Download - 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 !