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Mycobacterium General Characteristics

- Non-motile, non-spore-forming, thin aerobic (slightly microphilic) rods
- Slight, rare, easily disrupted branching
- Cell wall has N-glycomuramic acid with a high lipid content (mycolic acid) and resists decolorization
- Therefore, acid-fast
- Hydrophobic cell surface so they tend to clump up making nutrients sparce (thats why slow growing)

Complex

A term used in the clinical lab to describe two or more spp whose distinction is complicated and of little or no medical importance

M. tuberculosis complex

M. tuberculosis (Mtb), M. bovis, M. bovis (BCG), M. africanum, M. microti, M. canattii , M. pinnipedii

Slow growers

- Takes 3 weeks to grow
- TB complex
- M. tuberculosis, M. bovis, M. africanum

Rapid growers

- 7 days to grow
- Can't be TB complex

Photochromogens

- Colonies of NTM that develop pigment following exposure to light after being grown in the dark
- Takes more than 7 days to appear on solid media
- M. kansasii, M. marinum,

Scotochromogens

Colonies of NTM that develop pigment in the dark or light
- Take more than 7 days to appear on solid media
- M. scrofulaceum

Non-photochromogens

- Colonies of NTM that are nonpigmented regardless of whether they are grown in the dark or light
- Take more than 7 days to appear on solid media
- M. avium

Mycobacterium tuberculosis Epidemiology

- Humans are only natural reservoir
- 2002: 2 billion infected, 8.8 million new cases, 2 million deaths
- 2003 US: 15,000 cases
- High risk: homeless, substance abusers, prisoners, HIV
- Person to person by inhalation of droplet nuclei
- Drug resistant Mtb

Mycobacterium tuberculosis Clinical Disease

- Lung infections in immunocompetent pts(Middle or lower lung)
- Replication stops in 3-6 weeks
~5% active disease within 2 yrs, ~5-10% active disease
- Gradual onset
- Malaise, weight loss, cough, night sweats
- Sputum: Little or bloody and purulent
- Hemoptysis → tissue destruction (cavitary disease)
- Clinical signs: radiologic, positive skin test, lab detection
- Hematogenous spread (milliary tuberculosis)

Mycobacterium leprae Pathogenesis

- Leprosy (Hansen's disease): chronic disease of the skin mucous membranes and nerve tissue
- Has an immune system response
- Tuberculoid leprosy: strong cellular rxn, weak humoral rxn
with few mycobacteria
- Lepromatous leprosy: strong humoral rxn, weak cellular rxn with many mycobacteria

Mycobacterium leprae Epidemiology

- Has had a 90% decrease since 1985
- 620,672 cases in 2002
- Only 96 cases in US in 2002
- Endemic in armadilllos
- Person-to-person contact
- Route: inhalation or skin contact

Mycobacterium leprae Clinical Disease

- Chronic
- Skin, peripheral nerves
- Tuberculoid: Milder with hypopigmented lesions
- Lepromatous: Severe disfiguration

Mycobacterium avium Complex (MAC)

- M. avium, M. intracellulare
- Non-chromogens
- Ubiquitous in water, soil
- Transient colonization
- Most common mycobacterial infection in US
- Immunocompetent: chronic pulmonary disease
- Immunocompromised: disseminated, all organs, high numbers of mycobacteria

M. kansasii

- Mtb-like disease
- Photochromogen
- Infection more common in white males
- Natural resevoir is tap water
- Chronic pulmonary disease

M. scrofulaceum

- Affects cervical lymphatic tissue
- Scotochromogen
- Found in soil, water, raw milk

M. marinum

- Cutaneous dissease
- Photochromogen
- Natural resevoir is fresh and salt water due to infected fish or marine life
- Transmission by contact with contaminated water and organism entry by trauma or small breaks in the skin

Rapidly Growing Mycobacteria

- M. fortuitum, M. chelonae, M. abscessus
- Growth in less than 7 days
- Low virulence
- Negative staining with auramine-rhodamine fluorescent stain
- Trauma, iatrogenic procedures

Lab Diagnosis of Mycobacteria

- Assessment of cell-mediated immunity
- Turberculin skin test: injection of TB protein under skin, skin will react to the antigens by developing a firm red bump at the site within 2 days if positive (can't detect if latent or active)
- Lepromin skin test: determines type of leprosy by injection of inactivated leprosy bacillus
- QuantiFERON-TB test: detects latent TB

Mycobacteria Specimens for Culture

- Pulmonary Specimens: early morning sputum, induced sputa, gastric lavage, bronchoscopic aspirations
- Urine, stool, tissue, body fluids, blood wound aspirates
- Transport: Sterile leak-proof containers, biohazard bags
- Storage: refrigerate
- Process within 24 hours

Specimen Processing for Isolating Mycobacteria

- Class II biological safety cabinet
- BSL 3 Room: negative pressure room with washable walls, ceilings and one-pass air
- Caps on centrifuge tube holders
- Personal protective equipment (PPE)
- Respirator

Mycobacteria Nucleic Acid Amplification

- FDA-approved kits: Amplicore Mtb Direct Test (Roche Diagnostics), Ampified Mtb Direct Test (Gen-Probe)
- Non-FDA-approved kits: Home brew procedures
- Fast Track System

Mycobacteria Solid Media: Egg-based

- Lowenstein-Jensen medium
- Good growth of most mycobacteria
- No susceptibilities
- Hard to see growth

Mycobacteria Solid Media: Agar based

- Middlebrook 7H10 and Middlebrook 7H11
- Better defined
- Microcolony detection

Mycobacteria Liquid Media

- Reduced detection turn-around time (approx 10 days vs. agars 17 days)
- Higher isolation rates
- Difficult to ID from
- BACTEC, Septi-Chek AFB, MGIT (Mycobacteria Growth Indicator Tube)

Mycobacteria Incubation

- Incubate at 37°C
- 25-33°C (M. marinum, M. ulcerans)
- Atmosphere: 5-10% CO2 (high concentration)
- Liquid medium : 6 weeks
- Solid medium: 8 weeks
- Reading schedule (every day at the same time)

Phenotypic Identification of Mycobacteria

- Growth rate: rapid growers vs. slow growers
- Pigmentation & photoreactivity (Photochromogen, Scotochromogen, Nonphotochromogen)
- Colony morphology
- Biochemicals (Mtb: niacin & nitrate positive = yellow)

Genotypic Identification of Mycobacteria

- Fastest way to identify spp
- DNA probes
- 16S rDNA sequencing
- PCR-RFLP (fingerprint pattern)

Preliminary ID for Mtb

- Rough, buff-colored (tan), slow-grower
- 99.9% ID

Mycobacteria Treatment

- Most common two drug therapy: Isoniazide (INH) and rifampin for 9 months (if pyrazinamide is added in beginning, the total time is only 6 months)
- Resistance due to non-compliant patients
- Multidrug resistant Mtb (resistant to at least 1 frontline drug)
- Extensively multidrug resistant Mtb (more toxic to pt)
- Profoundly multidrug resistant Mtb (resistant to everything in which extensive surgery is the only thing to save the pt)

Mycobacteria Control

- Active surveillance,
- Prophylactic & therapeutic intervention
- Case monitoring

Chemoprophylaxis

- Prevention of infectious diseases using anti-microbial agents
- Administered prior to Mtb exposure
- Low CD4+ counts (M. avium)

Immunoprophylaxis

- The prevention of disease by the production of active or passive immunity (a.k.a. vaccination)
- M. bovis bacille Calmette-Guerin (BCG) (not used in US)

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