the first 5 years WHICH FUNGI MAY NOT SURVIVE WHEN USING THE CASTELLANI DISTILLED WATER METHOD FOR FUNGUS PRESERVATION? 1.) Candida krusei AND 2.) Saccaromyces cerevisiae WHAT ARE OTHER FUNGUS PRESERVATION METHODS OTHER THAN THE CASTELLANI DISTILLED WATER METHOD? 1.) Freezing 2.) Lyophilization 3.) Oil overlay

CHAPTER 2: LABORATORY PROCEDURES FOR FUNGAL CULTURE AND ISOLATION THERAPEUTIC AGENTS FOR MYCOSES Pyrimidine analogues

WHAT IS THE NAME OF THE PYRIMIDINE ANALOGUE? -the name is 5-Fluoreocytosine (5-PC) WHAT DOES 5-PC DO? 1.) It incorporates into RNA to cause production of nonsense protein 2.) Otherwise, it can convert into a potent thymidylate synthetase inhibitor, which stops DNA production WHAT IS 5-PC USED FOR? 1.) Candidiasis 2.) Cryptococcosis 3.) Chromoblastomycosis WHAT IS THE UNFORTUNATE DISADVANTAGE OF 5-PC? -organisms quickly develop resistance WHAT ARE THE 2 DRUGS THAT 5-PC IS SYNERGISTIC WITH AND WHAT DOES IT DO? 1.) 5-PC is synergistic with Amp B against most systemic Candida and C. neoformans -This synergistic relationship allows a lesser dose of Amp B, and lessening development of resistance 2.) 5-PC is also synergistic with fluconazole on C. albicans, giving an effect lasting 2.5 house longer per dose than either agent alone

CHAPTER 2: LABORATORY PROCEDURES FOR FUNGAL CULTURE AND ISOLATION CULTURING THE SPECIMEN PRIMARY FUNGAL MEDIA NONSELECTIVE MEDIA 3.) Subouraud Dextrose Agar (SDA) Give characteristics of SDA -see page 53, second box for SDA medium preparation and procedure

WHAT IS THE PH OF SDA AND ITS NUTRITION QUALITY? -this general medium has an acid pH of 5.6 and is nutritionally poor THE CONDITIONS OF SDA INHIBIT WHAT TYPE OF PATHOGENS AND ALLOW THE GROWTH OF WHAT KINDS OF PATHOGENS? 1.) Inhibits: -these conditions inhibit growth of many bacteria 2.) Allows the growth of: -fungal opportunists and pathogenic fungi can grow in SDA. WHICH PATHOGENIC FUNGI CAN NOT GROW ON SDA? 1.) Histoplasma capsulatum AND 2.) Some strains of Nocardia asteroides SDA MEDIUM PREPARATION AND PROCEDURE: -go to page 53 and type out or write out the whole box (second box), thoroughly study it

CHAPTER 2: LABORATORY PROCEDURES FOR FUNGAL CULTURE AND ISOLATION DIRECT MOUNT EXAMINATION 6.) India ink preparation give characteristics of the india ink preparation -see table on page 47 for india ink reagent preparation and procedure

WHAT IS THE PURPOSE OF THE INDIA INK PREPARATION? -this procedure is used to observe capsules around yeasts, especially Cryptococcus neoformans in CEREBROSPINAL FLUID SEDIMENT. ISSUES WITH INDIA INK PREPARATION AND HOW TO SOLVE IT 1.) Issue: -unfortunately, all bacteria and fungal organisms, encapsulated or not, will stand out distinctly against the black India ink background, because the ink will NOT penetrate the cell wall or capsule -some bacteria, such as KLEBSIELLA, possess capsules that may be mistaken for yeasts. 2.) How to solve issue: -be careful to search for capsules ONLY -bacteria possess capsules that may be mistaken for yeasts, BUT bacteria are one fourth the size of yeasts, and bacteria will NOT be budding SOME LABORATORIES USE A POSITIVE INDIA INK PREPARATION FOR WHAT? -definitive evidence for the presence of Cryptococcus neoformans. IT MUST BE EMPHASIZED THAT OTHER OTHER CRYPTOCOCCUS SPECIES AND OTHER GENUSES OF FUNGI MAY ALSO BE ENCAPSULATED, SUCH AS: 1- rhodotorula 2- torulopsis 3- sporobolomyces 4- trichosporon beigelii 5- prototheca stagnora (an alga that may be confused with crytococcus) WHAT OTHER TESTS MAY BE DONE BEFORE MAKING A DEFINITIVE DIAGNOSIS OF CRYPTOCOCCUS NEOFOMANS? 1-dark brown pigment production on caffeic acid agar 2-direct antigen testing 3-specifica carbohydrate assimilation reactions INDIA INK REAGENT PREPARATION AND PROCEDURE -see the box on page 47, and GO STUDY IT FROM THERE, type everything or write everything out fully when studying

CHAPTER 3: COMMON FUNGAL OPPORTUNISTS ASEPTATE OPPORTUNISTS Zygomycosis (mucormycosis, phycomycosis) characteristics

WHAT IS ZYGOMYCOSIS? -zygomycosis is an acute fungus infection caused by fungi from the phylum Zygomycota WHERE DO ZYGOMYCOTA FUNGI COMMONLY GROW? -they commonly grow on bread and fruits HOW IS ZYGOMYCOSIS INFECTION TRANSMITTED? -zygomycota spores become airborne and will only typically cause an infection in immunosuppressed patients WHO IS SUSCEPTIBLE TO ZYGOMYCOSIS INFECTION? 1.) Patient is in acidosis (as with uncontrolled diabetes or malnutrition) 2.) Patient is on corticosteroids 3.) Patient is on antibiotics 4.) Patient on antileukemic drugs -spores may infect the nasal sinuses and orbital (eye) area of susceptible individuals as listed above. DOES ZYGOMYCOSIS SPREAD RAPIDLY? -yes, zygomycosis spreads rapidly RAPIDLY FATAL MENINGOENCEPHALITIS -from the nasal sinuses and the orbital areas, infection spreads to the: 1.) Brain and 2.) Meninges -this produces a rapidly fatal meningoencephalitis WHERE ELSE MAY THE DISEASE DISSEMINATE TO? -the lungs and the gastrointestinal tract WHEN DOES DEATH OCCUR AFTER INITIAL SINUS/EYE INFECTION? -death ensues 2-10 days after the initial sinus/eye infection CHRONIC, SELF-LIMITING FORM OF ZYGOMYCOSIS -a chronic, self-limiting form of zygomycosis has been observed in subcutaneous lesions A MORE SERIOUS FORM OF ACUTE PROGRESSIVE CELLULITIS OF THE LEG -a more serious form of acute progressive cellulitis of the leg was reported in diabetic patients DIRECT MOUNTS OF ALL TYPES OF ZYGOMYCOSIS -direct mounts of specimens from all types of zygomycosis show branching and large ribbonlike aseptate hyphae, and round sporangia may be present. DIAGNOSIS IS CONFIRMED WITH -diagnosis is confirmed with repeated isolation of the organism WHEN SHOULD TREATMENT BE INITIATED? -the physician should begin treatment AS SOON as the disease is suspected, even before the confirmation of the diagnosis. ^^^-this is because zygomycosis of the sinuses, orbital area, and meninges is so rapidly fatal. -treatment with amphotericin B is usually successful if initiated promptly.

CHAPTER 2: LABORATORY PROCEDURES FOR FUNGAL CULTURE AND ISOLATION EXAMINING THE CULTURE Explain the modified slide culture method

WHAT MAKES THE MODIFIED SLIDE CULTURE METHOD DIFFERENT: 1.) this method uses water agar instead of distilled water for moisture 2.) this method uses a cover glass on the bottom instead of a microscope slide SEE BOX ON PAGE 63 FOR THE PROCEDURE FOR THE MODIFIED SLIDE CULTURE -seriously, go study it

CHAPTER 2: LABORATORY PROCEDURES FOR FUNGAL CULTURE AND ISOLATION PRIMARY FUNGAL MEDIA INCUBATION TEMPERATURE

WHAT TEMPERATURE SHOULD FUNGAL CULTURE BE GENERALLY INCUBATED AT? -should be generally incubated at 30 degrees celcius ROOM TEMPERATURE (25 DEGREES CELCIUS) INCUBATION TEMP FOR FUNGAL CULTURES: -room temperature is acceptable, although some organisms may multiply slower at this temperature 37 DEGREES CELCIUS INCUBATION TEMP FOR FUNGAL CULTURES -may inhibit some fungi SINCE SO FEW DIMORPHIC FUNGI ARE RECOVERED, WHAT IS THE PREFERRED METHOD? -it is preferable to first isolate them in the mold phase (room temperature [25 degrees celcius] or at 30 degrees celcius), then set up cultures at 37 degrees celcius for conversion to the yeast phase

CHAPTER 2: LABORATORY PROCEDURES FOR FUNGAL CULTURE AND ISOLATION DIRECT MOUNT EXAMINATION 4.) Gram stain What are the fungal elements that may be found? Give gram stain characteristics regarding fungi.

WHEN IS FUNGAL PRESENCE TYPICALLY FIRST NOTED? -often, fungal presence is first noted on the routine specimen gram stain performed for bacteriology HOW DO FUNGI STAIN ON THE GRAM STAIN? -fungi stain gram-positive, or blue WHAT FUNGAL FORMS MAY BE OBSERVED ON A GRAM STAIN, AND WHICH ARE MOST COMMON? -any fungal forms may be observed -yeasts and pseudohyphae are the most common HOW DO YEASTS APPEAR ON A GRAM STAIN? -yeasts are 2-3 times larger than gram-positive cocci and will usually be budding if they are causing an infection. ON A VAGINAL OR URETHRAL GRAM-STAINED SMEAR, WHAT MAY NONBUDDING YEAST BE MISTAKEN FOR? -a tailless spermatozoon. CRYTOCOCCUS NEOFORMAN GRAM-STAIN IDENTIFICATION ISSUES WITH RESPIRATORY AND CSF SPECIMENS: -on respiratory or CSF specimens, the capsule around Cryptococcus neoformans prevents definitive staining of the yeast itself, and thus the organism can be easily overlooked. -this point is especially important since the physician may not order histologic stains or fungus cultures, and the patient's disease would remain undiagnosed. -on a gram stain, C. neoformans appears either as a: 1- round, pale lavender cell with gram-positive, granular inclusions OR AS 2- a gram-negative fat body HOW DO HYPHAE APPEAR ON A GRAM STAIN? -hyphae are 2-3 times wider than gram-positive rods, and often hyphae will not stain solidly inside, eliciting a granular appearance

CHAPTER 2: LABORATORY PROCEDURES FOR FUNGAL CULTURE AND ISOLATION CULTURING THE SPECIMEN Primary Fungal Media Characteristics

WHEN IS PRIMARY FUNGAL MEDIA USED AND WHY? -primary fungal media is done at the same time that examining the direct mount is done -primary fungal media is when specific fungal media are inoculated (introduced into a culture media) to initially isolate any organisms WHAT IS PREFERRED FOR PRIMARY FUNGAL MEDIA AND WHY? -tubed media are preferred over plates because tubed media ensure that: 1.) tubed media will not dry out over the long incubation period 2.) tubed media reduced the chance for fungal reproductive structures to become airborne and contaminate the room and people WHICH SPECIMEN CAN NEVER USE PLATES FOR PRIMARY FUNGAL MEDIA? -NEVER use plates when Coccidioides immitis is suspected, as this fungus is extremely infectious and aerosols may be inhaled WHAT MUST ALWAYS BE DONE WHEN USING PRIMARY FUNGAL MEDIA? 1.) Always work under a biological safety cabinet 2.) Wear gloves 3.) Autoclave specimens and inoculated media when finished 4.) Disinfect the work area daily. CHART 2-2 -go to page 50-52 and study the fungi that may be isolated from various specimens and their suggested primary isolation media. PSEUDOMONAS IN A SPECIMEN -pseudomonas in a specimen can inhibit growth of the following from the same specimen : 1- Most Candida 2- T. glabrata 3- S. cerevisiae 4- A. fumigstus WHAT IS THE GOAL OF PRIMARY FUNGAL MEDIA AND HOW IS THIS DONE? -the goal of primary fungal media is to isolate all the possible pathogens -to isolate all possible pathogens, a combination of NONSELECTIVE AND SELECTIVE MEDIA (selective media is with antibiotics, and nonselective media is without antibiotics) is necessary. -also, because some systemic fungal pathogens are FASTIDIOUS, use media with BLOOD for extra nutrition.

CHAPTER 2: LABORATORY PROCEDURES FOR FUNGAL CULTURE AND ISOLATION DIRECT MOUNT EXAMINATION 5.) Acid-fast stain Give the characterizations See page 46 for the reagent preparation and procedure for modified acid-fast stain

WHICH FUNGUSLIKE BACTERIA IS FOUND ON THE ACID-FAST STAIN? -Nocardia -the funguslike bacteria Nocardia are PARTIALLY acid fast HOW DO NOCARDIA APPEAR ON THE ACID-FAST STAIN? -nocardia appear red against a blue background WHAT MAY NOCARDIA BE MISTAKEN FOR ON THE ACID-FAST STAIN? -nocardia may be mistaken for mycobacteria WHAT MAKES NOCARDIA DIFFERENT FROM MYCOBACTERIA ON THE ACID-FAST STAIN? -nocardia possess branching filaments as well as bacillary forms WHY IS THE NORMAL MYCOBACTERIAL ACID-FAST STAIN NOT THE BEST FOR NOCARDIA? -the acid-fast stain for mycobacteria may overdecolorize nocardia, giving false-negative results WHICH ACID-FAST SMEAR SHOULD BE MADE IF NOCARDIA IS SUSPECTED? -another smear should be made and stained with the modified Kinyoun acid-fast stain if Nocardia is suspected. NOCARDIA AND TUBERCULOSIS -nocardia may remain viable through the sputum digestion process for tuberculosis and will also grow on TB media. THE DISEASE CAUSED BY NOCARDIA RESEMBLE WHAT? -the disease caused by nocardia may resemble tuberculosis; hence, the diagnosis for nocardiosis must always be considered when working up a patient for TB REAGENT PREPARATION AND PROCEDURE FOR MODIFIED ACID-FAST STAIN -see the box on page 46, and GO STUDY IT FROM THERE, type everything or write everything out fully when studying

CHAPTER 4: SUPERFICIAL AND DERMATOPHYTIC FUNGI DERMATOPHYTES Growth rate

WHICH ORGANISMS FORM MATURE COLONIES IN 6-10 DAYS (INTERMEDIATE GROWERS)? 1.) Epidermophyton floccosum 2.) Microsporum canis 3.) Microsporum gypseum 4.) Trichophyton mentagrophytes WHICH ORGANISMS FORM MATURE COLONIES IN 11-21 DAYS (SLOW GROWERS)? 1.) Microsporum audouinii 2.) Trichophyton rubrum 3.) Trichophyton schoenleinii 4.) Trichophyton tonsurans 5.) Trichophyton verrucosum 6.) Trichophyton violaceum WHAT IS A A GOOD WAY TO REMEMBER WHICH DERMATOPHYTES ARE INTERMEDIATE GROWERS AND WHICH ONES ARE SLOW GROWERS? -all the Microsporum sp. listed in this module except for M. audouinii are intermediate growers, while all the Trichophyton sp. except T. mentagrophytes are slow growers

CHAPTER 4: SUPERFICIAL AND DERMATOPHYTIC FUNGI Introduction to superficial and dermatophytic fungi

WHY ARE SUPERFICIAL AND CUTANEOUS FUNGI OFTEN GROUPED TOGETHER? -because they infect the same outer areas of the body- skin, hair, and nails SUPERFICIAL MYCOSES DEFINITION -are noninvasive and basically asymptomatic -involves just the top of keratin-containing layers of skin or hair CUTANEOUS (DERMATOMYCOSES) MYCOSES DEFINITION -affects the deeper epidermal layers, producing more tissue destruction and symptoms

CHAPTER 3: COMMON FUNGAL OPPORTUNISTS FINAL EXAM QUESTIONS 1-14 ON PAGES 110-113 A 10-year-old boy was admitted to the hospital in diabetic ketoacidosis. Three days later, he complained of nasal sinus blockage. Stained histologic preparations of material removed from his sinuses showed the wide, ribbonlike hyphae seen in Figure 3-58. On SABHI agar, a white cottony mold rapidly grew. 11.) Which disease do you suspect, and why?

Zygomycosis. -Uncontrolled diabetes is the typical predisposing factor. -Infection characteristically begins in the nasal passages or eye, and aseptate hyphae indicate the fungus must be of the phylum Zygomycota.

CHAPTER 1: BASICS OF MYCOLOGY sporangiospores are formed in only what type of fungi?

aseptate fungi

CHAPTER 1: BASICS OF MYCOLOGY What is the field of conidiogenesis?

asexual (anamorph) conidium formation

CHAPTER 2: LABORATORY PROCEDURES FOR FUNGAL CULTURE AND ISOLATION FINAL EXAM QUESTIONS 1-7 ON PAGES 66-67 4.) Choose the correct answer: Lactophenol cotton blue stains the chitin/keratin in fungal cell walls

chitin

CHAPTER 1: BASICS OF MYCOLOGY What is another name for ringworm of skin, hair, or nail?

dermatophytosis

CHAPTER 1: BASICS OF MYCOLOGY What does the term ascogonium mean?

female sexual cell produced by the members of the phylum ascomycota

CHAPTER 1: BASICS OF MYCOLOGY See chart 1-2 to see taxonomic classification of clinically important fungi and funguslike bacteria

go look now

CHAPTER 2: LABORATORY PROCEDURES FOR FUNGAL CULTURE AND ISOLATION 1.) Blood and Bone Marrow FUNGAL SOURCES Bone marrow biopsy and culturing can be useful in the diagnosis of what type of infection because blood may give a sparse yield?

histoplasmosis

CHAPTER 3: COMMON FUNGAL OPPORTUNISTS SEPATATE OPPORTUNISTS HYALINE SEPTATE OPPORTUNISTS Opportunists with light-colored hyphae may cause what?

hyalohyphomycosis (infection caused by hyaline fungi

CHAPTER 1: BASICS OF MYCOLOGY Microscopically, fungal cells are observed as either ______ or as ________

hyphae (molds) or as yeasts.

CHAPTER 1: BASICS OF MYCOLOGY What does the term antheridium mean?

male sexual cell produced by the ascomycota

CHAPTER 1: BASICS OF MYCOLOGY What is another term for hyphae?

mold

CHAPTER 1: BASICS OF MYCOLOGY Conidium (plural is conidia) definition

nonmotile, asexual reproductive structure which usually separates from the parent. -is NOT produced by cleavage, conjugation, or free-cell formation.

CHAPTER 1: BASICS OF MYCOLOGY conidia are formed in only what type of fungi?

septate fungi

CHAPTER 1: BASICS OF MYCOLOGY What is the another name for sexual stage of fungi?

teleomorph stage

CHAPTER 3: COMMON FUNGAL OPPORTUNISTS STUDY QUESTIONS 1-3 ON PAGE 96 1.) Fill in the blank: If the conidia of Alternaria sp. are young and therefore not in chains, they may be mistaken for those of:

the opportunist, Stemphylium

CHAPTER 2: LABORATORY PROCEDURES FOR FUNGAL CULTURE AND ISOLATION FUNGAL SOURCES 4.) Respiratory: Bronchial Washings, Sputa, Throats, Transtracheal Aspirates Once bronchial washings, sputa, throat, and transtracheal aspirate specimens are in the laboratory, what are they directly examined for?

they are examined for: 1.) blood 2.) pus 3.) necrotic (death of body tissue) material and then these are plated

CHAPTER 1: BASICS OF MYCOLOGY Yeasts

yeasts consist of individual oval to round cells, which frequently bud to form daughter cells