Unit 2 Exam Review Flashcards
Inhibitory Mold Agar
Primary recovery of pathogenic fungi exclusive of dermatophytes
Sabhouroud Dextrose Agar
(Sab dex)
Primary recovery of saprobic and pathogenic fungi
Brain heart infusion agar
(with antibiotics) primary recovery of pathogenic fungi exclusive of dermatophytes
Cornmeal agar
Identification of C. albicans by chlamydospore production;
identification of Candida by microscopic morphology
Birdseed agar
Identification of C. neoformans
Dermatophyte test media
primary recovery of dermatophytes, recommended as screening medium only
PPLO media
agars and broths used for isolating and cultivating Mycoplasma when supplemented with nutritive enrichments
SP4
recommended for the isolation, differentiation and maintenance of mycoplasma
What is a yeast?
Unicellular organism
round to oval
2 to 60 um
Yeast microscopic morphologic features
limited utility to differentiate or identify;
have characteristics that suggest the ID or are pathogenic for a particular organism
What is a mold?
tubelike projections - hyphae mycelium - loose network of hyphae which penetrates the substrate to obtain nutrients for growth
Powerpoint definition of a yeast
single-celled fungus reproduces by budding to form blastoconidia
Colonies are moist or mucoid
may or may not produce pseudohyphae
Powerpoint definition of a mold
vegetative groth of filaments.
mushrooms consist of many filaments packed tightly together.
Reproduction by spores or conidia.
Conidia
born on specialized hyphae or conidiophores
What is the bio safety level of mold
BSL 2
How should Histoplasma capsulatum or Blastomyces dermatitidis be handled?
severe bio-hazard
handled with extreme caution in an appropriate pathogen handling cabinet.
How much specimen is required to ID?
The lab needs enough specimen to perform both microscopy and culture.
How was Blastomyces dermatitidis IDed in the past?
conversion from the mould form to the yeast form was necessary for dimorphic ID.
Chlamydophila psittaci ID
BSL3
indirect microimmunofluorescence
(hens egg yolk)
PCR
Is Chlamydophila psittaci endemic?
yes in all bird species.
Also seen in turkey processing workers and pigeon aficionados
Symptoms of Chlamydophila psittaci
pneumonia
severe headache
mental status changes
What is the treatment for Chlamydophila psittaci?
Tetracycline (fatality 20% if untreated)
Cutaneous
Involve the hair, skin, nails with no involvement of deeper tissues.
(Tinea, ringworm, piedra)
Subcutaneous
Only subcutaneous tissue with no dissemination
Systemic
Invade the lungs and become widely disseminated (agents usually dimorphic fungi)
Opportunistic
Immunocompromised patients (underlying diseases) Candidiasis, Aspergillosis
Dermatophytes
agents of ringworm, athlete’s foot, and so on
Examples of subcutaneous infections
chromoblastomycosis
mycetoma
phaeopyphomycotic cysts
Examples of systemic fungal infections
Blastoyces
Coccidioides
Histoplasm
Paracoccidioides
Infections usually involve the lungs but may also be widely disseminated and involve any organ system.
Most commonly encountered opportunistic pathogens
Aspergillus
Zygomycetes
Candida
Cryptococcus
Cutaneous Infections
Superficial mycoses
Tinea
Piedra
Candidosis
Dermatophytosis
Opportunistic Infections
Aspergillosis
Candidosis
Cryptococcosis
Zygomycosis
Trichosporonosis
What is the use of Calcafluor White Stain
Detection of fungi
What is the time required for calcafluor White Stain?
1 minute
What is the advantages of Calcafluor White Stain
Can be mixed with KOH
detects fungi rapidly due to bright fluorescence
What are the disadvantages of Calcafluor White Stain?
Fluorescence microscope
vaginal secretions are hard to interpret; background fluorescence prominent, but fungi exhibit more intense fluoresence
What is the use of KOH (Potassium hydroxide)
Clearing of specimen to make fungi more readily visible
What is the time required of KOH?
5 min; if clearing is not complete, and additional 5-10 min might be necessary
What are the advantages of KOH?
Rapid detection of fungal elements
What are the disadvantages of KOH?
Experience required clearing of some specimens require extended time
What is the use of India Ink?
Detection of C. neoformans in CSF
What is the time required for India Ink?
1 minute
What are the advantages of India Ink
When positive in CSF it is diagnostic of meningitis
What are the disadvantages of India Ink
Positive in less than 50% of cases of meningitis
not sensitive in non-HIV infected patients
What is the use of Darkfield stain?
Detection of syphilis or treponema pallidum
Calcoflour White appearance
cell walls of fungi will bind the stain which enhances fungal visibility in tissue.
Flurochrome used to detect fungi in clinical material and observe fungi grown in culture
What specimens is calcoflour white used on
microscopic exam of skin, hairs, nails and other specimens
Other uses of calcofluor white
used as whitening agents by paper industry to bind cellulose and chitin
What is the use of Lactophenol Cotton Blue
Staining and microscopic identification of fungus cultures
Germ Tube Test
Generally accepted and economical method to ID yeast within 3 hours
appear as hyphae like extensions of yeast cells w/o constriction at the point of origin from the yeast cell
Cryptococcal Antigen Test
Performed on CSF or serum
replaced use of India ink
What organism shares an antigen that is similar to C. neoformans
Trichosporon
What is the method of choice for diagnosing patients with cryptococcal meningitis?
detection of cryptococcal capsular polysaccaride antigen in spinal fluid
How often can cryptococcal antigen be detected in serums?
AIDS patients - 100%
non-AIDS - 60%
What should serum specimens be treated with and why?
Pronase detects antigen to avoid false negative results.
What should be kept for cryptococcal antigen testing and what is processed.
Supernatant for antigen testing and process the sediment.
What are the two most widely used nontreponemal serological tests used?
RPR and FTA-ABS
RPR - 80% positive in syphilis
How does a nontreponemal serological test function?
An agglutination test in which soluable antigen particles clump when they are aggregated by an antibody.
What are the 3 things a mold ID is based upon?
- Growth rate
- Colonial morphological features (Sab Dex Agar)
- Microscopic morphological features using lactophenol blue stain
What are identifiable microscopic morphological features?
Microconidia
macroconidia
phialides
Candida albicans gram stain
gram positive large oval bodies
Candida albicans on cornmeal agar
blastoconidia
pseudohyphae
chlamdoconidia
Candida albicans on media
yeast like growth on Sab Dex
Candida albicans growth requirements
25-30 degrees (optimal) can grow at 37 degrees
Candida albicans infections
Opportunistic to systemic
urogenital, respiratory, bloodstream
Candida albicans specimen
blood
urine
wounds
throat
Candida albicans ID
germ tube positive
chlamydoconidia observed
growth on cyclohexamide
ID this image
Candida albicans
ID this image
Candida albicans
Candida albicans
Gram stain of Rhizopus spp*
unbranched sporangiosphores with rhizoids that appear at the point where the stolon arises, at the base of the sporangiophore
What does Rhizopus spp* look like on media?
Blood enriched medium - colonies are extremely fast growing, wooly, and gray to brown to gray black. The reverse is white to pale
Rhizopus growth requirements
45C
Rhizopus infections
Wound infections, zygomycosis
Rhizopus specimens
nasal mucosa
sinuses
face and brain
Rhizopus spp* ID
LPCB
ID this image
Rhizopus microscopic LPCB prep
ID this image
Rhizopus on sab dex agar
gray wooly colony fills the sab-dex plate
Cryptococcus neoformans gram stain
gram positive cocci
Cryptococcus neoformans on cornmeal agar
following 72 hours incubation at 25C it produces globose yeast cells only (2.5-10um in diameter)
Penicillium gram stain
gram positive
Penicillium on media
fast growing, flat, filamentous, and velvety, wooly or cottony in texture. Initially white and become blueish gray, yellow or pinkish in time. The plate reverse is usually pale to yellowish.
Penicillium growth requirements
subculturing to BHI, incubate at 35C, 7 days, yeast like structures dividing by fission and hyphae with arthroconidia are formed
Penicillium infections
Opportunistic, infections are rare except with P. marneffei
Penicillium specimen
respiratory secretions, gastric washings, skin, urine, ear, cornea
Penicillium reservoirs
Humans with P. marneffei
Aspergillus fumigatus gram stain
gram positive, hyphae are septate and hyaline. Conidial heads are strongly comumnar in an undisturbed culture. Conidiophores are smooth-walled, uncolored, up to 300 um long
Aspergillus fumigatus colony appearance
smoky gray-green with a slight yellow reverse
Czapek agar
differential identification of aspergillus spp
Aspergillus fumigatus growth requirements
potato dextrose agar at 25C
How does Aspergillus fumigatus grow?
rapid growing mold (2 to 6 days) that produces a fluffy to granular, white to blue-green colony. Mature sporulating colonies most often exhibit the blue-green powdery appearance
Aspergillus fumigatus special growth requirements
Thermotolerant and grows at temps up to 55C. Cosmopolitan mould and has been found on many types of substrates, especially soil and decaying organic debris
Cryptococcus neoformans media
Bird Seed agar, colonies on Sabouraud dextrose at 25C are cream to beige and mucoid due the capsule surrounding the yeast cells
Cryptococcus neoformans growth requirements
72 hour @ at 25C
Cryptococcus neoformans infections
opportunistic, cryptococcosis
Cryptococcus specimen
all respiratory secretions
Cryptococcus neoformans ID
India Ink, for the direct microscopic examination of CSF for cryptococcus neoformans
Cryptococcus neoformans Unique
Urease positive but fails to grow on medium containing cycloheximide or at 40C. This species is found in nature in avian excreta, especially weathered pigeon droppings, which are believe to the the source of infection.
Cryptococcus neoformans reservoirs
Humans and domestic wild animals. In humans, C. neoformans affects immunocompromised hosts predominatly and is the most common cause of fungal meningitis worldwide, 7-10% patients with AIDS are infected
AIDS associated cryptococcus accounts for _____ of all cryptococcal infections reported annully.
50%
Cryptococcus neoformans usually occurs in HIV patients when their _______ is below ______
CD4 lymphocyte; 200/mm3
What is the predominant clinical presentation and symptoms with Cryptococcus neoformans
Meningitis; fever and headache
What percent of secondary cutaneous infections occur with disseminated cryptococcosis and indicate a poor prognosis
15
What do lesions look like when they begin?
small papules that ulcerate, but may also present as abscesses, erythematous nodules, or cellulitis
Aspergillus fumigatus infections
important human pathogen and is the most common cause of all forms aspergillosis
Aspergillus fumigatus specimen
nasal cultures
respiratory secretions
GI
genitourinary
skin
subcutaneous tissue
blood
bone
CNS
Aspergillus fumigatus unique
Most common aspergillosis
grows @ 40C and beyond
Hyphae in tissue may or may not display 45C angle branching. Fruiting heads may be seen in air cavities in the lung.
Aspergillus reservoirs
humans
What kind of organism is Microsporum canis?
Dermatophyte
Microsporum canis gram stain
gram positive
Microsporum canis colony morphology
white/cream over orange-yellow reverse
Microsporum canis growth rate
1 week
Microsporum canis Microscopic Identification
Thick walled spindle-shaped
rough-walled macroconidia - some with a curved tip
microconidia rarely seen
Microsporum canis infections
infections of the hair and skin; tinea
Microsporum canis specimen
hair and skin
Microsporum canis ID
5-7 days time required for ID
Microsporum canis unique
in vitro hair perforation test positive
Microsporum canis reservoirs
natural reservoir-cats and dogs. It causes tinea in humans, rarely mycetoma like lesions have been observed in immunocompromised hosts.
Coccididdes immitis gram stain
gram positive
coccididdes immitis sab dex agar
colonies are moist, membranous, and grayish. later producing white and cottony aerial mycelium. With age, colonies become tan to brown in color
Coccididdes immitis media
sabouraud dextrose agar
Coccididdes immitis infections
arthroconidia
Coccididdes immitis unique
Southwestern US
Valley fever
Coccididdes immitis reservoirs
mode of transmission - inhalation, humans. Comon sites of infection: lungs, skin, meninges
Rickettsia gram stain
small pleomorphic gram negative rods - reproduce inside host lyse host when released
rickettsia Special Growth
Obligate intracellular parasite; survive only briefly outside host
Rickettsia infections
Causes Rocky Mountain Spotted Fever, typus, scrub typhus and other spotted fevers
Rickettsia symptoms
fever, headache, rash
Rickettsia specimen
Recovered in blood or tissue and identified serologically; not reoutinely recovered from culture, labs need specialized techniques and precautions
Rickettsia ID
Weil-Felix test; agglutination of P. vulgaris by serum of infected patients and P. mirabilis **presumptive test
Rickettsia treatment
Tetracycline
Rickettsia unique
3 groups, spotted fever, typhus group and scrub typhus group
Rickettsia reservoirs
wild animals infected; we are incidental hosts through insect vectors
Chlamydia trachomatis gram stain
non-motile gram negative intracellular parasites; EB elementary body looks like a gram neg rod, but no peptidoglycan layer on cell wall
Chlamydia trachmatis infections
STD - Most common STD in US, major cause of PID. 4 million infections in US/year; ocular trachoma; lymphogranuloma venereum is very common venereal disease in Africa, Asia and SA. Specific LGV strain of C. trachomatis. Neonatal infections include conjunctivitis, pneumonia, enteric infections, mother to child during delivery
Chlamydia trachmatis Specimen
collection of swabs for culture or testing; cell cultures; McCoy, Hela, and monkey kidney cells - grow 48 hours, add iodine and view under scope
Chlamydia trachmatis ID
Frei’s test - intradermal skin test of LGV bubo - poor recovery, always positive for many years; DFA staining - elementary bodies; ELISA - Chlamydia antigen, gen-probe testing- PCR of urine specimens
Chlamydia trachmatis treatment
Erythromycin and tetracycline
chlamydia trachmatis reservoirs
exclusively human, sexual transmission 25% men and 70-80% women asymptomatic
Mycoplasma pneumoniae gram stain
spherical, griny, yellowish forms that are embedded in the agar, with a thin outer layer
Mycoplasma pneumoniae media
biphasic SP-4 triphasic system, PPLO broth or agar with yeast extract and horse serum, modified NYC medium
Mycoplasma pneumoniae growth requirements
agar and broths 37C, ambient air for up to 4 wk
Mycoplasma pneumoniae infections
Asymptomatic infection, upper respiratory tract infection in young children; lower respiratory tract infections in adults
Mycoplasma pneumoniae symptoms in young children
mild, nonspecific symptoms including runny nose, coryza, and cough, most without fever
Mycoplasma pneumoniae symptoms in adults
typically mild illness with non productive cough, fever, malaise, pharyngitis, myalgias
Mycoplasma pneumoniae extra infections
4 to 13% of patients develop pneumonia; complications include rash, arthritis, encephalitis, mycorditis, pericarditis, and hemolytic anemia
Mycoplasma pneumoniae ID
definitive ID of M. pneumoniae is accomplished by overlaying suspicious colonies with 0.5% guinea pig erythrocytes in phosphate-buffered saline instead of water. After 20 to 30 minutes at room temp, colonies are observed for adherence of red blood cells
Mycoplasma pneumoniae reservoirs
humans and mainly children
ID this organism
Aspergillus
Borrelia burgdorferi gram stain
spirochetes are not classified as either. When borrelia burgdorferi is gram stained, the cells stain a weak gram neg by default since safranin is the last dye used. Like most spirochetes, Borrelia does have an outer membrane that contains an LPS like substance, an inner membrane and a periplasmic space which contains a layer of peptidoglycan. Therefore it has a gram-negative bacterial type cell wall, despite its staining characteristics
Borrelia burgdorferi media
can be cultivated in vitro. Bacterium is fastidious and requires a complex growth medium. The media preferred is Barbour Stoenner Kelly (BSK). It contains 13 ingredients in a rabbit serum base.
Borrelia burgdorferi growth requirements
Optimal temp of 32C, in a microaerobic environment. Generation time is slow, about 12-24 hours
Borrelia burgdorferi infections
Causative agent of lyme disease, which is a sever protracted epidemic arthritis, spread by the deer tick, often appearing during the waning months of summer in northeastern US, certain central regions and on the west coast. Now in Europe, Japan, Russia and Australia. Multiple stages
What is Stage One of lyme disease
Tick bite elicits an enlarging erythematous path (erythema migrans)
What is Stage Two of lyme disease
Spirochetemia with the rise of arthritis, meningitis and debilitaiton
What is Stage Three of lyme disease
Chronic phase, skin and neurologic symptoms and chronic arthritis
Borrelia burgdorferi specimens
Peripheral blood is specimen of choice for direct detection of borrelia that cause relapsing fever. Specimens submitted for stain or culture include: blook, biopsy specimens, synovial and CSF.
Borrelia burgdorferi specimen collection requirements
no special requirements for collection, transort or processing
Borrelia burgdorferi vectors
ticks
Borrelia burgdorferi reservoirs
ticks and humasn
Treponena pallidum gram stain
stains neither positive or negative, not routinely cultured in lab
Treponena pallidum infections
causative agent of syphilis; exclusively infects huans, transmitted from one person to another; produces a chronic systemic infection, affecting about 30/100,000 in USA per year
Treponena pallidum ID
RPR 80% positive; Venereal disease research laboratory test 70% positive. Fluroscent Treponemal Antibody Absorbed test
Treponena pallidum unique
stains neither positive or negative, not routinely cultured in lab
Treponena pallidum reservoirs
Exclusively infects humans, transmitted from one person to another, transmitted via sexual contact; orther forms of transmission are extremely rare
Histoplasma capsulatum - DIMORPHIC gram stain
gram positive; large, rounded, single-celled tuberculate macroconidia formed on short, hyaline, undifferentiated conidiophores. Microscopically, numerous small round to oval budding yeast-like cells are observed
Histoplasma capsulatum media
Macroscopic - sabouraud’s dextrose agar at 25C, colonies are slow growing, white or buff brown, suede like to cottony with a pale yellow-brown reverse. Macroscopically on BHI blood agar incubated at 37C, colonies are smooth, moist, white and yeast like
Histoplasma capsulatum growth
Sab dex @ 25C
Histoplasma capsulatum infections
intracellular mycotic infection of the reticuloendothelial system caused by the inhalation of the fungus; histoplasmosis
Histoplasma capsulatum ID
postive ID required conversion of mold form to yeast phase by 37C growth on enriched, however culture ID by the exoantigen test is now method of choice
Histoplasma capsulatum unique
represent a sever biohazard to lab personnel and must be handled with extreme caution in an appropriate pathogen cabinet
Histoplasma capsulatum etiological agent
from soil enriched with excreta from chicken, starlings and bats
Histoplasma capsulatum reservoirs
world-wide, especially in USA, skporadic cases do occur in Australia
ID this organism
Aspergillus
ID this organism
Aspergillus
ID this organism
Aspergillus
ID this organism
Coccidiodes immitis
ID this organism
Coccidiodes immitis
ID this organism
Histoplasma capsulatum
ID this organism
Microsporum canis
ID this organism
Penicillium lactophenol cotton blue prep
ID this organism
Penicillium sab dex
What is Mycoplasma Broth Base (Frey) used for?
Used for the cultivation of avian mycoplasmas.
What is zygomycosis?
includes mucocutaneous, rhinocerebral, geniturinary, gastrointestinal, pulmonary and disseminated infections
Cryptococcus growth requirements
72 hours @ 25C
How is Blastomyces dermatitidis IDed now?
culture ID by the exoantigen test
