Mycobacterium Flashcards
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
Bactec Method
Broth-based analogue of proportion method
Procedure
– Control bottles: undiluted and 1:100 dilution
– Antibiotic bottles: undiluted
– Incubate and compare growth in antibiotic
bottles with growth in 1:100 control bottle
Requires 1 week vs. 4-6 for plate method
Validated for primary drugs only
Drug Susceptibility – the Future
Genes for resistance are being isolated
Direct or microarray sequencing to detect
resistance mutations
This approach is already widely used in HIV
`Isoniazid Resistance Genes
Most common resistance (9.1% of US
isolates in 1991)
Two gene loci identified in INH resistance
– katG: a catalase/peroxidase, probably
responsible for transforming INH to an active
drug
– inhA: involved in mycolic acid synthesis,
probably a direct target of INH action
Alterations in these 2 genes responsible for
at least 85% of INH resistance
Other Drug Resistance Genes
Rifampin resistance
– rpoB, the Beta subunit of RNA polymerase
alterations in this locus responsible for >95% of RMP resistance
Pyrazinamide resistance
– pncA, pyrazinamidase, cleaves pyrazinamide to pyrazinoic acid
– PZA inhibits a fatty acid synthetase; resistance mutations in this locus as well
Streptomycin resistance
– rpsL, S12 ribosomal protein
– rrs, 16S ribosomal RNA
Mycobacterial Disease by Organism
M. tuberculosis & complex M. avium complex M. kansasii Rapid growers M. leprae M. gordonae Other MOTT of special interest
M. tuberculosis – Primary Tb
Cough +/- sputum/hemoptysis – Pleural chest pain & dyspnea – Systemic symptoms Asymmetric hilar adenopathy – associated consolidation – +/- pleural effusion Untreated, progressive pulmonary & systemic disease – Pleural TB post-primary
M. tuberculosis – Reactivation TB
Risk factors – malnutrition, immunosuppression – ESRD, diabetes, other systemic illness Cough / Fever / Systemic symptoms – Hemoptysis in 1/3 Disease typically localized to upper lobes – apical and posterior segments – infiltration and cavitation
M. tuberculosis –With HIV
TB is a risk factor for progression and death in HIV
HIV is a risk factor for activation of TB
At CD4 counts >500, TB is typical reactivation disease
At low CD4 counts, systemic and atypical
disease common
Extrapulmonary TB
Spinal TB -- Pott’s Disease TB Osteomyelitis Miliary TB -- following bloodborne dissemination Lymphadenitis (usually cervical) GI and peritoneal Meningitis & other CNS
M. tb complex
M. bovis – responsible for as much as 40%
of TB in some areas
– Infects cattle, but also many other animals
– Disease resembles M. tb and is treated similarly
BCG – Used for immunization, may be
recovered incidentally or may cause
infection after use in topical treatment of
carcinoma of the bladder in situ
M. tuberculosis complex – Lab Hints
Slow-growing, rough colonies with serpentine cording
Usually identified by amplification, probe, or HPLC
Niacin producing and Nitrate reducing
– M. bovis is negative for Nitrate and is PZA resistant
– M. africanum is a bovis subspecies; M. microti is an animal pathogen. Both nitrate negative.
Drug resistance varies widely with geography and prior therapy
M. avium complex – Immunocompetent
Pulmonary disease primarily, in patients with underlying lung disease
multiple, cavitary lesions in smokers with COPD
nodular / bronchiectatic disease in nonsmoking, elderly women with no
underlying lung disease
Lymphadenitis in children
M. avium complex – Immunocompromised
Disseminated disease in HIV-infected – Up to 20% of infections polyclonal Febrile wasting syndrome – Usually with CD4 count <50 – Preventable with azithromycin or rifabutin prophylaxis – Frequent GI symptoms/involvement
M. avium complex – Lab Hints
Nonchromogenic, with multiple colony morphotypes on a single plate – smooth opague & domed – flat & transparent – some strains pigmented Niacin & nitrate (-) M. avium and M. intracellulare difficult to distinguish
M. kansasii – Clinical
Resembles TB both clinically and radiographically South & Central US, UK, Europe Prior pulmonary disease a risk factor Often isoniazid resistant
M. kansasii – Lab Hints
Photochromogen – intense pigment
Large, beaded acid-fast rods
Nitrate (+), niacin (-)
Rapid growers
Three major genogroups
Environmental organisms, ppportunistic/incidental pathogens
– Frequently associated with nosocomial and device-related infections: many species.
Evolving taxonomy; multiple-target gene sequencing required for full identification.
Rapid growers – Lab Hints
– Grow in <7d on mycobacterial media when subcultured
– Many strains grow well on SBA or chocolate agar
– Many are arylsulfatase positive
Rapid growers: M. fortuitum group
M. fortuitum
–Wound infections; furunculosis associated with nail salons and foot baths.
– Osteomyelitis by extension
M. peregrinum and senegalense
Seven more species within two subgroups
Rapid growers: M. chelonae-abscessus group
M. chelonae
– Disseminated cutaneous disease; multiple, chronic, draining nodules in compromised patients
M. abscessus
– Pulmonary infections: nodular/bronciectatic disease similar to MAC; also in CF patients;
– Disseminated cutaneous disease (rarer than with M. chelonae)
M. immunogenum
Rapid growers: M. smegmatis group
Occasional pathogens; pigmented
Arylsulfatase negative
Other Rapid Growers
M. mucogenicum
– Catheter and device-associated infections
Others
Leprosy
A chronic infection with M.
leprae
– ~ 1 million patients in therapy
– 2-3 million patients with permanent neurological damage
Acquired via contact with nasal secretions, probably through respiratory route
Dissemination to cutaneous regions
Leprosy – Pathogenesis
Manifestations depend on host response
Cellular response (tuberculoid leprosy) most effective in limiting disease
Reversal reactions related to increasing cellular response
Leprosy – Clinical
Specific guidelines exist for staging leprosy on the tuberculous-lepromatous axis
– tuberculoid > borderline tuberculoid > midborderline > borderline lepromatous
lepromatous
Peripheral nerve involvement is primary pathology
– Increased in lepromatous forms, and in lepromatous forms undergoing reversal reactions to tuberculoid
M. leprae – Lab Hints
Not cultivable
Diagnosed by tissue pathology
– skin biopsies from lesion edges & earlobes
– look for AFB with modified Wade-Fite stain
M. gordonae
The ‘tap-water chromogen’; has been described as a pathogen but is almost
always a contaminant.
Scotochromogenic and intensely pigmented
The most common contaminant isolated in AFB cultures
Unusual Isolation Requirements
M. marinum, M. hemophilum, and M. ulcerans have growth optima around 30oC -- all cause skin lesions M. hemophilum requires hemin for growth – can also cause systemic disease in compromised hosts M. genavense requires human blood for growth in vitro – systemic infections in HIVinfected patients
