Laboratory methods

Two to five milliliters (ml) of sputum and 6 ml of venous blood were collected from bacteriologically confirmed PTB cases. From cytological diagnosed TBLN cases, another fine needle aspiration cytology (FNAC) sample and 6 ml of venous blood were obtained. The FNAC samples were further confirmed by ZN microscopy and culture at Amhara Public Health Institute (APHI) before enrolment in the final analysis. Sputum bacilli load was measured using ZN acid fast bacilli (AFB) staining technique. The Mtb time to culture detection/positivity (TTD) was measured using Mycobacterium growth indicator tube (MGIT)-960 technique.

The collected blood sample from each participant was aliquoted into EDTA and serum separator tubes, each 3ml. The EDTA tube was shipped to APHI or FHCSH immunohematology laboratory for complete blood count and CD4 + T cell count. The hematological variables were measured using five part (BC-5800 Mindray, Shenzhen Mindray Bio-Medical Electronics Co., Ltd) or three part (sysmex xs-500i) auto hematology analyzer. The absolute CD4 + T cell count was measured at APHI using BD FACSPresto™ system which has over 96% agreement with gold standard methods (BD FACS Calibur and sysmex systems) [46].

The serum samples were separated and stored at -80^oc. Serum concentrations of total cholesterol (CHO), triglyceride (TAG), HDL-cholesterol (HDL-C) and creatinine (Cr) were determined by the closed system Dimension EXL 200 Integrated chemistry analyzer at Tibebe Ghion Comprehensive Specialized Hospital. We used the products of Siemens Healthineers reagents for the determination of CHO, TAG, HDL-C and Cr. Total cholesterol was determined by cholesterol oxidase- horseradish peroxidase methods with the analytical sensitivity of 1.295 mmol/L (0.0259*50 mg/dL) and 1.295 − 15.54 mmol/L measurement ranges while TAG was determined by glycerol-3-phosphate-oxidase-peroxidase technique with an analytical sensitivity of 0.1695 mmol/L (0.0113*15 mg/dL) and 0.1695–11.3mmol/L measurement ranges. HDL-cholesterol was determined by the modified cholesterol esterase and cholesterol oxidase methods with the analytical sensitivity of 0.0777mmol/L(0.0259*3 mg/dL) and 0.0777–3.885mmol/L measurement ranges while creatinine values was measured by the modified Jaffe technique with an analytical sensitivity of 13.26µmol/L (88.4*0.15 mg/dL) and 13.26–1768 µmol/L measurement ranges. The tests were performed in batches according to the manufacturers’ instruction. Before performing the laboratory analysis, the machine was calibrated and validated with standards. In addition internal quality control was performed to maintain the quality of generated data. The analysis was performed using the laboratory standard operating procedure.