Two patients with Mod-PTB had unilateral pleural disease, while one had lymph node involvement. Overall, 32 of 36 patients with PTB were AFB smear/culture and/or radiology positive. Four patients were diagnosed based on radiology, clinical diagnosis and response BTK inhibitor chemical structure to treatment. Tuberculous lymphadenitis (LNTB) was diagnosed by histopathological
staining of fine needle aspirate or excision biopsy with AFB smear and culture. Pleural TB was diagnosed on the basis of pleural fluid biochemical findings, AFB culture, histopathological findings on pleural biopsy, supportive radiological evidence on X-rays and/or contrast-enhanced CT scan and response to antituberculous treatment. Diagnosis of meningeal TB was based on CSF biochemical findings, supported by AFB culture and findings on contrast-enhanced CT and/or MRI [25]. Patients with L-ETB comprised those with LNTB (n = 19) and unilateral pleural disease (n = 12). PI3K inhibitor Patients with D-ETB comprised those with tuberculous meningitis (n = 1), bilateral pleurisy (n = 1),
abdominal (n = 2), spinal (n = 4) and miliary TB (n = 2). Bacille Calmette-Guerin (BCG)-vaccinated asymptomatic healthy volunteers who were staff of AKU were used as endemic controls (ECs). Tuberculin skin testing (TST) was assessed by administering five tuberculin units on the volar surface of the right arm subcutaneously and read by a Erastin cost single reader at 48 h. An induration of ≥10 mm was used as a cut-off for positive responses (TST+), which are considered to be indicative latent infection. TST+ (n = 21) and TST− (n = 21) ECs were included in the study. Reagents. Mycobacterium tuberculosis H37Rv whole cell sonicate (MTBs) and recombinant antigens ESAT6 and CFP10 were provided through the NIH Tuberculosis vaccine testing and reagent material contract (NO1-A1-40091) awarded to Colorado State University, USA. Whole blood assay. Heparinized venous blood was diluted 1 : 10 in RPMI-1640 medium each and set up in 96-well tissue
culture plates as per protocol [26]. Cells were stimulated with MTBs (10 μg/ml) and ESAT-6 and CFP10 (5 μg/ml each) and cultured for up to 5 days. Supernatants were collected for cytokine measurements at 2 and 5 days post-stimulation, spun to collect cellular debris and stored at −70 °C until tested. ELISA for IFNγ, CXCL9, CXCL10, CCL2 and IL10. IFNγ standards and monoclonal antibody pairs for capture and detection were obtained from Pharmingen, San Diego, CA, USA. Reagents for CCL2, CXCL9 and CXCL10 were obtained from R&D Systems, Minneapolis, MN, USA. All measurements were carried out according to the manufacturer’s recommendations and as described previously [16]. The limit of detection for each read-out was following: IFNγ, 15.6 pg/ml; IL10, 7.8 pg/ml; CXCL9, 69 pg/ml; CXCL10, 23 pg/ml and CCL2, 23 pg/ml, respectively. Statistical analysis.