-
What are fungi?
- Eukaryotic
- Lack chlorophyll
- Reproduce by spores
- Filamentous or unicellular
- Macroscopic or microscopic
- Few species cause human disease
- Many live as saprobes
- Humans usually accidental hosts
- Lack of susceptibility to antibiotics
-
Yeasts- Unicellular Fungi Colonies
-
Molds-Filamentous Fungi
Colonies
- Fluffy
- Cottony
- Woolly
- Powdery
-
Dimorphic Fungi
Can exhibit two forms
- Thermally dimorphic so temperature dependent
- 25-30 C = mold
- 35-37 C = yeast
-
Yeasts- General Descriptions
- Unicellular
- -Round to oval
- -Some have capsules
- Reproduction
- -Asexually by budding-blastoconidia formation
- -Sexually by ascospores or basidiospores
- Pseudohyphae
- -Elongated buds connected to one another
- -Not all species produce pseudohyphae
-
Yeasts- Candida albicans
- Part of our normal flora
- Superficial infections
- Invasive infections
-
- Yeasts- Candida albicans
- Colony morphology
-
- Yeasts- Candida albicans
- -Germ tube production
-
- Yeast- Candida albicans
- -Cornmeal morphology
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Yeasts- Other Candida species
- C. tropicalis
- C. krusei/inconspicua
- C. parapsilosis
- C. glabrata
-
-Exists in nature
-Causes disseminated disease
- Yeasts-Cryptococcus neoformans
- -Exists in nature
- -Causes disseminated disease
-
-
-
-
-
- Yeasts- C neoformans
- -Phenol oxidase detection
-
Other Cryptococcus species
- Urease positive
- Inositol assimilation positive
- Round yeast cells only on cornmeal agar
-
Causes disease in immunocompromised
Can colonize healthy individuals
Urease positive
Yeasts- Trichosporon beigelii
-
-
-
-
Causes Tinea versicolor-
(pityriasis versicolor)
Yeasts- Malasezzia furfur
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Part of normal human flora
Can cause disseminated infection
Microscopic exam
Yeasts- M furfur
-
Special growth requirements- lipophilic
so needs lipids or fatty
acids to grow in vitro
Yeasts- M furfur
-
Other yeasts
Base identification on morphologic and biochemical criteria
- Morphology determined on cornmeal agar
- Biochemical reactions- API’s or Vitek
-
Aseptate (nonseptate)
Septate
Molds- hyphae
-
- Molds- hyphal pigmentation
- a. Dematiaceous
- b. Hyaline
-
Have many different arrangements and sizes
Helps to identify the mold
Molds- spores
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Molds- Traditional Taxonomy
-4 Phyla
- Zygomycota
- Ascomycota
- Basidiomycota
- Deutromycota
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Aseptate hyphae
Rapid growers
“lid popper”
Asexual reproduction
sporangiospores
Zygomycota
-
-
Septate hyphae
Asexual reproduction
conidia
Ascomycota
-
Sexual reproduction
Ascospores within
cleistothecium
Ascomycota
-
Septate hyphae
Sexual reproduction –basidiospores
Plant pathogens or environmental organisms
Rarely cause human disease
Basidiomycota
-
Contains mushrooms,
rusts and smuts
Basidiomycota
-
No sexual stage
Septate hyphae
Classified based on conidia
Includes most saprobic and pathogenic fungi
Deutromycota
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Molds-Practical Classification
4 Categories
- Superficial or cutaneous mycoses
- Subcutaneous mycoses
- Systemic mycoses
- Opportunistic mycoses
-
Infect hair, skin and nails
Superficial Mycosis
-
Confined to subcutaneous layer without spreading to other sites
Subcutaneous Mycosis
-
Contains the dimorphic fungi
Primarily infects the lungs
They may disseminate to any organ system
Systemic Mycosis
-
Found primarily in immunocompromised patients
An ever-expanding list of organisms
All may cause disseminated disease
Opportunistic Mycoses
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Molds- A systematic approach to identification
- 1.Look at the hyphae
- -Aseptate or septate?
- 2.Look at color of hyphae/spores
- -Hyaline or dematiaceous?
- 3. Look at type and arrangement of spores
- -Single or multi-celled?
- -Single or in clusters?
- 4. Growth rate and temperature studies
- 5. Colony morphology
- -Color- fromt and reverse
- -texture
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Common Specimen Types
- Respiratory tract specimens
- Cerebrospinal fluid
- Blood
- Hair, skin and nail scrapings
- Urine
- Tissue, bone marrow and sterile body fluids
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Recommended Media
- All media should contain antibacterial agents unless specimen is from a sterile site
- Sabouraud with gentamicin and chloramphenicol
- Media with and without cycloheximide
- Mycosel
- Sabouraud
- Use a combination of above mentioned media types based on specimen
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Incubation Requirements
- Preferably 30 C
- Relative humidity 40-50%
- Hold 3 days to 6 weeks –depending on specimen type and request
- Gas permeable tape
- Examined at least 3 times weekly
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Direct Microscopic Exams of Specimens
-Gram stain
- -Potassium hydroxide preparations
- (KOH Prep)
- -Calcofluor White
- Fluorescent microscopy
- -Gomori methenamine silver (GMS)
- For histologic sections or direct specimens
- -Modified acid fast- Nocardia and AFB
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Laboratory Safety Considerations
- Molds
- Laminar flow hood and tape plates
- Yeasts
- Can be handled on bench top
-
Soil organisms and plant saprobes
More often seen on hands and feet
Most often seen in rural, tropical, agricultural areas
More often seen in men (occupational exposure)
Subcutaneous Mycosis
-
Three groups of subcutaneous infections
Chromoblastomycosis- warty lesions
Mycetoma- mycelial tumor
Phaeohyphomycosis- pigmented dark hyphae
-
-At site of trauma a warty, cauliflower-like lesion forms
-Microscopic- sclerotic bodies (copper pennies) seen in tissue
-Molds are all dematiaceous and form heaped grey to black colonies
Chromoblastomycosis
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Agents of chromoblastomycosis
- 1. Cladosporium carrionii
- Long chains of elliptical conidia on branching conidiophores
- 2. Phialophora verrucosa
- Flask-shaped phialides with distinct collarette, conidia in clusters at tip
- “vase of flowers”
- 3. Fonsecaea (pedrosa and compacta)
- Can have four arrangements of conidia
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At site of trauma a chronic infection occurs characterized by draining sinus tracts, granules, and tumor-like deformities of subcutaneous tissue
Mycetoma
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Bacterial cause- Nocardia, Streptomyces, and Actinomyces
Long filamentous gram positive rods
Actinomycotic Mycetoma
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Granules are many colors: yellow, white, red or black
Actinomycotic Mycetoma
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Fungal cause
Pseudoallescheria boydii/Scedosporium apiospermum
Exophialia jeanselmei
Madurella mycetomatis
Eumycotic Mycetoma
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Any infection caused by a dematiaceous fungi
Direct microscopic exam yields dark fungal elements
Phaeohyphomycosis
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Exophiala jeanselmei
Colony –yeastlike at first then wooly
Microscopic –balls of conidia on conidiophore
Growth at 37C but not at 40C
Grows with KNO3 media
Phaeohyphomycosis
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Phialophora richardsiae
Microscopic –phialides with distinct flared saucer-like collarettes
Phaeohyphomycosis
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Exophiala (Wangiella) dermatitidis
Colony-yeastlike when young then velvety black
Microscopic-ball of conidia appear to slide down conidiophores
Will grow at 40C
Doesn’t use KNO3 media
Phaeohyphomycosis
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Dimorphic Mycosis Causes deep seated infections
- Internal organs
- Lymph nodes
- Bone
- Subcutaneous tissue and skin
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Dimorphic Mycosis exist in two forms that are temp dependent
- Mold form (how it exists in nature)
- In vitro phase 25-30 C
- Yeast form (invasive tissue form)
- In vivo phase 35-37 C
- Can try to accomplish on enriched media with increased CO2
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Dimorphic Mycosis Usually considered slow-growing
- Requires 7-21 days for growth at 25-30 C
- Though exceptions do occur
- i.e. large number of organisms in specimen
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What are methods of ID for Dimorphic Mycosis
- In vitro conversion of mold form to yeast form
- Can be tedious and C. immitis has none
- Exo-antigen testing
- Gel immunodiffusion precipitin test
- Nucleic acid probes
- Sensitive, specific and rapid
- Very expensive
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Sporothrix schenkii
Source of infection
- Organism lives on vegetation
- Inoculation occurs through trauma
- Occupational hazard
- Farmers, gardeners, florists and miners
- “rose gardener’s disease”
- Possibly pulmonary route also
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Sporothrix cont’d
Primary lesion forms
Non-healing ulcer
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Sporothrix cont’d
Secondary lesions
- Nodular lesions
- Lymphatic channels and nodes
- Rarely disseminates systemically
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Sporothrix
Direct detection from sample
- Exudate from lesions
- Silver stain may show cigar shaped yeast cells
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Sporothrix culture from sample
- Culture –mold phase
- Leathery ,wrinkled colonies that darken with age
- Young colonies could be mistaken for yeast
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Sporothrix Microscopically
- – conidia borne in clusters at tips of conidiophore = rosette
- Leathery ,wrinkled colonies that darken with age
- Young colonies could be mistaken for yeast
- As culture ages may see “sleeve arrangement” – single conidia along hyphae
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Sporothrix Culture –yeast phase
- Try to convert at 37C on enriched media
- Cream colored yeast colony
- Cigar shaped yeast cells- cigar bodies
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What are the Endemic areas for Sporothrix?
- Found world-wide
- Outbreaks in gold mines
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Where is the source of infection for Histoplasma capsulatum?
- Organism lives in soil enriched with bird manure or bat feces
- Acquired via inhalation of conidia
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What is the etiology of Histoplasma?
- Pulmonary lesion- primary infection
- Chronic pulmonary disease- cavitations
- Secondary dissemination- lymphatics, organs and skin
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Histoplasma Direct detection
- Respiratory tract specimens
- Bone marrow, tissue or blood by Giemsa may yield round/oval yeasts within macrophages
- PCR on tissue
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Histoplasma Culture
- Mold phase –slow growing, usually 2-4 weeks
- Colony –white fluffy, turns brown with age
- Microscopic –macroconidia at first round and smooth but become tuberculate
-
Histoplasma Sepedonium
saprobic mold that looks similar but grows faster
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Histoplasma Culture
- Yeast phase –difficult to convert, not recommended
- Microscopic –small yeast cells
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Where is Histoplasma Endemic?
- Worldwide distribution
- Prevelant in Ohio, Mississippi, and Missouri River Valleys
- Outbreaks following cleaning of chicken coops, barns, pigeon roosts and spelunking
- In endemic areas- high percentage of population are skin test positive
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How is Blastomyces dermatitidis acquired?
- Acquired by inhalation of conidia
- Disease state very similar to Histoplasmosis
- More common in men associated with outdoor occupation
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What is characteristic of a primary Blastomyces infection?
- respiratory infection
- Dissemination to bones, soft tissue, and skin
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What would you see in a direct detection of Blastomyces using
Microscopy?
large thick walled yeast with single bud connected by broad base to parent cell
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Describe Blastomyces culture during mold phase:
- White, waxy to cottony colony
- Microscopic –single, round to pyriform conidia on conidiophores = “lollipop”
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Describe Blastomyces culture during yeast phase:
- Waxy, wrinkled colonies
- Relatively easy to convert
- Microscopically –large thick-walled yeast with broad base
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Blastomyces Endemic areas
- Seen in Americas and Africa
- Prevelant in Northern Ohio and Mississippi River Valleys and south eastern United States
- Diagnosed cases are usually the disseminated ones
- Mississippi River Valley Disease or Chicago Disease
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Coccidioides immitis
Source of infection
- Acquired by inhalation of arthroconidia
- Mold phase grows on vegetation producing airborne arthroconidia
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Coccidioides Etiology
- Primary infection is pulmonary disease (60% of which are asymptomatic and self limiting)
- Dissemination - <1% develop this in bone, CSF, organs or subcutaneous tissue
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Coccidioides Direct detection
- On sputum, bodyfluids or tissues, non-budding, thick-walled spherule containing endospores
- Spherules may burst and endospores may be confused with yeast
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Coccidioides Culture
- mold phase
- Cottony white colony
- Highly contagious!!!
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Coccidioides Culture Microscopically
- hyphae that form barrel-shaped arthroconidia that alternate with empty spaces
- No yeast phase in vitro only spherules in tissue or sample
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Coccidioides Endemic areas
- Prevelant in desert southwest United States, especially the San Joaquim Valley
- “Valley Fever”
- Outbreaks after windstorms
- Seen in other parts of the world due to travel- “snow birds”
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Paracoccidioides brasiliensis
- Dimorphic fungi
- Endemic in South America
- Diagnostic form –yeast phase known as “Mariners Wheel”
- Mold phase –similar to Blastomyces
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Others possible systemic mycoses
- Zygomycosis
- Candidiasis
- Aspergillosis
- Cryptococcosis
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What is the only genus in family Mycobacteriaceae?
Mycobacterium
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What are the only two species that are contagious in the family Mycobacteriaceae?
M. tuberculosis and M.leprae
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What are the characteristics of Mycobacteriaceae?
- Obligate aerobes
- Slightly curved rods
- Acid-Fast
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How many organisms are required for an acid fast smear?
Requires 5,000-50,000 orgs/ml to show a positive smear
-
What stains are used on acid fast organisms?
- Fluorochrome stains
- Carbol Fuchsin stains
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What makes an organism acid fast?
- Resistant to acid-alcohol decolorization due to Mycolic acids that have a lipid layer surrounding peptidoglycan layer of cell wall
- Involved in virulence
-
What is the generation time of mycobacteriacea?
Very long, 2 to 20 hours.
-
What is the optimal temperature for AFBs (acid-fast bacilli)?
- Optimal Temperature for most AFB is 35-37°C
- Some prefer 30°C
-
How is non tuberculosis mycobacterium classifeid?
- Pigment-Runyoun 1959 to classify NTM
- Nonphotochromogens
- Photochromogens
- Scotochromogens
- Rapid Growers
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What is M. tuberculosis plated on?
Lowenstein-Jensen
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What is the infective dose of M. tuberculosis?
1 bacilli
-
How do you control aerosols of M. tuberculosis?
- Negative Pressure Rooms
- Biosafety Cabinet
- Safety carriers for centrifuge
- Disinfectant soaked absorbent towels
- Incinerator/Disposable loops
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What is the PPD/Manoux test?
- Purified protein derivative
- Intradermal injection of antigenic protein particles from killed M. tuberculosis
- Delayed hypersensitivity reaction-localized swelling/redness
- Reveals only previous infection-does not differentiate an active infection from latent infection or a cured patient
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What is PPD made from?
antigenic protein particles from killed M. tuberculosis
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What are some side effects of PPD tests?
Delayed hypersensitivity reaction-localized swelling/redness
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Can the PPD/Manyoux test be used to differentiate an active infection from latent infection or a cured patient?
No. Reveals only previous infection-does not differentiate an active infection from latent infection or a cured patient
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Processing M. tuberculosis consists of what two parts?
- -Digestion-releases mycobacteria from specimen material
- N-acetyl-L-cysteine (NALC)
- -Decontamination-kills off contaminating bacteria
- 2% NaOH/4% NaOH
- 5% Oxalic Acid
- 4% H2SO4
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Digestion-releases mycobacteria from specimen material using which reagent?
N-acetyl-L-cysteine (NALC)
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Decontamination-kills off contaminating bacteria
- 2% NaOH/4% NaOH
- 5% Oxalic Acid
- 4% H2SO4
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What media is used to support the growth of mycobacteria?
- Egg based, Lecithin in eggs neutralizes many toxic factors in specimens
- Lowenstein-Jensen slants
- Malachite Green
- Gruft Modification with Antibiotics
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What ingredient in eggs is effective by neutralizing many toxic factors in specimens?
Lecithin
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What is the agar based medium used in culturing M. tuberculosis, that is sensitive to heat and light?
7H11-transparent media
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What liquid based media is used for culturing M. tuberculosis?
- Modified 7H9 broth
- Bactec
- Radiometric: ↑ CO2 concentration
- 9000MB: ↓ O2 depletion
- Biphasic
- Isolator
- Blood
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What is the incubation temp and time for culturing M. tuberculosis?
- Temperature
- 35-37°C for most cultures
- 30°C media added for skin and joint cultures
- CO2
- Mycobacteria require 5-10% CO2 esp. during early phase of growth
- Time
- 6-8 weeks
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What organisms does the M. tuberculosis complex consist of?
- M. tuberculosis
- Most common cause of clinical tuberculosis
- M. bovis
- Primarily a disease of cattle
- M. bovis has been irradicated from cattle in the U.S.
- M. bovis BCG - strain of attenuated M. bovis used as TB vaccine
- M. africanum, M. microti, & M. canettii
- Rare causes of clinical tuberculosis
-
What mycobacteria is the most common cause of clinical tuberculosis?
M. tuberculosis
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What mycobacteria is the primarily a disease of cattle; has been irradicated from cattle in the U.S.; and can be attenuated for use as a TB vaccine?
M. bovis
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What mycobacteria are rare causes of clinical tuberculosis?
M. africanum, M. microti, & M. canettii
-
How many of the world’s population is infected with TB
1/3
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How many of TB cases occur in racial and ethnic minorities?
81%
-
How many of TB cases are foreign-born individuals?
53.7%
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Describe the initial infection of TB:
- Bacilli are expelled when a person with active TB coughs, sneezes, sings, etc… Close contacts are at greatest risk.
- Bacilli are inhaled and reach the pulmonary alveoli.
- Immune response kills most of the bacilli leading to formation of granulomas.
-
Describe the latent infection of TB:
- Macrophages illicit a cell-mediated immune response and cause destruction of bacilli and formation of tubercules.
- ~90% of people infected will have latent infection only
-
Describe an active infection of TB:
- TB overcomes the immune system, allowing mycobacteria to multiply, leading to TB disease.
- Occurs most in those who lack a powerful cell-mediated immune response (e.g. kids, elderly, immunocompromised)
- 1-5% of primary infections result in active infections
-
Describe the reactivation of TB:
- Decrease in the body’s defenses leads to release of bacilli and active infecton.
- 5-9% of initial infections will result in reactivation TB.
-
What is the clinical presentation of TB?
Patients with active tuberculosis infection typically present with chronic low grade fever, night sweats, weight loss and most often a prolonged productive cough (>3 weeks).
-
What are some diagnostic tests for TB?
- Chest X-ray showing infiltrate or cavities in lungs which may be suggestive of TB.
- PPD
- AFB Smear/Culture
-
What are methods of identifying TB?
- Accuprobe
- Genprobe-Nucleic acid hybridization of a DNA probe to rRNA of the target organism.
- Conventional Biochemical Testing
- 68° Catalase Negative
- Nitrate Positive
- Niacin Positive
- PCR/NYS “Fast Track” Program
- Universal genotyping/DNA fingerprinting
- Spoligotyping/RFLP
-
What genetic test is used in identifying TB?
- Accuprobe
- Genprobe-Nucleic acid hybridization of a DNA probe to rRNA of the target organism.
-
What are conventional biochemical tests for TB?
- 68° Catalase Negative
- Nitrate Positive
- Niacin Positive
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WHat treatment is used for active TB?
- Active TB
- DOTS
- RIPE-for typical susceptible strains of Mtb in Non-HIV pts Rifampin (RIF), Isoniazid (INH), Pyrazinamide (PZA), and Ethambutol (EMB) daily for 2 months, then RIF and INH for 4 months
-
WHat treatment is used for latent TB?
INH daily for 9 months/Reduces risk of development of active TB from 10% to <1%
-
WHat treatment is used for exposure to TB?
INH for 9 months/may be discontinued if PPD remains Neg
-
What susceptibility testing is performed on TB?
- Broth or agar plates
- 1st line drugs-RIPE and Streptomycin
- 2nd line drugs-tested if any of 1st line show resistance
-
What are causes of TB resistance?
- In U.S. ~9% of Patients w/TB are resistant to at least 1 drug
- Inadequate treatment
- Mutation
- Health Care infrastructure
-
What are characteristics of MOTTs/NTMs (mycobacterium other than tuberculosis/non tuberculosis mycobacteria)?
- Ubiquitous in nature: soil, water etc…
- May colonize without causing disease
- Non-photochromogens
-
What is the most important MOTT (mycobacterium other than tuberculosis)?
M. avium complex
-
Which organism falls under the MOTT group of organisms and are non-photochromes?
M. avium complex
-
What is the transmission of M. avium complex?
Aerosolization/Ingestion leads to respiratory or intestinal colonization
-
What water-borne organism is implicated as a significant cause of death in AIDS patients?
M. avium complex
-
What is the primary species in M. avium complex?
M. avium and M. intracellulare
-
What are the patterns of pulmonary disease due to M. avium complex?
- Associated primarily with patients with underlying lung disease (COPD, chronic bronchitis, lung cancer etc…)
- Productive cough, weight loss, fever, bloody sputum (can be like TB)
-
What are the patterns of disseminated disease due to M. avium complex?
- Primarily AIDS pts - Severe immunosuppression greatest risk factor
- Fever, sweats, fatigue, diarrhea, SOB
- Wasting disease associated with CD4+ counts of <50 cells/ul
- Prior to availability of antiretroviral medication, 30% of all HIV+ patients developed disseminated disease. With highly active antiretroviral therapy incidence is only ~2%.
-
Lymphadenitis is primarily found in children due to which organisms?
M. avium complex
-
How is M. avium identified?
- Accuprobe
- Conventional biochemicals:68°Cat Positive/Tellurite Positive
-
What is the treatment for M. avium complex?
- Combination of clarithromycin/azithromycin and ethambutol with or without rifabutin
- Pulmonary MAC: 6 months/~90% chance of recovery
- Disseminated disease: Maintenance therapy for life
- Patients with low CD4+ counts get prophylactic TX w/HAART
- Lymphadenitis in kids: no treatment necessary/benign course
-
Which mycobacteria Requires hemin for growth; (chocolate plate or Middlebrook agar with “X” factor strip); Prefers 30°C and primarily causes skin and joint disease in AIDS patients
M. haemophilum
-
Which mycobacteria is most often seen in immunocompromised patients and the disease progression resembles TB-chronic pulmonary disease?
M. kansasii
-
What are the key biochemicals in identifying M. kansasii?
Photo Positive, Nitrate Positive, Accuprobe available
-
This organism is found in Fresh and Salt water and primarily causes cutaneous infections due to water-related trauma (fish tank, swimming pool, or natural water sources), prefers 30°C.
M. marinum
-
What organisms are considered schrotochromogens?
M. scrofulaceum and M. gordonae
-
What important pathogen is a cause of cervical lymphadenitis in children <5 years old and has been surpassed by M. avium complex as most common cause of lymphadenitis in developed countries?
M. scrofulaceum
-
This organism is very commonly found in water; Non-pathogenic
Most commonly encountered “contaminant” in AFB Lab; Used as QA monitor to evaluate decontamination procedure; ~5% of total isolates should be M. gordonae:
M. gordonae
-
What mycobacteria are considered rapid growers?
M. fortuitum complex, M. leprae,
-
Why is it important to differentiate M. fortuitum from M. chelonae? How are they differentiated?
M. chelonae is much more resistant
- M.fortuitum species group:Salt tolerance, Iron uptake, and Nitrate POS
- M. chelonae: Salt Tolerance, Iron Uptake, and Nitrate NEG
-
What mycobacteria causes Hansen's disease?
M. leprae
-
Which mycobacterium does not grow on routine Media; Requires Cell Culture because it is characteristic of an Intracellular “parasite”?
M. leprae
-
What is the transmission of M. leprae?
- Primary infection occurs through nose
- Affects superficial nerve endings causing anesthetic skin lesions
- If untreated can lead to sensory loss and paralysis
- Natural infection in the footpads of wild armadillos
- Primarily seen in Asia and Africa
- In U.S. occasionally seen in Texas/Louisiana
-
How do you identify M. leprae?
- Rapid Identification
- GLC/HPLC
- Analysis of cell wall mycolic acids
- HPLC used by many large laboratories and is very good at differentiating most documented species of Mycobacteria
- or
- Nucleic Acid sequencing
- 16S rDNA sequencing
- More widespread use has resulted in the recognition of many “new” genetically distinct species that have been previously uncharacterized.
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