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Mycobacteria Flashcards

(51 cards)

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1
Q

Reading TSTs

A

Reading TSTs
 5 mm is classified as positive in patients with: – HIV-positive – Recent contacts – fibrotic changes on chest radiograph consistent with old healed TB – organ transplants and other immunosuppression  10 mm is classified as positive in – Recent arrivals from high-prevalence countries – Injection drug users – Residents and employees of high-risk congregate settings – Mycobacteriology laboratory personnel – Persons with clinical conditions that place them at high risk – Children <4 years of age, or children and adolescents exposed to adults in high-risk categories  15 mm is classified as positive in – Persons with no known risk factors for TB  Targeted skin testing programs should only be conducted among high-risk groups

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2
Q

Characteristics of Mycobacteria
aerobic/anaerobic?

spore-forming?

related to?

hydrophilic/hydrophobic cell wall?

M. _____ and M. _____ are obligate human pathogens, others are environmental and zoonotic opportunist

A

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 opportunist

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3
Q

IGRAs

A

IGRAs
 Interferon-gamma release assays – Incubate patient lymphocytes with TB antigens and detect release of IFN- as a way of measuring exposure.  Measured by ELISA (Quantiferon) or by in-situ staining and counting cells (T SPOT-TB)

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4
Q

Mycobacterial Specimen Processing

Objectives and Procedure?

A

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
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5
Q

Scoring the AFB Smear

A

Scoring the AFB Smear

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6
Q
A
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7
Q

Mycobacteria Media?

A

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
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8
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A
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9
Q

The Runyon groups

A

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
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10
Q

Molecular Amplification
Detection of mycobacterial DNA or RNA

A

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
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11
Q

MTB Cultures: Rapid Broth Methods

A

Cultures: Rapid Broth Methods
Systems

  • MGIT fluorometric system, detects O2 consumption
  • Organon-Teknika & Bactec nonradiometric systems
  • Both detect CO2 production

Clinical Properties

  • Typically detect 66% of M. tb in 2 weeks, 90% in 4 weeks

Current practice is to use both rapid broth and solid media for all cultures

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12
Q

MTB Cultures: Incubation and Reading

A

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

Continuous-monitoring systems

  • MGIT and BacT/Alert
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13
Q

Niacin/nitrate Tests

A

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
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14
Q

Molecular Identification

PCR target?

Probes available?

TAT?

A

Molecular Identification
Accuprobe by Genprobe (now Hologic) Accuprobe

16s rRNA probe, chemiluminescent readout

Probes available for:

  • M. tuberculosis complex
  • M. avium complex
  • M. kansasii
  • M. gordonae

Same-day results from positive broths or colonies

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15
Q

DNA Sequencing for Mycobacterial Identification

A

DNA Sequencing for Mycobacterial Identification
Targets

  • 16S rRNA gene
  • Hsp 65
  • rpo B

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

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16
Q

MALDI-TOF

A

MALDI-TOF – The Future is Now!
Matrix-Assisted LaserDesorption/Ionization Timeof-Flight Mass Spectroscopy.

Analyzes high-copy proteins in the bacterial cell; mostly ribosomal proteins.

Sample is spotted onto plate, mixed with matrix, then fed into the instrument.

Low reagent cost (pennies), extremely fast ID (minutes).

Unlike for most bacteria, a fairly elaborate off-plate extraction is required for mycobacteria – tough targets!

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17
Q

Choice of MTB Identification Methods
Biochemicals

  • Molecular Probes
  • Sequencing
  • MALDI-TOF
A

Choice of Identification Methods
Biochemicals

  • In the developed world, these are mostly confirmatory and secondline methods – slow

Molecular Probes

  • First-line in relatively smallvolume labs, low capital cost, fairly simple methods
  • same-day

Sequencing

  • Used for final species ID of difficult strains, and in many academic medical centers.

MALDI-TOF

  • In development, not yet standardized or FDA-approved, but likely to become dominant
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18
Q

Principles of Susceptibility Testing
resistance in M. tuberculosis

A

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.

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19
Q

Proportion Method of MTB Susceptibility Testing

A

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
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20
Q

MTB Bactec Method

A

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
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21
Q

Isoniazid Resistance Genes

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A

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

22
Q

Other Drug Resistance Genes

Rifampin resistance

Pyrazinamide resistance

Streptomycin resistance

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A

Other Drug Resistance Genes

Rifampin resistance

  • rpoB, the b 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
23
Q

The GeneXpert TB: Rapid Detection, Resistance Screening

The Instrument and Test

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A

The GeneXpert TB: Rapid Detection, Resistance Screening

A tool for world TB control

The Instrument and Test
Cartridge-based integrated molecular system. Detects M. tuberculosis as well as rifampin resistanceconferring mutations

  • three specific primers
  • five unique molecular probes
24
Q

The GeneXpert TB

Workflow

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A

The GeneXpert TB

Workflow
Simple, rapid.

Deployable to limitedresource settings.

Still requires expensive reagents and maintenance. Under WHO-FIND program, a four module GeneXpert platform and linked computer costs about US$17K

With funding from PEPFAR, USAID, UNITAID, and the Bill & Melinda Gates Foundation, the cost per cartridge set at $9.98 from Aug 6, 2012, for the next 10 years.

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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
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Primary Tb
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
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TB histology
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Niacin producing and Nitrate reducing
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.
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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
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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
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Niacin/nitrate?
M. avium complex -- Lab Hints Nonchromogenic, with multiple colony morphotypes on a single plate * smooth opaque & domed * flat & transparent * some strains pigmented Niacin & nitrate (-) M. avium and M. intracellulare difficult to distinguish
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M. kansasii -- Clinical
M. kansasii -- Clinical Resembles TB both clinically and radiographically South & Central US, UK, Europe Prior pulmonary disease a risk factor Often isoniazid resistant
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Nitrate/niacin?
M. kansasii -- Lab Hints Photochromogen -- intense pigment Large, beaded acid-fast rods Nitrate (+), niacin (-)
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Rapid growers
Rapid growers Three major genogroups Environmental organisms, opportunistic/incidental pathogens * Frequently associated with nosocomial and device-related infections: many species. Evolving taxonomy; multiple-target gene sequencing required for full identification. Grow in \<7d on mycobacterial media when subcultured * Many strains grow well on SBA or chocolate agar * Many are arylsulfatase positive
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Treatment of Rapid-Grower Infections
Treatment of Rapid-Grower Infections Don’t typically respond to TB drugs More conventional antibacterial drugs including * Amikacin * Doxycycline * Imipenem * Fluoroquinolones * Trimethoprim/sulfa * Cefoxitin  Clarithromycin There’s a CLSI-approved broth microdilution method for these organisms.
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Rapid growers - 3 major genogroups
Rapid growers - Three major genogroups M. fortuitum group M. chelonae - abscessus group M. smegmatis group
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Rapid growers: M. _____ group Symptoms? 2 examples?
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
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M. fortuitum group Antibiotic-susceptible?
Rapid growers: M. fortuitum group The most antibiotic-susceptible group. – Amikacin (100 percent) – Ciprofloxacin, levofloxacin, and moxifloxacin (100 percent) – Sulfonamides (100 percent) – Imipenem (100 percent) – Linezolid (86 percent) – Cefoxitin (80 percent) – Clarithromycin (80 percent) – Doxycycline (50 percent) – Minocycline (50 percent)
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Rapid growers: M. ____ - ____ group
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 * These species are difficult to distinguish without mutilocus sequencing.
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M. chelonae susceptibilities
M. chelonae susceptibilities – Amikacin (80 percent) – Tobramycin (100 percent) – Clarithromycin (100 percent) – Moxifloxacin (75 percent) – Imipenem (not reproducible) – Linezolid (54 percent) – Clofazimine (25 percent) – Doxycycline (25 percent) – Ciprofloxacin, levofloxacin (20 percent) – Cefoxitin (resistant)
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M. abscessus susceptibilities
M. abscessus susceptibilities Clarithromycin (inducible resistance in most strains) – Clofazimine (90 percent) – Amikacin (90 percent) – Cefoxitin (70 percent) – Imipenem (not reproducible) – Linezolid (23 percent)
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Rapid growers: M. smegmatis group
Rapid growers: M. smegmatis group * Occasional pathogens; pigmented * Arylsulfatase negative
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Other Rapid Growers
Other Rapid Growers M. mucogenicum * Catheter and device-associated infections Others
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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
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Leprosy -- Pathogenesis
Leprosy -- Pathogenesis * Manifestations depend on host response * Cellular response (tuberculoid leprosy) most effective in limiting disease * Reversal reactions related to increasing cellular response
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Leprosy -- Clinical
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
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M. leprae -- Lab Hints
M. leprae -- Lab Hints Not cultivable Diagnosed by tissue pathology * skin biopsies from lesion edges & earlobes * look for AFB with modified Wade-Fite stain
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Leprosy Histopathology - Lepromatous * Cutaneous involvement, as with all forms of leprosy * Large numbers of dermal macrophages parasitized with numerous acid-fast bacilli
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Leprosy Histopathology - Tuberculoid * Skin with large numbers of non-caseating granulomas * Few, rare AFB
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M. gordonae
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
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Unusual Isolation Requirements * M. marinum , M. hemophilum , and M. ulcerans * M. hemophilum * M. genavense
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
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