T-SPOT.TB

A NEW WAY TO DIAGNOSE

LATENT TUBERCULOSIS INFECTION (LTBI)

Bui Diem Khue

Content

Active and Latent tuberculosis infection

Diagnosis of infection

Tuberculin skin test (TST) vs. Interferon-Gamma

Release Assays (IGRAs)

Principles of T-SPOT®.TB test (1 type of IGRAs)

Advantages and disadvantages of IGRAs

Tuberculosis Infection and

Tuberculosis Disease

Tuberculosis infection (latent TB

infection)

Tuberculosis disease (active disease)

-Occurs when people carry M. tuberculosis

bacilli in their body

-Bacteria are being controlled by the

infected person's immune system and so

are still in small numbers.

-No infectious

-No TB symptoms

-Positive TST or IGRA result

-Chest radiograph normal

-If done, respiratory specimens are smear

and culture negative

-Occurs when the bacterial load is

increased and overcomes the body's

immune defence

-May be infectious

-Have TB symptoms

-TST or IGRA result usually positive

-Chest radiograph is usually abnormal. (However, may be normal in persons with advanced

immunosuppression or extrapulmonary disease.)

-Respiratory specimens are usually (but not

always) smear or culture positive. (However, may

be negative in persons with extrapulmonary disease or minimal or

early pulmonary disease.)

The number of people with active TB at a given time is just

the tip of the iceberg, as many more are infected with TB

and are therefore at a risk of developing the disease.

Most of the cases of active disease occur through the

conversion of LTBI to active disease.

tuberculosis control and elimination strategies must aim

at diminishing the incidence and prevalence of latent

infection.

Diagnostics for

Tuberculosis: Global

Demand and Market

Potential/TDR, FIND

SA. WHO 2006: p.

21.

Diagnosis of infections (in vitro)

2 main routes

Direct detection of pathogen

Detect pathogen

itself

(e.g. Microscopy,

Culture)

Detect genome of pathogen

(e.g. PCR, In-situ

hybridisation)

Detect an immune

response to the pathogen

Detect antibodies to pathogen

(e.g. ELISA)

If the immune system recognises antigens

from the pathogen, the person must have been

infected by the pathogen.

T-cell responses to pathogens

Immune response = antibodies + T cells

•T cells control intracellular pathogens

•Many diseases where antibodies don’t work.

•T cells are key in these diseases

(e.g. HIV, Cancer, Allergy, Autoimmune

disease, HPV, Hepatitis)

Interferon-Gamma Release Assays (IGRAs)

Tuberculin skin test (TST) (in vivo)

TST is administered by injection

Tuberculin is made from proteins derived from inactive tubercle

bacilli

Most people who have TB infection will have a reaction at

injection site

Relates to delayed hypersensitivity reaction (delayed

hypersensitivity T cells)

Tuberculin skin test (TST)

One of the major drawbacks of the Tuberculin Skin

Test is the cross reaction that it has with BCG

vaccination.

Why ?

The Purified Protein Derivative that is injected in the skin test is

a crude mixture of around 200 peptides extracted from dead

MTB cells. Many of these proteins have common epitopes to

BCG (and environmental mycobacteria) so the skin test will

cross react with these.

How do the IGRAs

solve this drawback?

How do IGRAs work?

IGRAs measure a person’s immune reactivity to M.tuberculosis.

White blood cells from most persons that have been infected

with M. tuberculosis will release interferon-gamma (IFN-g)

when mixed with antigens derived from M. tuberculosis.

Picture from: http://www.stanford.edu/class/humbio103/ParaSites2006/TB_Diagnosis/quantiferon.gif

Types of IGRAs

• QuantiFERON®-TB Gold (QFT-G)

– CDC guidelines published in 2005

• QuantiFERON®-TB Gold In-Tube (QFT-GIT)

– Approved 10/2007

• T-Spot®.TB test (T-SPOT)

– Type of ELISpot assay

– Approved 7/2008

T-SPOT®.TB test

TB Skin Test Vs T-SPOT®.TB

The accuracy of the TB skin test varies and can be

affected by a previous BCG vaccination, a

weakened immune system and by other illnesses or

medical treatments.

Both tests identify LTBI by using the body's immune

response to TB proteins, whether the person is

showing signs and symptoms of TB disease or not.

TB Skin Test T-SPOT®.TB

False Positives

•BCG vaccination

•Environmental mycobacterium

False Negatives

•Skin reaction is a mixed immune

response (problem in immunosuppressed

patients)

•Often negative in active disease (75-

90% sensitivity, lower in

immunosuppressed).

Very specific:

•Specificity approaching 100%

•Not affected by BCG vaccination

•Does not cross-react with common

environmental mycobacterium

Very sensitive:

•Sensitivity ~95%

•Identifies infected subjects missed by TST

•Immunosuppression has little effect

•Performance maintained in infants

•High sensitivity in active TB, including

extrapulmonary TB

T-SPOT®.TB test bases on

Effector T cells

Interferon-gamma

ESAT-6 and CFP 10

Memory Cells vs. Effector Cells

Memory Cells Effector Cells

- Carry the body's long-term immunity or

'memory' of an infection-causing

pathogen

- Are present after any infection

the presence of memory T cells would

only indicate that someone had been

infected with the pathogen at some point

in the past

-Fight the pathogen directly and come

in various forms, designed to kill

different pathogens in different ways.

- Most are short-lived and die off when

the pathogen is cleared from the body.

the presence of effector T cells

indicates an ongoing infection.

Interferon gamma

Released by T cells

Fights the infecting organism which has been

recognized as foreign.

Specific epitopes on the infecting organism have to

be recognized by the T cells to initiate this release.

the T-SPOT.TB test uses very specific antigens

(ESAT-6 and CFP 10) to stimulate the effector T cells.

Picture from: http://pathhsw5m54.ucsf.edu/case32/images32/tbimmuno1.gif

ESAT-6 and CFP 10

BCG vaccine (and most environmental mycobacteria) lose an area from

M. bovis, which is known as the Region of Difference 1 (RD1). Nine

proteins are made by this genomic area including ESAT-6 and CFP 10.

What are the advantages of IGRAs?

Requires single patient visit to conduct test

Results can be available in 24 hours

Very few false negative results (sensitivity ~95%)

Improving reliable detection of truly infected individuals

No patient exclusions

Can be used in HIV, very young children,anti-TNF α, transplant, renal

dialysis, malnourished and other immunocompromised patient groups,

as well as in pregnancy

Virtually eliminates potentially toxic chemoprophylaxis due to

false positive results

Unaffected by BCG vaccination and common non-tuberculous

mycobacteria

What are the disadvantages and

limitations of IGRAs?

Blood samples must be processed within 8-30 hours after collection while white blood cells are still viable.

Errors in running and interpreting test can decrease accuracy

Limited data on its use in certain populations

Children younger than 5 years of age

Persons recently exposed to M. tuberculosis;

Immunocompromised persons; and

Serial testing.

Tests may be expensive.

Limited data on the use of IGRAs to predict who will progress to TB disease in the future.

References

Benh hoc lao – Y Ha Noi

www.oxfordimmunotec.com

www.cdc.gov/tb

And some other websites

THANKS FOR YOUR ATTENTION !