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Transmission and PathogenesisTransmission and Pathogenesisof Tuberculosisof Tuberculosis

John Bernardo, M.D.John Bernardo, M.D.Pulmonary CenterPulmonary Center

Boston University School of MedicineBoston University School of MedicineMassachusetts Department of Public HealthMassachusetts Department of Public Health

Division of TB Prevention and ControlDivision of TB Prevention and Control

2011

StevensonB t

Vivian Leigh

Brontes

Keats

How does one get TB???How does one get TB???

Transmission and Pathogenesis: Factors

• Source/environment

• Organism

• Host

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Transmission/Infection: SourceTransmission/Infection: Source

•• Respiratory route Respiratory route –– aerosol (most common)aerosol (most common)–– Usually unsuspected, “infectious” TBUsually unsuspected, “infectious” TB

•• IngestionIngestion–– Unpasteurized milkUnpasteurized milk

–– Uncooked meatUncooked meat

•• Solid organ transplantation Solid organ transplantation (Rose, G. Can J Infect Dis (Rose, G. Can J Infect Dis Med Microbiol. 2005)Med Microbiol. 2005)

The Environment

The Organism: Density and Virulence

HEPA Filter, Lobby, Pine Street Inn

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UV Lights

Dormitory, Pine Street Inn

Counselor- bed list- cough log

Bed Number

The Host:Susceptibility

TB pathogenesis TB virulence mechanisms Entry to macrophage Phagosomal failure to acidifyNo phago-lysosomal fusionAvoidance of apoptosis Intracellular replication

Macrophage

Immune subversion: Th2/cortisol

Host response PhagocytosisNO, ROS, Fe deprivationCytokinesApoptosis T cells: CMI & granuloma

Time Course: 8-10 weeks

TNF-, IL-1,IL-6, IL-8, IL-12,IFN-, MIP-1,MIP-1, RANTES,IP-10, MCP-1...

TGF-, IL-10

Lymphocyte

Initial StepsInitial Steps

• APCs present antigens to T-cells complexed in the groove of MHC II (HLA DR, DP, DQ)

• Most DTH responses are elicited through TCR-β– Activation requires additional signaling via CD4/CD8

molecules and co-stimulation - not physically associated with TCR

• For memory or effector T-cell activation, expression of inducible co-stimulator (ICOS)–L on APC that interacts with ICOS expressed on T cells is required

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Cytokines and TBCytokines and TB

• Family of secreted proteins that bind immune cells through specific signaling receptors

• IFNγ – key ingredientHigh levels found at rxn sites– High levels found at rxn sites

– Direct injection induces inflammatory rxn

• Also: IL-1, IL-2, IL-3, IL-4, IL-5, IL-6, IL-8, IL-10, IL-12, G-CSF, GM-CSF, PGE2, MCP-1, TGF-β, IFN-β identified in lesions

• TNF-α– Injection also induces inflammation

TH1 TH1 versusversus TH2 TH2 Mutually Exclusive?Mutually Exclusive?

• TH1 – IFN-γ: macrophage activation (DTH)

– IL2, TNF-α

• TH2– IL4, 5, 6: B cell activation (humoral immunity, tolerance)

• Induction depends on DC-generated cytokines:– IL12 TH1

– IL10 TH2

IFNIFN--γγ

• Produced by NK and TH1 cells• Major activator of macrophage-monocyte lineage

cells – Augments phagocytosis, respiratory burst– Upregulates MHCUpregulates MHC

• Upregulates T-cell activation markers (CD69, CD71), IL2Ra (CD25), HLA-DR– Promotes TH1 differentiation

• Suppresses TH2 differentiation– Downregulates TH2 cytokines – IL4, 5

TNFTNF--αα

• Produced by macrophages, TH1 (other cells)

• Induces macrophage, endothelial cell h kichemokines

• Up-regulates vascular endothelial cell adhesion molecules

• Induces inflammatory cytokines (IL1)

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Interleukin 12Interleukin 12

• Produced by APC

• Augments NK/T cell IFN-γ production

• Promotes TH1 cell-differentiation

• Enhances NK and CD8+ T cell cytolytic function

Primary TB in a Child

Containment

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“Latency” of M. tuberculosis

• Environment of granuloma favors altered metabolism:• Low pO2

• Reduced CHO• Reduced CHO• High Fat

• Replication time >>> 20hr.• Loss of acid fast staining properties• Mechanism(s) unknown

• genetic switch?

vascular endothelium

TNFα, IFNγ, …

vascu a e dot e u(chemokines, adhesion molecules)

5mm5mm 10 15mm

15mm

5mm 10mm 15mm

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Tuberculosis Cases by Skin Test ResultsTuberculosis Cases by Skin Test ResultsMassachusetts, 2006Massachusetts, 2006

Not Done13 (5%)

Not Significant27 (10%)

Unknown6 (2%)

Anergic3 (1%)

Significant210 (82%)

MDPH Bureau of Communicable Disease Control, ISISMDPH Bureau of Communicable Disease Control, ISIS (n = 259)(n = 259)

Tuberculin Skin Test in HIVTuberculin Skin Test in HIV

• Insensitive in low-prevalence populations• Reactivity varies with level of immunosuppression

– In early HIV reactivity is maintainedIn early HIV, reactivity is maintained– Smaller or no reaction in advanced HIV

• Cut point is reduced • Positive reaction ( 5mm; U.S. standard) should

raise suspicion for TB infection• Anergy testing generally is unreliable

Tuberculin Skin Test Responsesby CD4 Lymphocyte Count

10

12

14

16

D p

osit

ive

PPD > 10 mm

PPD > 5 mm

PPD > 0 mm

Markowitz, et al. Ann Int Med 1993, 119:185

0 - 199 200 - 399 400 - 599 600+ Total HIV +Total HIV -

CD4/ul

0

2

4

6

8

per

ce n

t P

PD

“Boosting”“Boosting”•• Principle: Principle: Loss of Immune MemoryLoss of Immune Memory

–– Number of sensitized TNumber of sensitized T--lymphocytes declines over lymphocytes declines over time following initial sensitizationtime following initial sensitization

–– A TST administered will:A TST administered will:1 Induce a reduced inflammatory response at site1 Induce a reduced inflammatory response at site1. Induce a reduced inflammatory response at site1. Induce a reduced inflammatory response at site

–– This may be interpreted as a This may be interpreted as a NegativeNegative TST (a TST (a falsefalse negative)negative)

andand

2. Recall prior sensitization 2. Recall prior sensitization –– expanding clone of sensitized expanding clone of sensitized

TT--cells within 3cells within 3--7d7d

–– A subsequent TST will reflect prior sensitizationA subsequent TST will reflect prior sensitization•• Prior sensitization: Prior sensitization: PositivePositive TSTTST

•• Solution: Solution: 22--step testing step testing in selected personsin selected persons

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DTH: Laboratory Correlates

• Lymphocyte proliferation (blast transformation)– H3-thymidine uptake on stimulation w antigen, mitogen

• Mixed lymphocyte reaction (MLR) – thymidine incorporation T-cell reaction to cell surface

antigens

• Cytokine generation– QuantiFERONs

– T Spot TB

InterferonInterferon--gamma Release gamma Release Assays (IGRA)Assays (IGRA)

• in vitro assays for Cell-Mediated Immunity to M. tuberculosis antigens– Utilize whole blood– Measure release of IFN-γ by circulating “effector” T-

l h f ll i i l i i h hlymphocytes following stimulation with PPD, other antigens

• QuantiFERON-TB (PPD): approved by FDA 2001as “… an aid to the diagnosis of TB infection.”

• QFT-Gold (specific antigens): FDA approved 2005• QFT-Gold in-Tube: approved 2007• T Spot-TB: approved 2008

IGRA Principles

Antigen recognition and secretion of IFNγ

Heparinised whole bloodHeparinised whole blood

ESATESAT--66 CFPCFP--1010 MitogenMitogenControlControl

Transfer undiluted whole bloodTransfer undiluted whole bloodinto wells of a culture plateinto wells of a culture plate

and add antigensand add antigens

Culture overnight at 37Culture overnight at 37ooCC

TB infected individuals TB infected individuals respond by secreting IFNrespond by secreting IFN--

Stage 1 Whole Blood CultureStage 1 Whole Blood Culture

QuantiFERONQuantiFERON®®--TB GOLD IT MethodTB GOLD IT Method

NilNilControlControl

ESATESAT--6/CFP6/CFP--1010

ESATESAT--6, CFP6, CFP--10, TB7.710, TB7.7

NilNil MitogenMitogen

*

Harvest Plasma from above Harvest Plasma from above settled cells and incubate settled cells and incubate

120 min in ‘Sandwich’ 120 min in ‘Sandwich’ ELISAELISA

Wash, add Substrate, Wash, add Substrate, incubate 30 minincubate 30 min

then stop reactionthen stop reaction

TMBTMB

COLORCOLOR

Measure OD andMeasure OD anddetermine IFNdetermine IFN-- levelslevels

Stage 2 IFNStage 2 IFN-- ELISAELISA

IFNIFN-- IU/mlIU/ml

OD

45

0n

mO

D 4

50

nm

Standard CurveStandard Curve

* Shaken – or Stirred?

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Species specificity of ESAT-6 & CFP-10

Environmental strains

Antigens

ESAT CFP

M abcessus - -M avium - -M branderi - -M celatum - -M chelonae - -M fortuitum - -M gordonii - -

Tuberculosis complex

Antigens

ESAT CFP

M tuberculosis + +

M africanum + +

M bovis + +

BCG substrain

gothenburg - - M gordoniiM intracellulare - -M kansasii + +M malmoense - -M marinum + +M oenavense - -M scrofulaceum - -M smegmatis - -M szulgai + +M terrae - -M vaccae - -M xenopi - -

g g

moreau - -

tice - -

tokyo - -

danish - -

glaxo - -

montreal - -

pasteur - -

QFTQFT--G inG in--TubeTubeSensitivity for LTBI?Sensitivity for LTBI?

Tested by TST/QFT-G (n=400)

TST-positive (n=40)

Will Develop TB in Lifetime (n=4)

QFT-positive (n=8-10)??

???What about Boosting?

The Answer?

• TBESC Study 19– Study of QFT-G-IT in household contacts

• To follow QFT-neg contacts for 1 yr • To assess acceptance of QFT result by contacts, re. perception

of need for treatment

– 2 national sites2 national sites• SF, Chicago

• Diel, et al.*– 601 household contacts x 103 wk

• 243 (40%) TST+; 66 (11%) QFT-pos• 6 developed TB; all were QFT-pos, declined tx

– BUT: • no indeterminates (in 292 tested w mitogen)• Small numbers

*AJRCCM, 2/08

Preventing TB: Treatment of Latent TB Infection

• Significant TST or IGRA test• Active disease excluded

– History, examination, CXR

id i id bidi i / di i• Consider coincident co-morbidities/medications – As affecting risks/benefits of treatment

• Treatment– Isoniazid (INH) 300mg/d x 9 mos (standard)– Rifampicin 600mg/d x 4 mos

• Reduces risk of disease by >90%

CDC: MMWR 49 (RR06), 6/9/2000

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Treatment of Latent TB Infection:Research Horizon

• New drug regimens– e.g. TBTC Study 26 (9m INH vs 3m INH/RPT 1x/wk)

• New drug developmentFl i l– Fluoroquinolones

– Diarylquinolines– Others …

• Vaccine development: Modulating the Host Response– To prevent infection– To prevent disease in infected persons– To use as adjuvant treatment in persons with disease

2011

Accelerating the Decline: Accelerating the Decline: The OpportunityThe Opportunity

224 Active TB cases

300,000 to 600,000 persons have TB Infection

Massachusetts, 2010

Most TB in Massachusetts Results from reactivation of TB infection (Genotyping: CDC)

ReactivationReactivation

• Latency and active Tuberculosis– A continuum?

• Immune Surveillance versus the mycobacteria

– Evidence for ongoing metabolism by organisms

• Reactivation => Loss of containment• Reactivation => Loss of containment– Risk increases with age

– Usually involves lungs as primary site of disease

– Poorly understood factors• HIV infection

• Steroids

• TNF inhibitors

• Role of IFN-γ and other cytokines?

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Vitamin D3?Vitamin D3?

Heliotherapy (sun therapy)Valley Echo, April, 1927

Common Risk Factors for Increased Likelihood of Progression from Latent Tuberculosis Infection to Active Disease.

Horsburgh CR Jr, Rubin EJ. N Engl J Med 2011;364:1441-1448

TBTC Study 26

• CDC-sponsored study of TLTBI in high-risk persons– Close contacts, recent converters, HIV, >2cm nodule

• 9 mos INH (270 doses; self-administered) vs3 INH + Rif ti k (12 d b DOT)3 mos INH + Rifapentine once a week (12 doses; by DOT)– Safety and efficacy

• At 23 TBTC sites, US, Canada, and overseas– 8,000 subjects enrolled; 2 year follow-up

• Non-inferiority (re-)design

Can we extrapolate findings to other groups?2011

Those who cannot rememberthe past are condemned to repeat it

G. Santayana, 1905

TB Elimination ControlX ?