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Micobacteria general characteristics - ___robic, micro appearance, important biochem
- Intracellular pathogens
- Aerobic (mostly obligate)
- micro: very thin, slightly curved, may branch
- gram +, but probably won't take stain
- biochem: acid fast cell wall (N-glycolymuramic acid, high lipid content)
- Non-spore forming (except M. marinum)
- SLOW GROWING
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What is the Mycobacterium tuberculosis Complex?
- A group of mycobacterium capable of causing tuberculosis (pulmonary or other)
- M. tuberculosis (most common)
- M. bovis
- M. africanum
- M. caprae
- M. microti
- M. canetti
- M. pinnipedii
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What are the basic categories of mycobacterium? What are their habitats?
- Mycobacterium Tuberculosis complex
- Nontuberculous mycobacteria - slow growing
- Nontuberculous mycobacteria - rapid growing
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MTC - colony appearance, infection
- Colony: slow growing (~6 wks), nonpigmented
- Infection: inhalation of a SINGLE organism can lead to infection
- M. tuberculosis - person-to-person via droplets
- M. bovis - associated w/ ingestion of milk from infected cows, penetrates mucosa
- *NOTE - BCG vaccine is attenuated M. bovis
- Individuals with HIV are more susceptible to TB
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Describe tuberculosis (disease - symptoms, global perspective)
- Chronic pulmonary disease (most cases)
- Can disseminate to every organ system
- Cavity formation and bronchial spread
- Rapid progression of pneumonia in AIDS patients (no cell-mediated immunity)
- Global: 2nd leading cause of death from single infections microorganism
- 1/3 world population has TB
- Common among poor, homeless, IV drug users
- 46% of notified TB patients had HIV test results
- *NOTE - US and CA rates are declining
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Describe tuberculosis transmission
- Respiratory spread: almost all cases
- droplet nuclei hang in the air
- infectious dose 10 bacilli
- AFB smear positive = active infection (contagious)
- *NOTE - highly contagious in AIDS patients w/o cavity formation (normal X-Ray)
- Ingestion: rare
- milk from infected cow
- Skin innoculation: very rare
- predominantly accident in lab
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Describe tuberculosis infection/repl
- Airborne droplet nuclei reach terminal air spaces and multiply (usually mid-lung)
- MTB ingested by alveolar M0
- small numbers eliminated
- large numbers allow growth within M0, then destroy M0
- more M0 invade site, carry MTB to lymph nodes
- nonimmune host: spreads directly into blood/body sites
- hypersensitivity develops 3-8wks after infection preventing further dissemination
- granulomas: "problem spots" in X rays, contain viable MTB for later reactivation
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What is NTB? Where is it normally found? How is it classified?
- All mycobacteria not found in the MTB complex
- Widely distributed in nature (water, soil)
- Runyon classification (old) (new is PCR)
- Group I - photochromogens
- Group II - Scotochromogens
- Group III - Nonphotochromogens
- *NOTE Group I-III = slow growers
- Group IV - Rapid growers (<7 days)
- *NOTE - M. leprae is noncultivatable
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What is the Photo Activation Test? Procedure? Results?
- USED TO DETERMINE Runyon Group for NTB
- Procedure: Innoculated 7H10 plates or tubes are immediately protected from light
- (caps must be loose for growth)
- Uncover tube then do light activation on it
- Results
- colonies become pigmented in light - Group I Photochromogens
- colonies pigmented in dark OR light - Group II - Scotochromogens
- colonies never pigment - Group III - Nonphotochromogens
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Photochromogens - group, details, examples
- Group I
- Slow growing
- become pigmented when exposed to light
- M. kansasii (most common)
- M. asiaticum
- M. marinum
- M. intermedium
- M. novocastrense
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Scotochromogens - group, details, examples
- Group II
- Slow growing
- Colonies pigment in dark OR light
- RARELY RECOVERED (contaminants)
- M. szulgai
- M. scrofulaceum
- M. interjectum
- M. heckeshornense
- M. tusciae
- M. kubicae
- M. gordonae
- M. cookii
- M. hiberniae
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Nonphotochromogens - group, details, examples
- Group III
- Slow growing
- Colonies produce no pigment in dark or light
- MOST are pathogenic
- M. avium complex (important)
- M. xenopi
- M. ulcerans
- M. malmoense
- M. genavense
- M. haemophilum (rare, needs heme disc and cool temp to grow)
- M. heidelbergense
- M. shimoidei
- M. simiae
- M. celatum
- M. conspicuum
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What is M. avium complex? Diseases/groups? Members?
- Most commonly isolated NTB species
- Incidence higher in immunosuppressed
- Respiratory disease in adults
- Lympadenitis in children
- Disseminated infection in HIV patients
- Consists mainly of M avium and M. intracellulare
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rapid growers - group, details, examples,
- Group IV
- Rapid growring (<7 days)
- can grow on routine media
- Weakly gram positive
- most common in posttraumatic wound infections
- M. abscessus
- M. fortuitum
- M. chelonae
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Mycobacterium leprae - disease, forms?
- Causes leprosy (Hansen disease)
- chronic disease of skin, membranes, nerve tissue
- bacilli multiplying in peripheral nerves cause sensory impairement
- Tuberculoid: normal
- no immune defect, localized to skin/nerves, few organisms present in lesions
- WBC stops spread
- Lepromatous: very bad
- extensive skin lesions, NUMEROUS acid-fast bacilli, dissemination
- organism unchecked, problematic
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M. leprae - isolation and ID
- Detection via stains and molecular amplification (can't culture)
- NOT CUTIVATABLE IN VITRO (cultivated in armadillo or mice footpads)
- *NOTE - these tests are not yet refined
- Clinical manifestations are best method of ID
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Describe blood cultures from mycobacterium w/ major use
- major use: M. avium-intracellulare
- automated detection system: specific media for direct culture from blood sample
- loaded into bactec instrument for each reading
- Isolator tube: functions as t.port/processing tube
- sample must be innoculated to media
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Describe Lower Respiratory Samples from mycobacterium w/ major use
- Expectorated sputum: early morning samples allow pooling
- 1 per day for 3 days, MINIMUM
- Induced sputum: use of expectorant if pt can't produce
- Brochoscopy: last resort if sampling problems occur (surgical)
- *NOTE - if positive for AFB 3 samples are enough, if negative 4-5 samples required
- Monthly collections during therapy allow monitoring of results
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Describe Gastric Aspirates from mycobacterium w/ major use
- Mainly used in children (can't produce sputum)
- early morning samples allow pooling of swallowed sputum
- 1 per day for 3 days
- AFB smears are not reliable (false pos from NTB)
- *NOTE - collection must be neutralized w/ NaCO3 within 4 hours
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Describe Body Fluids/CSF from mycobacterium w/ major use
- CSF: 10mL minimum
- Low # of organisms
- AFB smear RARELY positive
- Other fluids: collect as much as possible, then pellet
- RARE
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Describe Wounds, Tissues, Aspirates from mycobacterium w/ major use
- Biopsy: best sample
- 1cm cube
- often a punch biopsy
- Aspirates: good sample
- collect as much as possible
- Swabs: worst sample
- should not be used, may be rejected
- always contains excess microbes
- *NOTE - tissue samples must be ground prior to processing
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Describe stool samples from mycobacterium w/ major use
- Collect >1 gram
- MAC (avian) colonization: common in HIV pts
- MTB - VERY rarely
- *can be difficult to use due to contamination
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Describe urine asmples from mycobaterium w/ major use
- Collect first morning urine (organisms accumulate in bladder)
- 1 sampler per day for 3 days
- Pooled samples unacceptable
- Catheter required if clean catch impossible
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What are the direct detection methods for Acid-Fast bacilli?
- Acid-fast stains
- Fluorochrome - easy to read (glow)
- Ziehl-Neelsen - not usually used due to heat requirement
- Kinyoun - uses phenol instead of heat
- Nucleic acid probes
- Antigen protein detection
- Immunodiagnostic testing
- PCR/sequencing
- Chromatographic analysis
- Gas chromatography (mostly reference)
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How do acid fast stains work?
- Drive the stain (carbol fuchsin) into the cell
- Stain complexes with mycolic acids in cell wall
- Resists decolorization (3% HCl in alcohol)
- Counter stain with methylene blue
- *NOTE - the bright pink is unmistakable
- For fluorochrome stains
- auramine-rhodamine replaces carbol fuchsin
- acid decolorizer is 0.5% in alcohol
- counter stain is potassium permanganate
- orgaisms fluorese orange/yellow under UV microscope
- *NOTE - fluorochrome method more sensitive (used more), can review 30 fields instead of 300 fields
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How are fluorochrome stains reported?
- 20x ocular (standardized)
- none: no AFB seen
- suspicious: 1-2/30 fields
- 1+: 1-9/10 fields
- 2+: 1-9/1 field
- 3+: 10-90/1 field
- 4+: >90/1 field
- *NOTE - positive culture is diagnostic for TB, positive smear indicates any mycobacterium
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What methods are available for identification of mycobacteria?
- Acid-fast stain to confirm mycobacteria
- Phenotypic tests: growth rate, pigment production, colony morphology, biochemical testing (Niacin, Nitrate, Catalse, PZA)
- Molecular methods: accuprobe/genprobe
- high sensitivity and specificity
- chemiluminescent label on DNA probes to rRna
- available - MTBC, MAC, M. gordonae, M. kansasii
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Susceptibility testing for MTB?
- Proportional susceptibility method: plates incubated for 2-3 weeks with antimocrobial in 3/4 quadrants
- >1% growth = resistant
- Bactec 460 method: most common
- results available in 5-7 days
- broth (bactect) uses critical concentration for each drug
- >1% growth = resistant
- other systems available/developing
- 2nd line testing: IF isolate is resistant to rifampin, isonazid, pyrazinamide
- comprehensive panel of agents
- not common in hospital
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What is MDRTB and XDRTB?
- MDRTB: multiple drug-resistant TB
- resistant to isoniazid and rifampin (two most potent TB drugs)
- XDRTB: extensively drug-resistant TB (rare)
- resistant to isoniazid, rifampin, fluoroquinolone, and at least 1/3 injectable second-line drugs
- available treatments are much less effective
- special concern for HIV patients, more likely to develop TB disease, higher risk of death
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