Subjects with no signs of active TB based on X-ray, sputum examination and clinical evaluation and with a positive QFT test were defined as LTBI and offered preventive anti-tuberculous therapy ZD1839 purchase with isoniazid and rifampicin for 3 months. The decision to treat was made by the clinician and the QFT test was known at the time of decision. Blood samples for flow cytometry analyses were obtained before start of any anti-tuberculous therapy, and for the LTBI group also at the end of therapy. Seventeen were followed with repetitive blood sampling at the end of therapy, whereas three were lost to
follow up. 13/20 were still QFT positive, 4/20 had turned negative whereas in 3/20 no QFT test was performed. Because of logistic difficulties, we were not able to collect blood samples from the active TB group at the end of therapy or to perform longitudinal blood sampling from QFT-negative subjects not starting preventive therapy. Written informed consent was obtained from all participants. The study was approved by the Regional Committee for Ethics in Medical Research (REK) in Bergen, Norway. QuantiFERON-TB
GOLD in-tube assay. The assay was performed according to the manufacturer’s instructions (Cellestis International Pty Ltd., Chadstone, Vic., Australia). One ml of whole blood selleck was added to each of the three QFT tubes containing TB antigen (ESAT-6, CFP-10 and TB 7.7 [p4]), mitogen-positive control [phytohemagglutinin (PHA)] and a negative control, respectively. The tubes were incubated at 37 °C for 16–24 h, centrifuged and plasma removed. The amount
Protein tyrosine phosphatase of interferon-gamma (IFN-γ) in plasma was quantified by enzyme-linked immunosorbent assay (ELISA). The QFT Analysis Software (Cellestis International Pty Ltd) was used to analyse raw data (optical density values) and calculate results. The level of IFN-γ was corrected for background by subtracting the IU/ml value obtained for the respective negative control. The cut-off value for positive test was ≥0.35 IU/ml. Flow cytometry analyses. Peripheral blood mononuclear cells (PBMCs) were isolated from heparinized whole blood using density gradient centrifugation (LymphoprepTM, Fresenius Kabi Norge AS, Halden, Norway), cryopreserved in 10% dimethyl sulfoxide (DMSO)/90% foetal calf serum (FCS) and stored in liquid nitrogen before analysis. Cryovials were thawed, washed and resuspended in RPMI media with 10% FCS to a final concentration of 4.106cells/ml.
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