Quantitative investigation of factors relevant to the T cell spot test for tuberculosis infection in active tuberculosis

This retrospective study analyzed 360 pathogenically positive TB patients who were hospitalized in Ankang Central Hospital, Shaanxi, China, and the data were accessed between October 1, 2016 and June 30, 2018. We collected data on 29 variables associated with five aspects, including the patients’ general status, bacteriology, imaging, routine examination, protein electrophoresis and immunology. The subject screening process is illustrated in (Fig. 1).

This study was approved by the ethics committee of Ankang Central Hospital (ECACH-2016011). Informed consent was not required because the study did not put the patients at risk. Prior to analysis, the patients’ identities were protected by anonymity and by the use of codes.

The inclusion criteria were as follows [21]: (1) the MTB-specific deoxyribonucleic acid or ribonucleic acid test was positive, and patients had suspected symptoms of TB; (2) patients had one sputum smear that was positive for acid-fast staining or positive sputum culture, and thoracic imaging showed lesions conforming to ATB and/or suspected symptoms of TB; (3) patients were subjected to the T-SPOT.TB test, and the results were accessible.

The exclusion criteria were as follows: (1) patients who were taking immunosuppressants; (2) patients who did not undergo IGRA examination; (3) patients who were positive for HIV infection; (4) patients with nontuberculous mycobacteria infection; (5) patients with combined cirrhosis; (6) patients with combined tumor.

The T-SPOT.TB diagnosis kit was provided by Shanghai Fosun Long March Medical Science Co., LTD (Oxford Immunotec Ltd., United Kingdom). Specimens were examined within 2 h of collection at room temperature (18–25 °C) in a sterile environment. (1) RPMI 1640 medium was mixed with an equal volume of a whole blood sample. (2) Three milliliters of Ficoll human lymphocyte isolation solution was added to the first centrifuge tube; 6–9 ml of diluted whole blood was then slowly added to the solution, and the tube was centrifuged at 1000 x g for 22 min. (3) Approximately 5 ml of the layer containing PBMCs was transferred to a second centrifuge tube, and the mixture was brought to a volume of 10 ml with RPMI 1640; the tube was then inverted and centrifuged at 600 x g for 7 min. (4) The supernatant was discarded, the pellet was resuspended in 1 ml of medium, and RPMI 1640 was added to bring the final volume to 10 ml. After mixing by inverting the tube, the sample was centrifuged at 350 x g for 7 min. (5) The supernatant was discarded, and the pellet was resuspended in AIM-V medium at a density of 2.5 × 10⁶ cells/ml. (6) Fifty microliters of negative control (ALM-V), antigen A (early secreted antigenic target 6 kDa, ESAT-6), antigen B (culture filtrate protein 10 kDa, CFP-10) and positive control (PHA) were added sequentially to an IFN-γ antibody-coated microplate; 100 μl of diluted cells were then added to each well, and the plate was placed in a CO₂ incubator (37 °C, 5% CO₂) for 20 h. (7) After removal of the medium, each well was washed four times with 200 μl of PBS and incubated with 50 μl of secondary antibody working solution (1200) for 1 h at 2–8 °C. (8) After removal of the secondary antibody, each well was washed four times with PBS, 50 μl of substrate chromogen solution was added to each well, and the plate was allowed to stand for 10 min in the dark. The reaction was terminated by washing the plate with distilled water. (9) SFCs were automatically counted using a plate reader (ES-15, Shanghai Fosun Long March Medical Science Co., Ltd., Shanghai, China). When the counts could not be determined using the plate reader, the SFCs were manually counted using a microscope. The results were recorded and interpreted as recommended by the kit manufacturer: ESAT-6 or CFP-10 ≥ 6 SFCs was regarded as T-SPOT.TB positive. T-SPOT.TB was negative if both ESAT-6 and CFP-10 were < 6 SFCs. The test results were uncertain if there were > 10 SFCs in the blank control well or < 20 SFCs in the positive control well. In cases of uncertain results, blood samples were taken again for another test.

Sputum or body fluid specimens were first examined using Auramine O fluorescence staining (KRJ/TTR500 automatic smear staining machine, Xiangyang Courager Medical Apparatus, Xiangyang, China) and were then subjected to MTB nucleic acid amplification (RNA constant temperature amplification, Roche LightCycler 480 II Real-time PCR Cycler; Rendu Biotechnology [Shanghai, China]; PCR-fluorescence method, Applied Biosystems 7500 Real-Time PCR System; DAAN Gene of Sun Yat-sen University [Guangzhou, China]) and rapid drug tolerance gene analysis (DNA microarray chip method; CapitalBio Corporation, Chengdu, China). Finally, the specimens were subjected to MTB culture (Roche fixed culture test), bacterial species identification and drug sensitivity testing (Ratio methods, BaSo Diagnostics INC., ZHUHAI). Using the Roche solid culture as the standard and no repeated counting, bacterial load was reported in accordance with the “Diagnostic Criteria and Principles of Management of Infectious Pulmonary Tuberculosis” [21] (see Additional file 1). The laboratory meets the national P3 requirements and accepts the quality control and management of the national reference laboratory. All operators received special training in the testing methods and in operation of the apparatus.

BMI was calculated by dividing the individual’s body weight (kg) by the square of his or her height (m). Body weight and height were measured at the time of admission. Behavioral risk factors (such as smoking and exposure to dust) and medical history were collected by physicians. The diagnostic criteria for diabetes were random blood glucose ≥11.1 mmol/L or fasting blood glucose ≥7.0 mmol/L [22]. The severity of lung imaging was graded according to the criteria set by the National Tuberculosis Association of America [23] (see Additional file 2). Whole blood cell counts were determined using a Sysmex XN-9000 automatic blood fluid analyzer and supporting reagents. Serum protein electrophoresis was performed with the Sebia Capillarys 2 Flex Piercing detection platform and supporting reagents (Pare Technologique Leonard de Vinci, CP8010-Lisses 91,008 EVRY CEDEX, France). Lymphocyte subsets were detected using the Mindray flow cytometer BriCyte E6 and a kit from BD.

The data were analyzed using SPSS software (version 22.0, IBM Corp., Armonk, NY, USA). Non-normally distributed data were expressed as medians and interquartile ranges. Wilcoxon’s rank sum test was used to compare two-sample dichotomous variables. The rank data and measurement data were analyzed using the Spearman relationship test, and the statistically significant variables were subjected to optimal scaling regression analysis. The false negative rate of T-SPOT.TB was analyzed by the exact logistic regression model of R software (version 3.4.4; https://​www.​r-project.​org). Tolerance ≥0.1 is considered as collinearity between variables. The missing values of the independent variables were replaced by the series mean. Adobe Photoshop CS6 software (version 13.0 × 32, Adobe Systems Incorporated, San Jose, CA, USA) was used for graphing. P < 0.05 was considered statistically significant.