Validation of Interferon Gamma Release Assays (IGRA) using frozen Peripheral Blood Mononuclear Cells (PBMC)

Approximately a third of the world population has been infected with
Mycobacterium tuberculosis, of which the large majority has a latent
tuberculosis infection (LTBI). Approximately 5% of immunocompetent persons with
LTBI will develop active tuberculosis within two years and another 5% during
their lifetime. However, these estimates have been made using the Mantoux or
Tuberculin Skin tests (TST), which frequently gives false positive results. The
percentage of progression to active TB can therefore be higher. In
immunodeficiënt patients this percentage is many times higher, approximately
10% per year [1.2]

Since the eighties of the last century efforts have been made to develop a
specific test for LTBI, based on the stimulation of interferon gamma production
by lymfocytes. This led to the first publications concerning commercialised
IGRA tests for medical application in 2004. [2, 3] The test principle of the
IGRA is in vitro exposure of whole blood (Quantiferon TB-gold, Cellestis, QFT)
or PBMC (T-spot TB, Oxford immunotec, T-spot) to Mycobacterium tuberculosis
specific antigens. If Mycobacterium tuberculosis specific lymfocyts are present
production of interferon gamma is stimulated. Interferon levels are
subsequently detected by means of ELISA. [2, 4] Which of both tests is superior
in diagnosing LTBI is yet unclear. Some studiessuggest that T-spot has a higher
sensitivity in immunocompromised patients.

Unfortunately there is no gold standard for diagnosing LTBI and the value of
IGRA has not yet been set. To determine wether the IGRA can reliably predict
the progression of LTBI to active tuberculosis, longitudinal studies are
necessary. [4] The Amsterdam Drug Users cohort is a unique opportunity for
carrying out such a study. First because of the presence of stored PBMC, and
secondly for its excellent clinical follow-up.

Within the framework of research and development on the GGD Amsterdam a project
application for examining the predicting value of the IGRA has been remunerated
at participants to the Amsterdam drug user cohort. This study has the name
"Value of Interferon Gamma Release Assay (IGRA) in Predicting Active
Mycobacterium tuberculosis Infection in HIV negative and HIV positive
Participants in the Amsterdam Drug Users Cohort".

The present protocol concerns a validation study and serves as proof of
principle before we applie for the stored samples of the Amsterdam drug users
cohort, as the IGRA has only been validated on fresh blood samples. To prevent
losing this valuable material in a test that has not been validated, we first
carry out this validation study. The results of this study will help to decide
whether or not to continue with the planned study and if so, which IGRA test is
most suitable.

1. World Health Organisation. Global Tuberculosis Control: Surveillance,
planning, financiën. WHO report 2005, Geneva, Zwitserland (2005).
2. Rothel JS, Andersen P. Diagnosis of latent Mycobacterium tuberculosis
infection: is the demise of the Mantoux test imminent? Expert Rev. Anti Infect
Ther. 2005; 3: 981-993.
3. Pai M, Liley LW, Colford JM, et al. Interferon gamma assays in the
immunodiagnosis of tuberculosis: a systematic review. Lancet Infect Dis. 2004;
4: 761-776.
4. Menzies D, Pai M, Comstock G. Meta-analysis: new tests for the diagnosis of
latent tuberculosis infection: areas of uncertainty and recommendations for
research. Ann int med 2007; 146: 340-354.

Determining the impact of freezing PBMC on the test result of the IGRA.

The QFT, which is routinely being performed at the Streeklaboartorium, and the
T-spot TB test, which is also commercially available, will be validated on
frozen PBMC's. At the department for tuberculosis control of the GGD
approximately 20 volunteers (patients) with and without a positieve TST and
with and without a BCG vaccination in the past will be asked to participate in
this study. Volunteers will be recruited from known tuberculosis patients who
come for follow up or patients with active tuberculosis (15 IGRA positive
samples) and from patients who come for contact tracing (low suspicion for
tuberculosis for 5 IGRA negative samples). Subsequent to informed consent, 22
ml venous blood will be drawn in 5 tubes. Fresh blood will immediately be
analysed according to the guidelines of the manufacturer; a second set of
samples will be frozen in liquid nitrogen in collaboration with the Acadamical
Medical Center (AMC). After 1 month samples will be thawed and analysed. On the
basis of this validation study it will be decided which test qualifies for the
further research. If both tests show a good performance on frozen PBMC's both
will be continued, since it is unclear which IGRA test performs best [5]. If
the results with the stored PBMC do not deviate qualitatively from the results
on fresh blood the comparison will be repeated when PBMC's have have been
frozen for 1 year.

5. Franken WPJ, Koster BFPJ, Bossink AWJ, et al. Follow-up study of
tuberculosis-exposed supermarketcustomers with negative tuberculin skin test
results in association with positive gamma interferon release assay results.
Clinical and vaccine immunology 2007; 14: 1239-41.

15 minutes Will be sufficent for informed consent and venous puncture. There is
no need for extra research or procedures. There are no riscs in case of
pregnancy. After drawing blood a haematoma can occur .