Mycobacteriology Update 2018

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Mycobacteriology
Update 2018
Margie Morgan, PhD

Mycobacteria
 Acid Fast Bacilli (AFB)
 Cell walls contain complex mycolic acids and free
lipids, many properties of the ...

Identification of the Mycobacteria
 For decades, identification algorithm started with the
determination of pigment in th...

Mycobacteria Taxonomy >170 species
 TB complex:
 Mycobacterium tuberculosis
 M. bovis and the Bacillus Calmette-Guerin ...

Mycobacteria that cause disease?
 Mycobacterium tuberculosis – the major pathogen of this genus
The others: MOTT
 M. avi...

Mycobacteria that rarely if ever cause
disease! If so, in immune compromised!
 Mycobacterium gordonae
 M. gastri
 M. ce...

Algorithm for identification of MOTT
- historically speaking
 Runyon System
 Classify species not in the TB complex (MOT...

Light Test – Determine does it produce a yellow
pigment after being exposed to 8 hours of light?
bulb.
Group I Photochromo...

Runyon Classification System Results
 Group I - Photochromogen – turns yellow when
exposed to light, no color in the dark...

Runyon Classification cont’d
 Group III – Non-photochromogen – No pigment
produced in the light or dark
 M. avium-intrac...

Safety in the
AFB Laboratory
 Level 3 biosafety precautions required in AFB laboratories that
process, identify and perfo...

Specimen collection
 Sputum – 3 early morning collection, or
1 early morning, 2 at least 8 hours apart
 Bronchial lavage...

Specimen Processing
Start to Finish!
 5 ml of specimen pipetted into conical tube
 Decontaminate and Liquify specimen fo...

Why Decontaminate Specimens?
 For the slow growing mycobacteria to be cultured
 Must eliminate competing bacteria/yeast ...

Specimen Decontamination/Digestion
Most often used :
 4% NaOH / N-acetyl-L-cysteine
 Used in Special circumstances:
 Ox...

Specimen Centrifugation
 Centrifugation at 3000 X g (fast) with safety cups
 Speed of centrifugation is important - AFB ...

Manual Plating/ Culture Media
 Middlebrook – Synthetic media
 Clear solid agar and liquid media
 Synthetic = chemical i...

Automated Detection of AFB
Bactec MGIT 960, Bacti-Alert and VersaTREK
Liquid Middlebrook 7H9 tubed media for growth
Bact...

Acid Fast Staining for Mycobacteria -
only concentrated specimen should be stained
 Carbol Fuchsin based stains
 Carbol ...

Fluorochrome based stain
Auramine Rhodamine –
 Fluorescent stain, organisms stain fluorescent
yellow with black backgrou...

Acid Fast staining of the Mycobacteria
Mycobacterium avium complex
Organisms are routinely shorter than TB
(Kinyoun stain)...

Direct Detection of TB from Respiratory
Specimens by Molecular Amplification
 FDA cleared to detect TB complex organisms ...

PPD (Purified Protein Derivative)
Mantoux test/TB skin test
 Detects past or current TB exposure
 False positive reactio...

Cell Mitogen assays – Interferon
Gamma Release Assays
 QuantiFERON-TB-Gold (QFT) and T-spot
assays
 Whole blood tests to...

Mycobacterium tuberculosis
 Optimal Temp 37˚ C, Grows in 12 –25 days
 Buff colored, dry cauliflower-like colony
 Manual...

Mycobacterium
tuberculosis
Demonstrates Cord factor –
due to high lipid content in cell
wall, rods stick together and
deve...

Tuberculosis
 Classic Disease
 Slowly progressive pulmonary infection cough, weight loss, and
low grade fever
 Lesion w...

Pathology of M. tuberculosis
Lung with pulmonary TB has apical cavitations with
numerous areas of caseous necrosis and mas...

TB in HIV/AIDS patients
 Worldwide -TB is the most common opportunistic infection
affecting HIV/AIDS patients
 AIDS pati...

M. tuberculosis outside lung
 Scrofula -Unilateral lymphadenitis (cervical lymph node)
most often seen in immune compromi...

Susceptibility testing of TB
 Two methods for testing
 (1) Agar dilution (indirect proportion method)
 antibiotics embe...

Mycobacterium bovis
 Produces disease in cattle and other animals
 Spread to humans by raw milk ingestion
 Disease in h...

Mycobacterium ulcerans
 Bairnsdale or Buruli ulcer boil that progresses into a
painless skin ulcer
 Can progress into av...

Mycobacterium kansasii
 Culture 37* C, 10-20 days
 Photochromogen
 Niacin accumulation test = negative
 Nitrate reduct...

Mycobacterium marinum
 Optimum temp for growth is 30˚ C
 Grows poorly or not at all at 37°C
 Grows in 5-14 days
 Photo...

Mycobacterium marinum
 Disease: Tender, red or blue/red subcutaneous nodules
develop following trauma
 Biopsy (skin) spe...

Mycobacterium szulgai
 Grows at 37 ˚C in 12 - 25 days
 Scotochromogen at 37˚C / Photochromogen at 25˚C
 Only AFB that h...

Mycobacterium xenopi
 Optimum temp 42˚C,
 Capable of growing in hot water systems
 Grows in 14 - 28 days in culture
 S...

M. avium-intracellulare complex
 M avium & M intracellulare most common but complex
includes eight species of environment...

M. avium-intracellulare complex
 Opportunistic infection
High organism load in intestine, liver, spleen and bone marrow
...

M avium-complex in lymph node tissue
(Kinyoun stain)
Granulomatous inflammation
lung tissue (H&E stain)
Bowel -Lamina prop...

M. avium complex clinical correlation
 HIV/AIDS
 Disseminated infection in end stage AIDS disease
 Nonspecific low grad...

 Low virulence, found in nature
 Cause: Infections in soft tissue, venous catheter sites
and shunts @ 20 species – 5 spe...

Miscellaneous
 M. gordonae –
 Rare! Cause of Infection
 Scotochromogen
 Major laboratory water contaminant in cultures...

Miscellaneous pathogenic species
 Mycobacterium haemophilum
 Requires addition of hemoglobin or hemin to
culture media f...

Mycobacterium leprae
 Leprosy – Hansen’s disease
 Begins with anesthetic skin lesions,
peripheral neuropathy with nerve ...

Tuberculoid leprosy
Lapromatous leprosy
Skin biopsy - AFB seen in nerve fiber
AFB in “cigar packets”

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