Pathogenesis of Mycobacterioses, especially Tuberculosis

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Pathogenesis of Mycobacterioses, especially
Tuberculosis

• Marcel A. Behr
• marcel.behr_at_mcgill.ca
• www.molepi.mcgill.ca

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Mycobacterial pathogenesis overview

• Lecture 1
• Introduction and definitions
• Epidemiology
• Pathogenesis of TB infection and disease
• Clinical Manifestations
• Treatment and control
• Lecture 2
• Host factors
• Virulence factors
• Immunity and Vaccination

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Mycobacteria Background

• Most Mycobacteria non-pathogenic
• soil water organisms
• more named each year (sampling)
• Pathogenic Mycobacteria
• Can be environmental-humans accidental host
• E.g. Mycobacterium avium
• Can be obigate pathogens with no known
  environmental reservoir
• E.g. Mycobacterium leprae

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Mycobacteria are not limited to the tropics
Leprosy in Norway, 1851-1920 Rates low in cities
where TB rates high
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Pathogenic Mycobacteria Properties

• Most slow growing, doubling on order of day (c.f.
  E coli 30 min.)
• Gram-positive, but dont gram stain
• Mycolic acid cell wall
• acid fast staining
• Wall protects bacteria from environment,
  molecular biology
• Wall immunostimulatory Freunds

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Non-tubercuous Mycobacteria pathogenic to humans

• Mycobacterium avium sp. avium
• Avian tuberculosis
• In humans
• disease in AIDS
• Chronic pneumonia
• Lymph node disease in children
• M. avium sp. paratuberculosis
• Inflammatory bowel disease in ruminants, primates
  (Johnes disease)
• In humans implicated by some in Crohns
• M. leprae
• The agent of leprosy

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Non-tuberculous mycobacterial infections in
Swedish children
Rates increasing where TB gone, BCG stopped
BCG discontinued
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Mycobacterium tuberculosis

• M. tuberculosis complex
• Includes M. tuberculosis, M. bovis, M. caprae, M.
  microti, M. africanum
• Identical by 16s rRNA sequence
• Homology in other genes usually 99 or more
  (thus, one species)
• Agents of tuberculosis (TB) in a variety of
  mammalian species

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Clinical Definitions

• Tuberculous infection is the carrier state, aka
  latent infection
• clinically latent, either because bacteria latent
  or bacterial replication death
• non-infectious, tuberculin positive
• Tuberculosis is diseased state
• actively replicating bacteria
• contagious, culture positive

GET IN STAY IN
GET OUT SPREAD
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Clinical Definitions

• Tuberculous infection is the carrier state, aka
  latent infection
• clinically latent, either because bacteria latent
  or bacterial replication death
• non-infectious, tuberculin positive
• Tuberculosis is diseased state
• actively replicating bacteria
• contagious, culture positive

Bacterial survival
Bacterial pathogenesis
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TB Epidemiology

• Originally from animals, ca. 15,000 B.C.
• With pasteurization, most TB now due to human -
  human Respiratory aerosols
• 1/3 worlds population carry M. tuberculosis
• not infectious
• 8 million cases / year
• contagious
• 20 million active TB cases
• 2 million deaths / year (tied with AIDS 1)

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TB and AIDS Epidemiology

• Infection rates often high to both
• Most notably sub-Saharan Africa, South-East Asia
• M. tuberculosis accelerates progression of HIV to
  AIDS
• TB cause of death in about 25 AIDS
• HIV infection is single strongest risk factor for
  progression of TB infection to TB disease
• Evil synergy where resources most limited

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Pathogenesis of Mycobacterial infections

• Best studied for M. tuberculosis
• Initial insult likely function of
• which Mycobacterial species
• dose and site of infection (gut / lungs)
• immune status of host
• age
• constitutive immunity (host genetics)
• acquired immunity (naïve vs. primed, HIV,
  nutrition, etc.)

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Outcomes after TB exposure

• Exposure, no infection
• ? Frequency (2/3 TB contacts dont test )
• ? Bacteria dead at contact
• Exposure, infection, never disease
• 90 wont get disease if immune status OK
• 10x more common than disease
• Exposure, infection, disease, /- death
• Variable latent period
• 5 in 5 years, 5 in rest of life

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Overview of TB
90 no sequellae
Primary infection (tuberculin positive)
5 primary TB (within 2 years)
GET IN
5 reactivation (later in life)
GET OUT
STAY IN
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Pathogenesis of TB - 1

• Aerosol travels to alveoli of lungs
• M. tuberculosis engulfed in alveolar macrophages
• if activated (e.g. healthy adult), host may clear
  bacteria, or at least contain infection
• tuberculin positive
• if unactivated (e.g. infant), bacteria survive
  and replicate in macrophages
• attract more cells (PMNs, T-cells), damage
  tissue, and form granulomatous tubercle

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Pathogenesis of TB - 2

• Tubercle can remain silent (abN X-ray)
• Granulomatous response may fail to contain
  bacteria
• Immediate lymphatic / hematogenous spread
  (primary TB)
• Granulomatous response can result in tissue
  damage
• N.B. TNF - alpha production
• Granuloma may remain wall off for years to
  decades, then allow release of viable bacteria
  reactivation

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Pathogenesis of TB - 3

• Infection starts in lungs, but may spread to
  anywhere in body (15 cases)
• Extrapulmonary TB generally not contagious,
  therefore dead-end for bacteria
• Primary TB, Post-primary TB, Reactivation
  TB as pathophysiological concepts
• Clinically not always evident
• For treatment, irrelevant

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Clinical Manifestations of TB

• General
• fever, weight loss, weakness, consumption
• result from inflammatory response
• Organ specific
• pneumonia cough, sputum /- blood
• scrofula swollen lymph nodes
• genitourinary sterile pyuria
• bone back pain, fracture, hump-back
• meningitis headache, obtundation
• miliary TB no obvious source

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Chest radiograph in pulmonary TB
Abnormalities often seen in upper lobe or
superior segments of lower lobe May have unusual
appearance in HIV-positive persons Cannot
confirm diagnosis of TB but useful screen,
because this is most contagious form
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Strategies for TB control

• Preventive vaccination
• Immunize prior to exposure with goal to
• Prevent establishment of infection
• Prevent infection from developing to disease
• Immunize post exposure to
• Prevent established infection from progressing
• Antibiotic treatment
• Can target active TB or latent infection

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Antibiotic treatment of TB

• TB disease
• Untreated TB disease generally lethal can
  survive years
• Therefore, TB treatment can
• Reduce individual mortality
• Prevent spread of infection
• TB infection
• 10 will get disease
• Therefore, treatment of latent infection can
  prevent progression to active disease

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How to treat active TB1 - diagnosis

• Canada culture, PCR
• Can detect small numbers of bacteria
• This translates into capacity to detect disease
  early
• Developing world microscopy only
• Only positive when large numbers of bacteria
• Many patients have progressed to substantial
  disease burden when first diagnosed
• More complicated to treat (they are sicker)
• Further spread already achieved

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How to treat active TB2 antibiotic therapy

• Diseased individual 105 bacteria / ml sputum x
  1 litre liquified lung
• If spontaneous mutation rate to isoniazid 10-6
  bacteria, INH alone should select for resistance
• Clinical response followed later by relapse with
  drug-resistant TB
• Add rifampin (10-8 bacteria), resistance to both
  unlikely

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Difficulties with treatment of active TB

• Multi-drug treatment required
• Only learn of antibiotic susceptibility after
  about 6 weeks
• Therefore, usually begin with 4 drugs
• Short-course antibiotic regimen is 6 months
• Resistance can result from
• Failure to take all medications
• Failure to be provided with steady supply of
  medications
• Resistant forms can of course spread

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How to treat latent TB infection1 - diagnosis

• Canada tuberculin skin test
• Detects immune response (DTH) to bacteria
• Limitations
• Can have weak false positive response due to
  infection with other Mycobacteria
• Can be transiently positive after BCG vaccination
• Can be negative in persons with weakened immune
  system (i.e. those at higher risk of progressing)
• Developing world who has time for this?
• 1/3 of all people positive
• Can we realistically treat 2 billion people?

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How to treat latent TB infection2 antibiotic
therapy

• Infected individual should have small bacterial
  burden
• Unclear how many actively replicating
• Treatment usually consists of isoniazid alone for
  9 months
• No clinical response to follow
• Easiest to target those most likely to progress
  to active TB
• Recent infection (5 in 5 years)
• Immune compromised (HIV, transplant)

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Difficulties with treatment of latent TB infection

• Hard to ensure adherence in someone who
  essentially feels well
• Never get antibiotic susceptibility
• Therefore, hope INH sensitive
• Resistant forms require antibiotics of unproven
  efficacy in this setting

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Summary

• Epidemiology
• 1 infectious disease
• Pathogenesis
• Establishment of infection
• Progression to disease
• Clinical Manifestations
• Pulmonary TB contagious, good for the bacteria
  but bad for the host
• Treatment and control
• Antiobiotics alone not doing it

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Mycobacterial pathogenesis overview

• Lecture 1
• Introduction and definitions
• Epidemiology
• Pathogenesis of TB infection and disease
• Clinical Manifestations
• Treatment and control
• Lecture 2
• Textbook issues
• Host factors
• Virulence factors
• Immunity and Vaccination

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Textbook

• p295 TB is more contagious in infants and
  children than it is in adults
• 32 of children who rode bus were infected
• Infection rate normal
• 51 of 81 infected children developed active TB
• Progression to active disease in gt ½
• Does this reflect that TB is more contagious or
  more likely to cause active disease in children?
• p305 The demise of the Nramp1 hypothesis
• Still alive
• p306 M. microti is a BCG alternative.
  Ubiquitous environmental Mycobacteria that
  interfere with immune response include M. avium,
  M. terrae., M. scrofulaceum

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Overview of TB
90 no sequellae
WHY?
Primary infection (tuberculin positive)
5 primary TB (within 2 years)
GET IN
5 reactivation (later in life)
GET OUT
STAY IN
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TB pathogenesis two genomes do battle
M. tuberculosis
H. sapiens
Virulent vs. attenuated
Susceptible vs. resistant
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90
TB infection (2 billion people)
Active TB (2-3 million deaths / year)
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TB pathogenesis three genomes do battle
M. tuberculosis
H. sapiens
Virulent vs. attenuated
Susceptible vs. resistant
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TB infection (2 billion people)
Active TB (2-3 million deaths / year)
HIV
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What is normal outcome of interaction?

• Host 90 of infections do not result in disease
• Therefore, disease is anomaly
• Bacteria infection without disease is dead end
• Therefore, disease is necessity
• This does not set up well for compromise
• Best case scenario, have disease in minority

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Determining host factors

• Classic epidemiologic studies
• Genetic epidemiologic studies
• Candidate gene approach
• Agnostic search (whole genome scan)
• Rare genetic diseases
• Animal models
• Acquired immunedeficiency
• Natural experiments, e.g. HIV

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Determining host factors

• Classic epidemiologic studies
• Genetic epidemiologic studies
• Candidate gene approach
• Agnostic search (whole genome scan)
• Rare genetic diseases
• Animal models
• Acquired immunedeficiency
• Natural experiments, e.g. HIV

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Host factors Epidemiology

• Extremes of age (infants, elderly)
• More likely to develop active TB
• Male sex
• Men outnumber women throughout world
• HIV infection
• Stongest risk factor in causing reactivation of
  previous latent infection
• Also accelerates rate from new infection to rapid
  primary disease
• Smoking, Drinking, Drugs
• Appear to increase risk of TB, but marker of
  exposure or risk factor?

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Host factors Genetic Epi

• Candidate genes from animal models
• Nramp1 a player in the Gambia, Northern Alberta
• Stronger effect in primary disease
• Vitamin D receptor mutations
• Mannose binding protein
• Unidentified gene on X chromosome

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Host factors Rare genetic defects

• Occasional families observed with rapidly
  disseminating disease
• Can be disease due to BCG vaccine
• Can include other non-pathogenic Mycobacteria
• Also, occasionally Listeria, Salmonella
• Lessions
• Gamma-interferon receptor
• IL-12
• IL-12 receptor

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Bacterial factors

• Bacterial factors
• Infection
• Persistence
• Provocation of disease state to continue
  transmission cycle

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Problem with defining TB virulence

• M. tuberculosis more virulent for humans than
  cows
• M. bovis more virulent for cows than humans
• Which one is more virulent?
• Both of these more virulent for humans than other
  Mycobacteria
• What makes M. tuberculosis complex more virulent?

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Problems with studying TB virulence

• Biohazard
• need proper facilities
• Slow generation time
• need patience
• Targeted gene disruption very difficult
• homologous recombination works poorly

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Difficulties in studying TB virulence animal
models

• Human studies unethical
• Monkeys closest disease to humans
• expensive, also ethics issues
• Rabbits close in pathology
• get cavitary lung lesions
• Mice cheapest, easier
• specific immune defects and reagents
• help understand lack of containment of primary
  infection
• Cell culture easiest
• Only one cell, therefore immunologically
  simplified

ease
relevance
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Difficulties in studying TB virulence - 3

• Models help understand specific process, but
  dont necessarily emulate natural pathogenesis
• No animal model for latency
• No animal model for late reactivation
• No animal model for effect of BCG vaccination on
  exposure much later in life

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Key Steps in TB Pathogenesis

• Bacteria get into the cell
• Bacterial survival in phagocytes
• Avoidance of activated macrophage response
• Bacteria thrive in phagocytes
• How to make the macrophage your home
• Bacteria apparently wait it out
• Latency if inactive, then inert?
• Tissue destruction
• Whos fault is this?

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Entry and survival in Phagocytes Theories

• Alternate entry into macrophage
• Multiple methods in suggest that the bacteria
  wants to go there
• Not so much phagocytosis as invasion
• Impaired acidification of phagosome
• impaired action of lysosomal enzymes at higher pH
  
• Delayed fusion of phagosome-lysosome
• Neutralization of oxygen radicals
• catalase, phenolic glycolipids
• Escape from phagosome into cytoplasm?

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Avoidance of activated macrophage response

• Lipoarabinomannan (cell wall glycolipid)
• inhibits T cell proliferation
• blocks IFN-gamma activation in macrophage cell
  lines
• Ag85 blocks fibronectin (which stimulates
  T-cells)
• Other factors?

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Latency

• Big debate
• Con
• no evidence this exists
• bad lesson from HIV latency paradigm
• Pro
• most bacteria move slow in soil
• Mycobacteria have all the genetic apparatus for
  anaerobic life, genetically similar to spore
  forming organisms
• vegetative growth perhaps the abnormal condition

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Latency - advantages

• Great way to see the world
• hedge against disasters
• Metabolically inert may mean antigenically inert
• hide from immune system in phagocyte
• Metabolically inert likely means not susceptible
  to antibiotic therapy
• treatment of latent infection 60-90 effective

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Tissue destruction

• Mycobacteria immunostimulatory
• host response kills the patient
• host response also renders the patient
  contagious, liberating the bacteria
• Mycobacteria stimulate TNF-alpha, which does the
  damage lung becomes an atomizer
• Cant explain species specificity unless can
  explain degree of TNF stimulation
• Certain antigens can evoke DTH response
• Mycolic acids from cell wall toxic when injected
  into animals

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Genomic analysis of virulent and attenuated
strains

• BCG vaccines are lab attenuated
• In vitro growth for 5 decades
• Reduced virulence with in vitro passage
• Circulating isolates in S.F. have enough to cause
  disease
• Compare the genomes of circulating clones and BCG
  vaccines, by hybridizing to Genechip

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Affymetrix genechip
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Affymetrix genechip detail
20 tiles for one gene
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A Probe Set (DNA Chip)
Perfect Match
Mismatch
AGGCTATCGCACTCCAGTGG
Perfect Match
AGGCTATCGTACTCCAGTGG
Mismatch
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Genechip properties

• With 100,000 probes on chip
• Can have 20 probes per gene
• Permits one to scan for genomic deletions smaller
  than complete genes
• Rapidly can catalogue small genomic deletions
• Not single nucleotide polymorphisms,
  rearrangements, duplications

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Lessons from comparative genomics

• BCG vaccines have lost more genome in 5 decades
  than circulating clones in ? 5 centuries
• BCG vaccines are missing regulatory genes and
  antigens
• Circulating clones are missing insertion elements
  and phages
• Could loss of antigens be responsible for loss of
  virulence?

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Targeted analysis of RD1

• One region (RD1) absent from all BCG strains,
  present in all virulent strains
• 9.5 kB, 9 open reading frames, none with known
  function
• 2 antigenic proteins
• Goal
• Delete RD1 from H37Rv by allelic exchange
• Look for virulence phenotype
• If attenuated, explore why

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Growth in macrophages
Bacteria / well (x 10(3))
Alamar blue reducing activity ( of day 0)
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Growth in C57BL6 mice
TB TB?RD1 BCG
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RD1 analysis lessons

• Deletion clearly associated with attenuation of
  virulence
• Region has 2 best candidates for immunodiagnosis
  parts of tuberculin
• Antigens appear to be virulence factors in M.
  tuberculosis
• During growth in vitro, the organismn
  preferentially drops antigens, perhaps because
  they are no longer needed (and expensive to
  maintain)
• in vivo, organism conserves antigens

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TB immunology

• Important to understand pathogenesis
• Role of host
• Genetics, e.g. Nramp1
• Environment, e.g. previous Mycobacterial
  exposures
• Role of bacteria, e.g. antigens
• Critical to developing new vaccines

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Use of immunology to develop new vaccine

• Live, attenuated vaccine based on premise that
  one wants to mimic natural infection
• Disease lite
• Does natural infection with TB produce memory?
• Latent infection reduced TB rates
• Active TB treated reinfected and disease again
• Suggestion of concomitant immunity
• What type of immune response is desired?

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Properties of vaccine needed

• Live vaccines more protective than killed
• New vaccine either should be
• Attenuated strain
• Antigen provided in live vector (e.g. adenovirus)
• Some clever immunologist figures it out
• Old vaccine preferentially missing antigens
• Effect of vaccination will vary with
• Host genetics
• Host age at vaccination
• Host environemnt other Th1/Th2 stimuli
  encountered, e.g. helminths, HIV, malnutrition

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TB pathogenesis - summary

• M. tuberculosis is a bacterium
• H. sapiens is a host
• TB is the interface of the two
• Conventionally, each side studied in depth
  holding other variable steady
• Previously pathology due to host response
• bacterial factors gaining increasing attention
• Future approaches to understanding require
  studying interaction of two