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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) -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 Tuberculosis disease (active disease) -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 an immune response to the pathogen

Detect pathogen itself (e.g. Microscopy, Culture)

Detect genome of pathogen (e.g. PCR, In-situ hybridisation)

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 dont 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 persons 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

.TB T-SPOT

test

TB Skin Test Vs

.TB T-SPOT

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

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

False Negatives Skin reaction is a mixed immune response (problem in immunosuppressed patients) Often negative in active disease (7590% sensitivity, lower in immunosuppressed).

.TB T-SPOT

test bases on

Effector T cells Interferon-gamma ESAT-6 and CFP 10

Memory Cells vs. Effector Cells


Memory Cells - Carry the body's long-term immunity or 'memory' of an infection-causing pathogen - Are present after any infection Effector Cells -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.

the presence of memory T cells would only indicate that someone had been infected with the pathogen at some point in the past

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 Can be used in HIV, very young children,anti-TNF , transplant, renal dialysis, malnourished and other immunocompromised patient groups, as well as in pregnancy

No patient exclusions

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 !

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