06- Mycology & Mycobacteriology (Exam #6) Flashcards
How are fungi different from bacteria?
- Fungi are eukaryotic; they possess a true nucleus, with a nuclear membrane and mitochondria.
- Bacteria are prokaryotic, lacking these structures.
How are fungi different from plants?
- Like plants, fungi are eukaryotic.
- Unlike plants, fungi lack chlorophyll and must absorb nutrients from the environment.
- Fungal cell walls are made of chitin, whereas those of plants contain cellulose.
Define:
Saprobe/Saprophytic
- In contrast to classic pathogens, these fungi are found in the environment living on nonliving material.
- Isolation of these organisms would only be important in an immuncompromised patient
Fungal Diseases
It is helpful to organize the fungal diseases by the depth of the skin that they infect.
Describe
Processing Specimens for Fungal Culture
- Because of the additional hazard of airborne conidia, a class 2 biological safety cabinet should be used to reduce exposure of personnel to fungal elements.
- All specimens for mycology should be transported and processed as soon as possible. Because many pathogenic fungi grow slowly, any delay in processing compromises specimen quality and decreases the probability of isolating the causative agent as a result of overgrowth by contaminants.
- Although almost any tissue or body fluid can be submitted for fungal culture, the most common specimens are respiratory secretions, hair, skin, nails, tissue, blood, bone marrow, and CSF.
Describe
Direct Microscopic Examination of Specimens
-
10-20% KOH
Specimen is placed on a slide with a drop of KOH and a coverslip. The slide is heated and allowed to cool for ~ 15 mins
KOH breaks down keratin and skin layers to reveal fungi if present -
KOH with Calcofluor White
Calcofluor white binds to chitin and will fluoresce apple green -
India Ink
Added to CSF, to quickly detect capsulated yeast (Cryptococcus species)
Which microscopic method is seen in the picture?
KOH
Which microscopic method is seen in the picture?
India Ink
Which microscopic method is seen in the picture?
Calcofluor white
Describe
Primary Plating Media
-
Potato dextrose agar (PDA) is the recommended plating medium for the cultivation, enumeration, and identification of yeasts and molds from products, as well as from clinical specimens. The potato infusion encourages the growth of and sporulation by fungi.
Some labs use a similar agar = Sabouraud Dextrose Agar -
Potato dextrose with chloramphenicol (PDACH)
Same as above, but with chloramphenicol to inhibit bacteria - Mycosel/ Mycobiotic Agar are selective media used to isolate pathogenic fungi, dermatophytes, and systemic pathogens. The cycloheximide suppresses the growth of saprophytic fungi, whereas the chloramphenicol inhibits bacterial contaminants.
Describe
Primary Plating Media
Describe
Incubation of Fungal Cultures
- Fungal Culture: 30 degrees Celcius for 4 weeks (28 days)
- Fungal Screen: 30 degrees Celcius for 10 days
What test methods are used to ID yeasts?
- Germ Tube Test
- Urease Test
- Chrom Agar
- Carbohydrate Assimilation Tests
Describe
The Germ Tube Test
- Purpose: Rapid identification test for Candida albicans
- Principle: Candida albicans and Candida dubliniensis produces tube-like projections with parallel walls and no constrictions at the jucture of the parent cell.
-
Procedure:
1. Using an isolated colony make a light suspension of the organisms 1 mL of bovine albumin
2. Incubate for 2 hours
3. Prepare a wet preparation of the suspension
4. Examine on 10X and confirm on 40X
What is the purpose of the urease test?
Rapid screening test for urea positive yeast including Cryptococcus neoformans. Also positive for Rhodoturula
Principle: The test relies upon the release of ammonia during urea hydrolysis to change the pH of the medium to alkaline, converting the light orange phenol red indicator to pink.
Procedure:
Pipet 200 um of urea broth into a well on a microtiter plate
Using a wooden applicator stick, heavily inoculate the urea broth with the test isolate
Incubate in ambiently at 35-37 degrees C for 2-4 hours
What is the purpose of the Carbohydrate Assimilation Test (API 20C)?
Assimilation tests identify which carbohydrates a yeast can use aerobically as a sole carbon source.
How to ID Mold
Macroscopic:
1. Describe forward and reverse colony color
Microscopic:
1. Describe hyphae as hyaline or dematiaceous
2. Describe hyphae as septate or pauciseptate
3. Describe reproductive structures
How to ID Mold
Describe the appearance of mold.
Most molds have a fuzzy or woolly appearance
What is the name of the structures that give mold it’s fuzzy or wooly like appearance?
Mycelia.
Mycelia are made up of many long strands of tubelike structures called hyphae, which are either aerial or vegetative.
Aerial mycelia extend above the surface of the colony and are responsible for the fuzzy appearance. In addition, aerial mycelia support the reproductive structures that produce conidia. Conidia, in many cases, are used to identify different fungal genera. The vegetative mycelia extend downward into the medium to absorb nutrients.
How to ID Mold (Macroscopic)
Step 1: Describe the
Base = reverse side (light or dark)
Lid = forward side (colony color)
Mold ID (Microscopic)
How to ID Mold: Microscopic
Step 2:
What procedures are used to identify the structures of mold?
How to ID Mold: Microscopic
Step 2 Continued:
Reading the lactophenol cotton blue describe the hyphae as Hylaine or Phaeoid.
- Hyaline (moniliaceous) hyphae are nonpigmented or lightly pigmented
- Phaeoid (dematiaceous) hyphae are darkly pigmented because of the presence of melanin in the cell wall.
How to ID Mold: Microscopic
Step 3:
Describe the different types of hyphae.
- Septate hyphae show frequent cross-walls occurring perpendicular to the outer walls of the hyphae
- Pauciseptate show sparsely septate hyphae have few cross-walls at irregular intervals.
How to ID Mold: Microscopic
Step 4:
Identify reproductive structures.
- Fungi can reproduce asexually (imperfect) or sexually (perfect). Asexual reproduction results in the formation of conidia (singular, conidium) following mitosis. Asexual reproduction is carried out by specialized fruiting structures known as conidiogenous cells. These structures form conidia, which contain all the genetic material necessary to create a new fungal colony.
- In the clinical laboratory, most mold identifications are based on the structures formed as a result of asexual reproduction. Sexual reproduction requires the joining of two compatible nuclei, followed by meiosis . A fungus that reproduces sexually is known as a teleomorph. Occasionally, these fungi will also reproduce asexually. When this occurs, the asexual form is termed the anamorph. If more than one anamorph is present for the same teleomorph, the anamorphic strains are termed synanamorphs.
How to ID Mold (Microscopic)
Step 4:
Identify reproductive structures continued:
- Two common conidiogenous cells are the phialides and annellides.
- Phialides are vaselike structures
- Annellides are ringed structures
- Both form their conidia blastically (budding) like many yeasts; the parent cell enlarges and a septum forms to separate the conidial cell.
General Mycology
How do yeast reproduce?
- Single vegetative cell
- Reproduce by budding or fission
Yeasts reproduce by budding or fission. Budding involves maturation of the bud to an independent structure called what?
Blastoconidium (aka daughter cell)
This process involves lysis of the yeast cell wall so that a blastoconidium can form. As this structure enlarges, the nucleus of the parent cell undergoes mitosis. Once the new nucleus is passed into the daughter cell, a septum forms and the daughter cell breaks free. During fission, two cells of equal size are formed. These cells continue to grow from the tips of the cell and divide only after a medial fission is formed.
The term spore should be reserved for referring to what type of reproduction?
Meiosis (i.e., sexual reproduction), such as ascospores, oospores, or zygospores,
The term conidia should be reserved for referring to what type of reproduction?
Mitosis (i.e, asexual reproduction)
Asexual reproduction is carried out by specialized fruiting structures known as what?
Conidiogenous cells.
These structures form conidia, which contain all the genetic material necessary to create a new fungal colony.
Two common conidiogenous cells are the phialides and annellides. Phialides are vaselike structures that produce phialoconidia (Fig. 27.6), whereas annellides are ringed structures that produce annelloconidia. Both form their conidia blastically (budding) like many yeasts; the parent cell enlarges and a septum forms to separate the conidial cell.
Name another form of asexual reproduction that are formed by fragmentation of fertile hyphae.
Arthroconidia
This type of conidiation is characteristic of the mold form of Coccidioides immitis and Geotrichum species, among others.
Chlamydoconidia (aka chlamydospores) are formed from preexistent cells in the
hyphae, which become thickened and often enlarged. This type of conidiation is associated with which yeast?
Candida albicans
What type of hyphae are darkly pigmented due to the presence of melanin?
Phaeoid or dematiaceous
- Surface & reverse side of the plate are both dark
Look at the picture
Septate or aseptate hyphae?
Septate hyphae
Septate hyphae show frequent cross-walls occurring perpendicular to the outer walls of the hyphae,
Look at the picture
Septate or aseptate hyphae?
Aseptate hyphae
The term aseptate, which means absence of septations, has historically been used to describe the hyphae of the Zygomycetes.
Name the rootlike structures that might be seen in some of the Zygomycetes, and their presence and placement can assist with identification.
Rhizoids
What is mycoses?
Infections caused by fungi
Identify the structures in the picture
Which picture displays true hyphae? Pseudohyphae?
- Pseudohyphae and hyphae are commonly called the “filamentous” morphologies and are elongated in form, and are attached end to end.
- Pseudohyphal cells generally have constrictions at the septal junctions.
- True Hyphal cells generally possess true septa lacking constrictions and can create a germ tube.
A positive germ tube is a presumptive ID for what yeast?
Candida albicans
Candida dubliniensis is also positive but is isolated infrequently.
The procedure for inoculating a germ tube requires what type of suspension and is incubated for how many hours?
A light suspension incubated for 2 hours
Which Candida spp is most commonly isolated from humans, regardless of site?
Candida albicans
A common cause of thrush (an infection of the mouth or tongue).
Colonies from what Candida spp frequently form filamentous edges (“feet”) around the edges?
Candida albicans
Which Candida spp is:
* Frequently isolated from blood & urine
* Grows much more slowly than other Candida spp
* Positive for rapid trehalose assimilation
Candida glabrata
Which Candida spp is:
* An emerging pathogen associated w/ outbreaks healthcare facilities
* Causes serious invasive infection
* Multidrug resistant
Candida auris
Both C. albicans & C. dubliniensis are germ tube positive & green on ChromAgar, how can incubation temperature be used to differentiate between the two?
C. albicans can grow at 42 deg C.
C. dubliniensis can not.
What is cornmeal agar used for?
The patterns of growth on cornmeal agar are helpful in making a presumptive identification. The different types of morphology include blastoconidia, chlamydoconidia, pseudohyphae, and arthroconidia.
How is urease used to help ID yeast?
Almost all clinically encountered Candida spp. are urease negative, whereas all Cryptococcus and Rhodotorula organisms are urease positive.
Identify the colonies based on their color on Candida CHROMagar.
Which species of yeast tend to be mucoid due to capsule formation?
Cryptococcus spp.
The capsule of Cryptococcus that surrounds the budding yeast in CSF can be detected with the aid of what stain?
India ink
The cryptococcal capsular polysaccharide antigen can be detected in either serum or body fluid (usually CSF), this is commonly referred to as?
CAD (Cryptococcal Antigen Detection)
Cryptococcus only produce what type of conidia?
Cryptococcus spp. are noted for producing blastoconidia only, without producing true hyphae or pseudohyphae on cornmeal agar.
What is Cryptoccocus neoformans associated with?
Bird droppings (pigeon & chicken)
What is Cryptoccocus gattii associated with?
Eucalyptus trees
What yeast is the cause of tinea versicolor?
Malassezia furfur
It is also thought to be a cause of dandruff.
Describe the appearance of Malassezia furfur in the KOH
KOH preparations reveal budding yeasts, approximately 4 to 8 µm, along with septate, sometimes branched, hyphal elements. This microscopic appearance has gained M. furfur the nickname the spaghetti and meatballs fungus.
Because of M. furfur’s special nutritional requirements, routine fungal cultures are negative for growth. What does it require?
M. furfur requires lipids for growth and typical yeastlike colonies may be observed only after the culture medium has been overlaid with olive oil.
Identify
This organism is noted for their bright salmon pink color. They resemble the cryptococci because they bear a capsule and are urease positive. Some species are also nitrate positive. They are not common agents of disease but have been known to cause opportunistic infections.
Rhodotorula spp.
This yeast-like fungus can cause superficial mycoses where white piedra occurs producing white-to-tan gelatinous, pearly nodules surrounding the hair shaft.
Trichosporon spp.
Originally classified as protozoa, this fungus in immunocompetent individuals, is asymptomatic; however, in immunocompromised patients (AIDS), serious life-threatening pneumonia can develop.
Pneumocytis jirovecii
- The life cycle of Pneumocystis sp. has three stages— the trophozoite, which is 1 to 5 µm in size and is irregularly shaped; the precyst, 5 to 8 µm; and the cyst, which is a thick-walled sphere of about 8 µm containing up to eight intracystic bodies.
- Transmission of the organism is known to occur through the respiratory route, with the cyst being the infective stage. The spores or intracystic bodies are released from the cyst in the lung, and these trophic forms multiply asexually by binary fission on the surface of the epithelial cells (pneumocyte) lining the lung. Sexual reproduction by trophozoites also occurs, first producing a precyst and then the cyst containing spores or intracystic bodies.
Explain
Laboratory Diagnosis of Pneumocystis
- Infected patients typically present w/ respiratory symptoms
- Respiratory samples show typical exudate and organisms are easily seen with histologic stains such as Giemsa & Gomri methanamine silver.
- Calcofluor white can be used to screen specimens for Pneumocystis and other fungi.
Define
Dimorphic fungi
Dimorphic fungi include a mold phase and a yeast or spherule phase.
Which state is observed in vivo or when a dimporhic fungi is grown at 37 deg C. with increased concentration of CO2?
The yeast (or tissue) state.
Which state is observed when a dimorphic fungi is grown at room temp, in ambient conditions?
The mold state.
List the thermally dimorphic fungal species associated with human disease:
- Blastomyces dermatitidis
- Coccidioides immitis
- Coccidioides posadasii
- Emmonsia spp.
- Histoplasma capsulatum
- Paracoccidioides brasiliensis
- Sporothrix schenckii
- Talaromyces (Penicillium) marneffei
Define
Polymorphic fungi
- Polymorphic fungi** have both yeast and mold forms in the same culture.**
- This characteristic occurs despite growth conditions and is best observed in Exophiala spp., in which the yeast phase is typically observed initially, followed by the mold phase as the colony ages.
Thermally Dimorphic Fungi
- Found in the Ohio & Mississippi River valleys
- In soil contaminated by chicken and in caves containing bat droppings (guano)
- Primary infection is pulmonary
Histoplasma capsulatum
Thermally Dimorphic Fungi
- Found in Ohio & Mississippi river valleys & areas bordering the Great Lakes
- Found in soil, acquired through inhalation
Blastomyces dermatitidis
Thermally Dimorphic Fungi
Which fungi is probably the most virulent of all human mycotic agents?
Coccidioides spp
- Infectious arthroconidia are present in soil
- Aquired through inhalation (arthroconidia in lab also highly infectious)
After inhalation, the barrel-shaped arthroconidia round up as they convert to spherules
Thermally Dimorphic Fungi
Where is Coccidioides immitis found?
The San Joaquin Valley of California
Coccidioides has become the 2nd most common opportunistic infection in AIDS pts who have resided in Arizona.
Thermally Dimorphic Fungi
Found in the rainforests of Central & South America
Paracoccidioides brasiliensis
Thermally Dimorphic Fungi
Acquired by penetrating injury from a contaminated plant.
“rose gardener disease”
Sporothrix schenckii
Acquired by penetrating injury from a contaminated plant.
“rose gardener disease”
Sporothrix schenckii
Thermally Dimorphic Fungi
Which fungi is a common cause of systemic infection in immunocompromised patients who have visited the endemic region of Southeast Asia. Infections are usually disseminated, with multiple organ involvement. The fungus can be isolated from cutaneous lesions, which are frequently present in infected individuals. Disseminated disease is typically fatal.
Talaromyces (Penicillium) marneffei
Another characteristic useful in identification is pigmentation.
What type of hyphae are nonpigmented or lightly pigmented?
Hyaline or moniliaceous
- Surface of colonies may be white or colored
- Reverse side of the plate is usually light
Hyaline/brightly colored molds (opportunistic)
- These types of mold are ubiquitous environmental saprophytes and can frequently be isolated from a number of hospital sites, including ventilation systems, and food.
- They are the second most commonly isolated fungus after Candida spp.
- Causes pulmonary infections in immunocompromised patients
Aspergillus spp.
- Their conidia are constantly inhaled, but they are generally readily cleared in healthy, immunocompetent individuals.
- Mortality from infections caused by the aspergilli remains high, especially in the immunocompromised host. (Aspergilloma or “fungus ball”)
- Most commonly affects the respiratory tract
Hyaline/brightly colored molds (opportunistic)
Identify the structures of the fungi (Aspergillus)
- Aspergilli may be uniseriate or biseriate.
- Uniseriate species are those whose phialides attach directly to the vesicle at the end of the conidiophore.
- Biseriate species possess a supporting structure called a metula.
- Metulae attach directly to the vesicle, and attached to each of the metulae are phialides .
- Conidia are produced from the phialides.
- Other characteristics include an erect conidiophore arising from a foot cell within the vegetative hyphae.
It is also important to note whether or not phialoconidia remain in long chains or are easily disturbed into individual phialoconidia. Chains of conidia can be aligned in very straight, parallel columns or in a radiating pattern around the vesicle, and the conidia may be rough or smooth.
Hyaline/brightly colored molds (opportunistic)
- Short conidiophores bearing uniseriate phialides covering the uppper two-thirds of the vesicle
- Colonies blue green with white apron and light reverse
Aspergillus fumigatus
Hyaline/brightly colored molds (opportunistic)
- Spiny conidiophores with uniserate and biseriate phialides covering the entire vesicle
- Yellow green to olive colonies w/ light reverse
Aspergillus flavus
Hyaline/brightly colored molds (opportunistic)
- Long conidiophores bearing biseriate phialides covering the entire vesicle
- Dark brown to black colonies w/ a light reverse
Aspergillus niger
Hyaline/brightly colored molds (opportunistic)
- Short conidiophores with biseriate phialides covering the upper two-thirds of the vesicle
- Colonies are cinnamon brown on the surface w/ a yellow or orange reverse
Aspergillus terreus
Hyaline/brightly colored molds (opportunistic)
- Usually considered a contaminant
- Branched or unbranched conidiophores that have secondary branches known as metulae. Phialides bear chains of conidia. “brushlike phialides”
- Colony surface is powdery bluish green with a white border
Penicillium spp.
Hyaline/brightly colored molds (opportunistic)
- “Canoe-shaped”, multicellular macroconidia w/ 3-6 cells each, often clumping together
- Colony is usually violet
Fusarium spp.
Causes infections similar to Asperillus spp, in addition to **fungal keratitis **and opportunistic in fection in burn wounds
Hyaline/brightly colored molds (opportunistic)
- Common contaminant but also causes nail infections
- Single or branched conidiophores that give rise to annellides (similar to phialides); conidia are rough and “lemon drop” shaped
- Colony is powdery light brown or tan
Scopulariopsis spp
Hyaline/brightly colored molds (opportunistic)
- Conidiophores bearing** long tapered phialides** and chains of spindle-shaped conidia
- Colony is lavender pigmented and velvety
Purpureocillium lilacinum
Hyaline/brightly colored molds (opportunistic)
- Usually a contaminant but also seen in sinusitis & eye infections
- Colony surface is yellowish brown, pink, white, yellow-green, but never bright green or blue-green
- Branching conidiophores w/ elongated, flask-shaped phialids arranged in pairs or brushlike groups. Long chains of oval or spindle-shaped microconidia emanate from the phialides
Paecilomyces variotii
Hyaline/brightly colored molds (opportunistic)
- Can cause mycetoma
- Produces long, narrow, unbranched phialides bearing clusters of single or 2-celled microconidia
- Colonies can be gray, white, yellowish or pale rose
Acremonium spp
Hyaline/brightly colored molds (opportunistic)
- Implicated in pulmonary disease in immunocompromised patients.
- Microscopic evaluation reveals abundant arthroconidia formed from vegetative hyphae that occur singly or may be branched
- Colonies appear white to cream and yeastlike and can be confused with Trichosporon spp.
Geotrichum spp
Hyaline/brightly colored molds (opportunistic)
- Similar to Pencillium, but the conidia do not remain in chains but clump together to form large balls
- Center of colony become dark green for most isolates
Gliocladium spp
Hyaline/brightly colored molds (opportunistic)
- Single, cutoff microconidia directly on hyphae or on the tips of simple conidiophores
- Colony varies but is usually lighter in color- white, yellow, or tan.
Chrysosporium spp
Hyaline/brightly colored molds
- Considered a contaminant
- Colonies are often yellow w/ age
- Unbranched or branched conidiophores bearing large thick walled conidia similar to Histoplasma capsulatum
Sepedonium spp
What are dermatophytes?
Fungi that are keratinophilic; that is, they are adapted to grow on hair, nails, and cutaneous layers of skin that contain the scleroprotein keratin.
Dermatophytes typically form two sizes of reproductive cells called what?
Macroconidium or Microconidium
What diseases are associated w/ dermatophytes?
Superficial mycoses are infections confined to the outermost layer of skin or hair. Because infections of skin, hair, and nails were at one time believed to be the result of burrowing worms that formed ring-shaped patterns in the skin, the term tinea (Latin, meaning “worm”) was applied to each disease, along with the Latin term for the body site.
Identify the dermatophyte:
- Infects the skin & nails
- Most frequently isolated dermatophyte infecting humans worldwide
- Colony surface is white to buff; red pigment causes reverse side to appear red
- Tear-shaped microconidia form singly along the hyphae, giving a “birds on a wire” appearance
- Pencil-like macroconidia may be present
- Urease negative
Trichophyton rubrum
Identify the dermatophyte:
- Common cause of athlete’s foot
- Morphology varies; surface may be buff or white or yellowish
- Reverse can be colorless, yellow, tan or red
- Microconidia are very round “grapelike” and arranged in clusters
- Macroconidia may be seen and are cigar shaped
- Urease positive
Trichophyton mentagrophytes
Identify the dermatophyte:
- Major pathogen for ringworm of the scalp
- Microconidia that are extremely variable in shape, ranging from a round shape to a peg shape.
- Colonies usually form a rust-colored pigment on the colony’s reverse.
Trichophyton tonsurans
Identify the dermatophyte:
- Infects the scalp & skin; usually acquired from infected dogs & cats
- Surface is white with yellow pigment at edge; reverse is deep yellow
- Macroconidia are spindle shaped & rough “echinulate” and taper to a knoblike end
* Each macroconidia contains > 6 cells, seperated by transverse septate
Microsporum canis
Identify the dermatophyte:
- May infect scalp & skin but more often infects animals
- Colony surface is powdery to granular, buff, tan or cinnamon.
- Macroconidia are oval-shaped, rough walled, with rounded ends and transverse septae
- Each macroconidium contains no more than 6 cells
Microsporum gypseum
Identify the dermatophyte:
- Former cause of tinea captis epidemics in children
- Terminal chlamydospores on hyphae that often have a “comblike” appearance
Microsporum audouinii
Identify the dermatophyte:
- Infects skin & nails
- Macroconidia are smooth, club shaped structures w/ rounded ends “beaver tail”
- Each macroconidium contains 2-6 cells
- Never any microconidia
Epidermophyton floccosum
How are dematiaceous molds characterized?
- By production of melanin pigment in both the hyphae and conidia
What 3 types of infections do dematiaceous molds cause?
- Chromoblastomycosis
- Mycetoma
- Phaeophyomycosis
Describe the Subcuataneous Infection
Chromoblastomycosis
- Visualize a chrome-plate (chromo) fungus blasting cauliflower warts on the skin
- It is a subcutaneous infection caused by a variety of copper colored soil saprophytes (Phialaphora, Cladosporium, and Fonsecaea) found on rotting wood.
- Infection occurs following a puncture wound. Initially, a small, violet wartlike lesion develops.
- Over monthes to years, additional violet-colored wartlike lesions arise nearby. Clusters of these lesions resemble cauliflower.
- Skin scrapings w/ KOH reveal copper-colored sclerotic bodies.
Describe the Subcuataneous Infection
Mycetoma
- AKA Madura Foot
- A chronic, granulomatous infection of the subcutaneous and cutaneous tissues that arises at the site of inoculation. The disease is characterized by swelling, with characteristic exudate draining to the skin surface through sinus tracts. Mycetomas occur primarily in tropical and subtropical areas but are also seen in temperate zones. The disease is endemic in India, Africa, and South America.
Describe the Subcuataneous Infection
Phaeohyphomycosis
A mycotic disease caused by darkly pigmented fungi or fungi that have melanin in their cell walls. The term “phaeohyphomycosis” was coined to distinguish several clinical infections caused by phaeoid fungi from those distinct clinical entities known as chromoblastomycosis. In tissue, these fungi may form yeastlike cells that are solitary or in short chains or hyphae that are septate, branched, or unbranched and often swollen to toruloid (irregular or beaded).
Rapid Growing Dematiaceous Molds
Chains of club-shaped conidia with transverse & longitudinal septations in the conidia and have alternating blunt and pointed ends
Alternaria species
Rapid Growing Dematiaceous Molds
Septate hyphae w/ conidiophores bearing dark multicell conidia on sympodial condiophores. Conidia have angular cross walls and spikey (echinulate) surfaces
Ulocladium species
Rapid Growing Dematiaceous Molds
Crescent-shaped conidia with three to five cells of unequal size and an enlarged central cell
Curvularia species
Rapid Growing Dematiaceous Molds
Short branching conidiophores that have “shield cells”
Cladosporium species
Rapid Growing Dematiaceous Molds
Each conidium contains 3-5 septations. Production of germ tubes from both ends of the conidia
Bipolaris species
Rapid Growing Dematiaceous Molds
Produces a singular germ tube along the sides of the conidia
Drechslera species
Rapid Growing Dematiaceous Molds
Conidia have 7-10 septa
Exserohilum species
Rapid Growing Dematiaceous Molds
Conidia are club shaped
Helminthosporium species
Rapid Growing Dematiaceous Molds
Perithecia are pineapple shaped and are ornamented with straight or curl
Chaetomium species
Slow Growing Dematiaceous Molds >7 days
- Sexual state (telemorph) that produces large, dark cleistothecia
- Common cause of mycetoma
Pseudallescheria boydii complex
Slow Growing Dematiaceous Molds >7 days
- Asexual state (anamorph)
- No cleistothecia but produces conidiophores that produce conidia singly or in small groups
Scedosporium boydii complex
Slow Growing Dematiaceous Molds >7 days
- Septate hyphae with annelides having swollen bases and elongated necks. Conidia form small clusters at the apex (conidiogenous cells)
Scedosporium prolificans
Slow Growing Dematiaceous Molds >7 days
- Young culture consists of many yeast like budding cells. Septate hyphae with numerous annellides that are sledner, tubular, sometimes branched and characteristically tapered to a narrow elongated tip.
- The conidia gather in clusters at the end of the conidiophore.
Exophiala jeanselmei complex
Slow Growing Dematiaceous Molds >7 days
- Hyphae are septate and dark with alteral and terminal conidiophores of various sizes.
- Conidiophores produce long branching chains of brown oval conidia
Cladophialophora species
Slow Growing Dematiaceous Molds >7 days
Septate hypae with vase shaped phialides and oval conidia that accumulate at the apex of the phialide.
Phialophora species
Slow Growing Dematiaceous Molds >7 days
Four types of conidiation can develop on the conidiophore
Fonsecaea pedrosoi
What type of molds are commonly referred to as a “lid lifters”?
Hyaline pauciseptate Hyphae
Mucorales (Zygomycetes)
- Mucorales are common environmental isolates associated with soil and plants. They contaminate grains, breads, and fruits and are most often associated with infections of the sinuses, lungs, and skin of immunocompromised patients. Diabetes is a significant risk factor for these infections.
- Fungi in the order Mucorales are usually found in patients with diabetes and ketoacidosis. In this patient population, the infection usually begins in the sinuses, where conidia are inhaled and take up residence. From the sinuses, infection rapidly spreads to the orbits, face, palate, and brain. This presentation is known as rhinocerebral mucormycosis.
Identify the structures of Mucorales
Identify the Mucorales (Zygomycetes)
Rhizoids and unbranched sporangiophores that arise directly over the rhizoids.
Rhizopus species
The most common Mucorales that causes human disease
Identify the Mucorales (Zygomycetes)
Produce rhizoids but the sporangiosphores arise at points between rhizoids, rather than over the rhizoids.
Lichtheimia species
Identify the Mucorales (Zygomycetes)
No rhizoids present
Mucor species
Identify the Mucorales (Zygomycetes)
Branched sporangiophores are topped by large vesicles. The vesicles are covered w/ spines (denticles), each of which supports a single spore contained within a round sporangiolum.
Cunninghamella species
Identify the Mucorales (Zygomycetes)
Each sporangiophore contains stacks of sporangiospores
Syncephalastrum species
Mycobacterium
What does the term “acid-fast” mean?
- Mycobacteria have the cell wall structure of gram-positive bacteria but contain mycolic acids, which repel crystal violet/iodine, thus are usually gram invisible.
- Acid-fast staining uses heat and/or phenol to allow a fuchsin dye to penetrate the hydrophobic barrier and stain the mycolic acids in the cell wall (red stain).
- The cell wall lipids of the mycobacterium do not dissolve when the acid-alcohol is applied, and thus the red stain does not wash off holding fast to their red stain while bacteria that are not acid fast lose the red stain and take on the blue.
Describe
Aerobic Actinomycetes as it relates to acid-fast staining.
Describe
What specimen type is appropriate for mycobacteria?
- Bronchial washings & sputum
Although a variety of clinical specimens may be submitted to the laboratory to recover mycobacteria, respiratory secretions, such as sputum and bronchial aspirates, are the most common. An early-morning specimen should be collected on three consecutive days.
Describe
Safety precautions used in an AFB lab
- Mycobacteriology (AFB) lab should be a seperate room from the main lab with a non-recirulating ventilation system and negative pressure
- All infectious materials should be tightly covered (parafilm)
- All manipulations of cultures and specimens must be done with gloves and coats/gowns in a biological safety cabinet
- N95 (fit testing) must be worn when performing procedures outside of the biological safety cabinet
- Loops, pumpmatics, sticks, swabs, needles must be disinfected after use (vesphene)
- All waste must be sterilized (autoclaved)
Describe
Mycobacteria Specimen Processing
- Because Mycobacteria grow slower, specimens that may contain commensal bacteria should be decontaminated and then concentrated before plating onto media.
- The purposes of the digestion-decontamination process are as follows:
- To liquefy the sample through digestion of the proteinaceous material
- To allow the chemical decontaminating agent to contact and kill the nonmycobacterial organisms - A combination of a liquefying agent, such as N-acetyl-L-cysteine (NALC), plus sodium hydroxide (NaOH) is commonly used.
- Oxalic acid, 5%, can also be used to decontaminate specimens contaminated with P. aeruginosa or cystic fibrosis patients
- Concentration centrifugation speeds must be at least 3000 times the gravitational constant (g) to maximize recovery.
How can AFB survive the NALC & NaOH decontamination procedure?
Because mycobacteria have a high lipid content on the cell wall
Mycobacteria Specimen Staining
What is the screening stain?
Auramine stain or auramine-rhodamine fluorochrome stain
* Primary Stain: Auramine O (15 min.)
* Decolorizer: HCL in ETOH (2 min.)
* Counterstain: Potassium permanganate (2 min.)
* Slides are examined using a 20X objective on a light microscope viewing a minimum of 30 fields before a slide is called negative. Confirm on 40X.
All slides must heat fix for 1 hour before staining
Mycobacteria Specimen Staining
What is the confirmatory stain? (only performed if the screen is positive)
Ziehl-Neelsen (hot method) or Kinyoun (cold method)
* Primary Stain: Carbolfuchsin (5 min.)
* Decolorizer: HCL in ETOH (2 min.)
* Counterstain: Methylene blue (1 min.)
* Slides are examined using a 100X immersion objective on a light microscope for 15 minutes, viewing a minimum of 300 fields before a slide is called negative.
All slides must heat fix for 1 hour before staining
Describe
Mycobacteria Specimen Plating
- Every patient needs: solid nonselective, solid selective, and liquid media
- Incubated at 35-37 degrees C for **6 weeks **/42 days
- Culture is gold standard but it is slow. It is required for AST. Growth is typically faster in broth.
- Malachite green is added to media to prevent the growth of contaminating organisms
Describe
Nocardia Specimen Processing
- Body fluids (>2mLs) concentrated by centrifugation
- Tissues will be ground
- Respiratory specimens are inoculated as is
- BAL (>10mLs) concentrated by centrifugation
- Miscellaneous specimens
-swabs are suboptimal
-prefer aspirates
Describe
Nocardia Specimen Staining
Modifed Acid-Fast Stain Stain
* Modified Ziehl-Neelsen or Kinyoun
* Primary Stain: Carbolfuchsin (5 min.)
* Decolorizer: 1% Sulfuric Acid (2 min.)
* Counterstain: Methylene blue (1 min.)
Slides are examined using a 100X immersion objective on a light microscope for 15 minutes, viewing a minimum of 300 fields before a slide is called negative.
Describe
Nocardia Specimen Plating
Inoculate 2 Buffered Charcoal Yeast Extract (BCYE ) agar by four quadrant streak
* can use selective BCYE with PAC
* P= Polymyxin B
* A= Anisomycin
* C= Cefamandole
Incubate in O2
* one at 35-37 degrees C
* one at 30 degrees C
28 days (4 weeks)
* read twice a week
Describe
Less Common Aerobic Actinomycetes
What Stains What With What!!
AFB processing Picmonic overview
General Mycobacteria Characteristics
- Nonmotile
- Gram-positive cell wall with mycolic acid
- Mycolic acid = high lipid content
- High lipid content= poor staining with basic dyes ex. Gram stain
- Obligate aerobes
- Slow growing when compared to bacteria
- pathogenic members can take 2-6 weeks to grow
Describe the pathogenesis of Mycobacterium tuberculosis (MTB)
- TB is one of the oldest documented communicable diseases and remains a leading cause of morbidity and death globally. As many as one third of the world’s population may be infected with the bacteria causing TB.
- Tubercle bacilli are acquired from persons with active disease who are excreting viable bacilli by coughing, sneezing, or talking. Airborne droplets containing bacteria, 1 to 5 µm in size, enter the respiratory tract of an exposed individual and reach the lung alveoli.
- M. tuberculosis cells are phagocytized by alveolar macrophages and multiply intracellularlly.
- The pathologic features of TB are the result of a hypersensitivity reaction to mycobacterial antigen.
- If there is little antigen and a strong hypersensitivity reaction, a hard tubercle, or granuloma, may be formed. The granuloma is an organization of lymphocytes, macrophages, fibroblasts, and capillaries, along with fibrosis and encapsulation. With granuloma formation, the bacteria can ultimately be killed, and healing occurs, along with calcification, with scar formation as a reminder of the past infection.
- If the antigen load and hypersensitivity reaction are both high, tissue necrosis from the enzymes of degenerating macrophages can occur; the tissue response is less organized, and granulomas are surrounded by fibrin that protects the bacteria from phagocytosis by macrophages.
- After healing of the primary infection, the bacilli are not totally eradicated but can remain viable, but dormant, in granulomas for months or years. In infected individuals, there is a potential for reactivation of TB when the bacteria are released from the granulomas. This happens as a result of immunosuppression from old age or other causes.
How are species of mycobacteria classified?
- Rapid grower VS. Slow Grower
- Temperature preference and colony pigmentation (Nonchromogen, scotochromogen and photochromogen)
- Series of biochemical tests
- Nonchromogen: No pigment produced in colony
- Scotochromogen: Produce orange or deep yellow pigment in dark or light
- Photochromogen: Develops pigment exposed to light and develops no pigment in the dark
3 Major Mycobacterium groups
- M. tuberculosis (MTB)
- Nontuberculous mycobacteria (NTM)
- Mycobacterium avium complex (MAC)
Describe NTM Runyon Classification
Identify the mycobacteria:
Colonies of this slowly growing species are typically raised, with a dry, rough appearance. The colonies are nonpigmented and classically described as being buff and rough
Mycobacterium tuberculosis complex
Identify the mycobacteria
- Slow-growing NTM Photochromogens with Pulmonary Involvment
- Infection that resembles TB in patients with immunosuppression or underlying pulmonary disease
Mycobacterium kansasii
Slow-growing NTM Photochromogen that is associated with wound exposure to freshwater fish tanks or salt water and causes localized cutaneous infection (“fishtank granuloma”)
Mycobacterium marinum
Optimally grows at 32 degrees C but will also grow at 25 degrees C
Slow-growing NTM Schotochromogens most commonly associated with cervical lymphadentitis.
Mycobacterium scrofulaceum
Slow-growing NTM that is a scotochromogen at 37 deg C and a Photochromogen at 22 deg C
Mycobacterium szulgai
Slow-growing NTM Nonchromogens with Pulmonary Involvment
Optimal growth is 42 degrees C
Mycobacterium xenopi
Slow-growing NTM Nonchromogens with Pulmonary Involvment
- Environmental sources, especially natural waters, seem to be the reservoir for most human infections.
- Pulmonary disease resulting from this infection can have a variety of clinical presentations. The most common form is a slowly progressive cavitary disease in middle-aged men with a history of smoking and other underlying pulmonary disease. The disease resembles the clinical picture of TB— cough, fatigue, weight loss, low-grade fever, and night sweats.
Mycobacterium avium complex (MAC)
**The most common NTM that causes pulmonary infections, especially in AIDS patients. **
Name 2 Slow-growing NTM Nonchromogens that Cause Skin Infections
- Mycobacterium ulcerans (Buruli ulcer)
- Mycobacterium haemophilum (Also seen in lymphadenitis
Needs hemin (factor X)
Both optimally grow at 32 degrees C
This slow-growing NTM Schotochromogens is a common
Contaminant
Mycobacterium gordonae
Name 3 Rapid growing NTM
- Mycobacterium chelonae - causes skin infections but can disseminate
- Mycobacterium fortuitum group - causes skin infections but can disseminate
- Mycobacterium abscessus group - causes pulmonary dissease in CF patients
- Causative agent of Hansen disease (leprosy), an infection of skin, mucous membranes, and peripheral nerves.
- Found in the U.S., found in Hawaii, Texas and Lousiana (where it is harbored by armadillos)
Mycobacterium leprae
- The most important mode of transmission is not known, but the disease can be transmitted by direct contact and inhalation of aerosols from skin lesions. Shedding from the nasal passage is another route of transmission
- The two major forms of the disease are tuberculoid leprosy and lepromatous leprosy.
- Symptoms of tuberculoid leprosy include skin lesions and nerve involvement that can produce areas with loss of sensation.
- Lepromatous leprosy is characterized by disfiguring skin lesions and progressive, symmetric nerve damage. Lesions of the mucous membranes of the nose can lead to destruction of the cartilaginous septum, resulting in nasal and facial deformities.
- Laboratory diagnosis of Hansen disease depends on the microscopic demonstration of AFB from skin biopsy specimens. M. leprae does not grow for culture.
Describe the picture
The acid-fast rod M. leprae is seen here cooling off on an ice cube. Leprosy involves the cooler areas of the body. It damages the skin (sparing warm areas such as the armpit, groin and perineum), the superficial nerves, eyes, nose and testes.
Which strain of Mycobacterium tuberculosis complex is the vaccination strain?
M. bovis (Calmette-Guerin [BCG])
M. bovis is found primarily in cattle, but it can infect other mammals, including humans.
MDR or Multi-drug resistant TB is defined as being resistant to which drugs?
Rifampin and isoniazid
XDR or extremely drug resistant TB is defined as being resistant to which drugs?
Rifampin and isoniazid, a flouroquinolone and an injectable agent (such as an aminoglycoside)
Clinical diagnosis of primary TB is usually limited to signs and symptoms and a positive PPD skin test result. What does PPD stand for?
Purified Protein Derivative (PPD)
- Intradermal injection of antigenic protien particles from killed M. tuberculosis results in localized skin swelling and redness. This test reveals whether or not a person has been infected with TB.
- This is important because many infected individuals will not manifest a clinical infection for years.
- When a positive PPD test occurs, you can treat and eradicate the disease before it significantly damages the lungs or other organs.
Describe the gram stain and what is it consistent with?
Beaded, branching gram-positive bacilli consistent with nocardia
What other stain will detect nocardia spp?
Modified acid-fast stain
Describe the colony morphology of Nocardia
- Colonies of Nocardia spp. might have a chalky, matte, velvety, or powdery appearance and may be white, yellow, pink, orange, peach, tan, or gray pigmented
- They can have a dry, crumbly appearance similar to breadcrumbs
- Most grow in 3-6 days
- Musty basement smell
- BCYE is ideal for primary culture
What kind of soft tissue infections can Nocardia cause?
Mycetomas, forming macroscopic granules similar to the sulfur granules of actinomycoses
What is the most common type of Nocardia infection?
May cause pulmonary infection and disseminated infection, often involving the central nervous system
What antibiotic is Nocardia resistant to?
Penicillin
Describe Rhodococcus equi
- Gram-positive cocci, coccobacilli, or coryneform rods
- Colonies are typically salmon colored & slimy
- Often found in histiocytes
- Affects immunocompromised pts, most commonly causing pulmonary & bloodstream infections that may go to the other locations, eg, lymph nodes, skin
- Modified acid-fast positive
