Mycobacterium Flashcards
(51 cards)
Characteristics of Mycobacteria
acid-fast, aerobic, non-spore-forming bacilli
– related to Nocardia, Corynebacterium,
Rhodococcus
slow-growing
– require specialized media
– hydrophobic cell wall
cell-mediated immunity
– serology unreliable
M. tuberculosis and M. leprae are obligate
human pathogens, others are environmental
and zoonotic opportunists
Detecting Exposures
PPD/TST
IGRAs
Problems with the TST
False-Positive
- BCG immunization
- Non-TB mycobacterial infection
False-Negative - Technique - Anergy - Very recent infection - Infants <6 months old -Immunocompromise, including live virus vaccination and overwhelming TB -Latent TB of long standing (decades): booster effect
IGRAs
Interferon-gamma release assays – Incubate patient lymphocytes with TB antigens and detect release of IFN-γ Measured by ELISA or by in-situ staining and counting cells Three wells / tubes – Control – Mitogen – Recombinant MTB antigen May replace the PPD – Single blood draw rather than 2 visits – No subjectivity in interpretation – Expensive, limited data so far Operationally Complex – Requires viable, functional PBMCs; rapid transportation and processing.
Collecting Specimens
Pulmonary TB – multiple (x3) sputa AM sputum is optimal No assessment of specimen quality – morning gastric aspirate in children – bronchoscopy specimens Immunosuppressed patients – atypical presentations; culture blood, urine, stool, bone marrow
Specimen Processing
Objectives -- Sputum (and stool) – eliminate contaminating flora – digest solid material and release mycobacteria – concentrate mycobacteria Procedure – NaOH ± N-acetyl-cysteine – centrifuge – Neutralize, add albumin to stabilize, continue with staining and culture
Staining Methods
Kinyoun or Ziehl-Neelson – conventional microscopy fluorochrome – requires fluorescent microscope – allows more rapid screening – AFB appear as golden fluorescent rods semiquantitate by counting bacilli/hpf
AFB Stain – Clinical Characteristics
TAT: usually done daily Sensitivity in pulmonary TB – 20-60% sensitivity (per specimen) – ~90% for 3 or more sputa Specificity – ~90% in US populations – higher in high-incidence areas – depends primarily on incidence of non-TB disease
Molecular Amplification
Detection of mycobacterial DNA or RNA
– PCR & TMA
Clinical properties:
– TAT: daily or a few times/week
– analytically: 10-100X more sensitive than smear
– clinically: ~80-90% sensitivity (per specimen)
– Provides species identification of M. tb only
– false + from contamination or therapy
Polymerase Chain Reaction (PCR)
- Target DNA + Primer oligonucleotides (present in excess)
- Split DNA strands (95oC 5 min), then allow primers to bind (40-70oC)
- DNA polymerase extends the primers (40-80oC) to produce two new double-stranded molecules
- Repeat the split-bind-extend cycle
- This ‘short product’ amplifies exponentially in subsequent split-bind-extend cycles, driven by the temperature changes in a ‘thermal cycler’.
Transcription-Mediated Amplification (TMA)
- Target DNA OR RNA + Primer oligonucleotides (in excess, contains RNA pol site)
- Reverse transcriptase extends primer, making DNA copy (from either RNA or DNA template)
- RT also replaces RNA template with DNA
- RNA polymerase uses the new binding site to make 10-1000 RNA molecules that can feed back into reaction
Molecular Tests for TB
Gen-Probe AMTD – Now approved for smear-positive and smear-negative specimens – rRNA target Amplicor M. tuberculosis assay – Approved for smear-positives – DNA target Other systems in development Clinically similar, choice depends on technical, economic, operational details
Cultures: Solid Media
Media types
– Egg-based: Lowenstein-Jensen (L-J)
& derivatives
– Synthetic: Middlebrook 7H10-11 plates (and analogous broths)
Clinical Properties
– Detect 66% of M. tb in 4 weeks, 90% in 6 weeks
Cultures: Rapid Broth Methods
Systems
– Bactec radiometric (460) system
– Organon-Teknika & Bactec nonradiometric
systems
– All detect CO2 production
– MGIT fluorometric system, detects O2 consumption
Clinical Properties
– Radiometric Bactec detects 66% of M. tb in 2 weeks, 90% in 4 weeks
– Newer systems similar
Current practice is to use both rapid broth and solid media for all cultures
Cultures: Incubation and Reading
5-10% CO2 stimulates primary growth Solid media – place in gas-permeable bags – read 2x/week to 4 weeks, then weekly to 8 – 37oC except for skin cultures at 30-32 – hemin, blood, or SBA for suspected M. hemophilum BACTEC – read 2-3x/week x 3 weeks, weekly to 8 – GI >10 is positive Continuous-monitoring systems – MGIT and BacT/Alert Current practice is to use both rapid broth and solid media for all cultures
Biochemical Identification
Many biochemical tests classically used for
identification of mycobacteria
– Labor-intensive, 4-6 weeks to ID
Molecular and HPLC now used for most clinical purposes
Growth characteristics (pigmentation) still used as a primary screening/grouping method
Niacin production & nitrate reduction used for TB speciation
Growth & Pigmentation
The Runyon groups (M. tb NOT counted)
– I. Photochromogenic: M. kansasii
– II. Scotochromogens (always pigmented): M. gordonae
– III. Nonchromogens: M. avium complex
– IV. Rapid growers: M. chelonae & fortuitum
complexes
Niacin/nitrate Tests
Used to confirm identification of M.
tuberculosis made by other methods
M. tb complex also contains M. bovis, BCG,
and M. africanum
M. tuberculosis is niacin/nitrate positive; M.
bovis and M. africanum are negative
All produce a catalase that’s labile at 68oC;
most other mycobacteria produce heatstable
catalase
Molecular Identification
Accuprobe by Genprobe 16s rRNA probe, chemiluminescent readout Probes available for: – M. tuberculosis complex – M. avium complex – M. kansasii – M. gordonae Same-day results
Identification of Mycobacteria by HPLC
Mycolic acids extracted, derivatized, and run on HPLC
Patterns species-specific
Same-day, but needs more growth than probes
DNA Sequencing for Mycobacterial Identification
Targets
– 16S rRNA gene
–Hsp65
– rpoB
Microseq system is FDA-approved for 16S.
No single target is sufficient to identify all
mycobacteria to the species level.
Expensive, labor-intensive, but likely to
expand as methods improve.
Choice of Identification Methods
Biochemicals
– In the developed world, these are mostly confirmatory and second-line methods – slow
Molecular Probes
– First-line in relatively small-volume labs, low capital cost, fairly simple methods – same-day
HPLC
– Method of choice for high-volume labs; more demanding but nearly as fast and much less expensive than probes
New Molecular Approaches
– In development, not standardized, but likely to take over in the next decade or so
Principles of Susceptibility Testing
resistance in M. tuberculosis – no transmissible/plasmid-mediated resistance – spontaneous mutation (1 in 105-107) and selection – Resistance mutations have been characterized for the primary drugs slow-growing organism criteria – >1% resistance has been set as the threshold susceptibility testing in MOTT unstandardized except for rapidgrowers
Proportion Method
Inoculate media with defined # of M. tb cfu
Control media: undiluted and diluted 1:100
Antibiotic media: undiluted
Compare control 1:100 with antibiotic colony counts
Drugs
– Primary: isoniazid, rifampin, ethambutol, streptomycin, pyrazinamide
– Secondary: quinolones, ethionamide, PAS, cycloserine, others
