The association between magnitude of T-SPOT.TB and clinical characteristics in active pulmonary tuberculosis: a retrospective multicenter investigation

Background: The aim of this study was to evaluate the association between magnitude of T-SPOT.TB and clinical characteristics in active pulmonary tuberculosis. Methods: In this retrospective multicenter investigation, the medical records’ archives of PTB patients were reviewed. Data including gender, age group, bacterial culture, sputum-smear microscopy, lung cavity and range of PTB were extracted from the records. The Jonckheere-Terpstra test and chi-square test for trend were used to evaluate the association between magnitude of T-SPOT.TB and clinical characteristics in active pulmonary tuberculosis. Results: A total of 1252 PTB patients were included in this study.Chi-square trend tests revealed signicant positive trends between bacterial culture and magnitude of T-SPOT.TB A (χ2 =18.978, P <0.001) or T-SPOT.TB B (χ2 =19.677, P <0.001). There were signicant positive trends between AFB and magnitude of T-SPOT.TB A (TJT =345784, P <0.001) or T-SPOT.TB B (TJT =336017, P <0.001). There was a signicant positive trend between range of lung cavity and magnitude of T-SPOT.TB A (TJT =326079.5, P =0.023). There were signicant negative trends between age group and magnitude of T-SPOT.TB A (TJT =204306, P <0.001) or T-SPOT.TB B (TJT =206572, P <0.001). Conclusions: The magnitude of T-SPOT.TB in PTB patients may reect the condition of the patients and could be helpful for clinicians to assess the degree of infection and lung tissue damage.

China to evaluate the association between magnitude of T-SPOT.TB and clinical characteristics in active PTB.

Study design and participants
The study was performed at 5 specialized TB hospitals in China. Diagnosis of TB refers to Clinical diagnosis standard of TB issued by Chinese Medical Association [5]. The PTB patients who underwent valid T-SPOT.TB assays, bacterial culture, sputum-smear microscopy and X-ray examination from December 2012 to November 2015 in the ve hospitals and met the study criteria were retrospectively evaluated. The ve hospitals are situated in the south, north, east, and center of China. At each study hospital, trained health workers extracted data from the medical records of inpatients. Records were collected in terms of sputum smear/culture, range of pulmonary tuberculosis, range of lung cavity, age, gender, etc. All cases had negative results on serological tests for HIV. All patients had not had immune diseases or received immunosuppressant before.

T-SPOT.TB assay
The T-SPOT.TB test (Oxford Immunotec Ltd., UK) was performed using peripheral blood mononuclear cells (PBMCs) separated from heparinized blood samples according to the manufacturer's instructions.
Brie y, PBMCs were isolated and incubated with two antigens in parallel (peptides derived from early secreted antigenic target-6 ESAT-6 and from culture ltrate protein CFP-10). The procedure was performed in plates pre-coated with anti-IFN-γ antibodies at 37°C for 16-20 h. After application of alkaline phosphatase-conjugated secondary antibody and chromogenic substrate, the number of spot-forming units (SFUs) was counted using an automated ELISpot counter. The test result of T-SPOT.TB assay include Panel A (containing peptide antigens derived from ESAT-6) and Panel B (containing peptide antigens derived from CFP-10). The results were interpreted as recommended by the test kit manufacturer [6]. The magnitude of T-SPOT.TB A and B were divided into the following ve groups according to the number of Spot-Forming Units (SFUs): 0-5 SFUs group, 6-19 SFUs group, 20-39 SFUs group, 40-69 SFUs group and ≥70 SFUs group,which the positive magnitudes of T-SPOT.TB A and B were classi ed by quartile and integers.

Statistical analysis
We took some measures to guarantee the data quality, including a standardized study protocol and standardized training of research staff. Descriptive statistics including frequency distribution and median and interquartile were used to report study ndings. The T-SPOT.TB results were evaluated in the subgroups of gender, age, bacterial culture, sputum-smear microscopy, range of PTB disease, range of lung cavity. The scatterplot were used to describe the trends between T-SPOT.TB and clinical characteristics. Jonckheere-Terpstra test(JT),a nonparametric trend test appropriate for ordinal variables, has higher test e ciency on the premise of overall priori ranking.So the trends and association between ordinal variables were assessed using the Jonckheere-Terpstra test and Spearman rank order correlation test. The differences and trends between bacterial culture, gender and T-SPOT.TB were assessed using the chi-square test and chi-square test for trend. All data were collected in datasheets in MS O ce Excel (Microsoft, Redmond, WA, USA) and all analyses were conducted using SPSS software for Windows, version 13 (Chicago, USA). A P value <0.05 was considered as statistically signi cant in all analyses.

Results
Demographic and clinical characteristics of the PTB patients A total of 1252 eligible PTB patients were included during the study period with a median age of 42 (25-59) years, and 67.7% of whom were male. The proportion with culture-positive PTB was 57.2%. The demographics and clinical characteristics of the study population are presented in Table 1.
The association and trends between sputum-smear microscopy and T-SPOT.TB There was a low but signi cant correlation between sputum-smear microscopy (AFB) and T-SPOT.TB A (r s =0.141, P <0.001) or T-SPOT.TB B (r s =0.121, P <0.001), respectively.

Discussion
Despite substantial progress to combat TB, TB remains a major global health problem in the world. Recently, interferon-γ assays have emerged as immunodiagnostic tools to detect tuberculous infection. T-SPOT.TB assays use the M. tuberculosis-speci c antigenic peptides, ESAT-6 andCFP-10, which are not pres ent in Bacille Calmette-Guérin (BCG) strains and most nontuberculous mycobacteria, making them highly spe ci c for detecting TB infection [7]. These proteins are encoded by genes located within the Liquefaction of solid caseous tuberculous lesions and the subsequent cavity formation are probably the most dangerous processes in the pathogenesis of human pulmonary tuberculosis. Our results suggested there was a signi cant positive trend between range of lung cavity and T-SPOT.TB A (T JT =326079.5, P =0.023). Furthermore, with increasing magnitudes of T-SPOT.TB A, the positive magnitude of lung cavity increased. Since the magnitudes of T-SPOT.TB A re ect the T cell response to ESAT-6, the nding is consistent with a role of this antigen in virulence and pathogenicity. Wards et al. [24] reported that guinea pigs inoculated with either an ESAT-6 knockout strain of M. tuberculosis or its virulent parent had positive skin test reactions to PPD but only the animals inoculated with the parent strain had positive skin test reactions to ESAT-6. Gross pathology, histopathology and mycobacterial culture of tissues indicated that the knockout strain was less virulent than its parent. In contrast to our ndings with T-SPOT.TB A, there was almost no association between the range of lung cavity and T-SPOT.TB B. This could imply that there is no role of the T cell response to CFP-10 in cavity development. It may be relevant that Berhet FX et al. [25] reported that recombinant CFP-10, produced in E. coli, did not seem to provide a major target for the humoral immune response that had been generated during human tuberculosis. Thus the magnitude of T-SPOT.TB A , but not T-SPOT.TB B, may indirectly re ect the cavity range of PTB patients, which would be helpful for clinicians in assessing the degree of lung tissue damage in such patients.
Our study showed that the magnitudes of T-SPOT.TB in males were not signi cantly different from those in females (T-SPOT.TB A, χ 2 =0.295, P =0.990; T-SPOT.TB B, χ 2 =7.357, P =0.118). A previous study in China also showed that the positive rate of T-SPOT.TB was not different between male and female in active tuberculosis [26]. Thus we thought that the T-SPOT.TB results may not be in uenced by gender in PTB patients.
Our study found here that with increasing age of patient the magnitude of T-SPOT.TB gradually decreased, consistent with the known fact that, due to a decline in the strength of immune responses, older individuals tend to be more susceptible to TB[27-29]. Kobashi et al. [29] reported that the positive rate for both combined and separate ESAT-6 and CFP-10 antigens of QuantiFERON TB-2G tested in the elderly patients was signi cantly lower than that in younger patients. In another study conducted by Kobashi et al. [30], increasing age associated with false-negative results in ELISPOT assays. Concern has been expressed that a general decline of immune responsiveness may decrease the sensitivity of IGRAs in aged populations [31]. Accordingly, we suggested that the severity of tuberculosis is not readily assessed in elderly patients by T-SPOT.TB assays.
This study had some limitations. One of the main limitations was the restricted nature of the sample population, which could have affected the results of our analyses when considering the multiple patient levels. The purpose of the study was to analyze the association of magnitude of T-SPOT.TB and clinical characteristics in active pulmonary tuberculosis, but the PTB patients were all inpatients and did not include outpatients. Thus, there was bias in the results. on the other hand, the data were all retrospectively collected and analyzed, which might have had impacts on results.

Conclusions
In conclusion, despite these limitations, to our knowledge, this study is unique in being a multicenter large-scale investigation to evaluate the association between magnitude of T-SPOT.TB and clinical characteristics in active pulmonary tuberculosis in China. Our study revealed some associations. Thus, with increasing magnitudes of T-SPOT.TB, the positive magnitudes of the bacterial culture and AFB increased. With increase in the magnitude of the T-SPOT.TB A, the range of lung cavity increased. Hence, the magnitude of the T-SPOT.TB in PTB patients may re ect the condition of the patients and could be helpful for clinicians in assessing the degree of infection and lung tissue damage. This was an observational retrospective study. Given that the medical information of patients was recorded anonymously by case history, which would not bring any risk to the participants, the Ethics Committee of Beijing Chest Hospital, Capital Medical University approved this retrospective study, with a waiver of informed consent from the patients.

Consent to publish
All authors read and approved the nal paper for publication.

Availability of data and materials
All supporting data in this study can be provided by the corresponding author upon reasonable request.

Competing interests
The authors declare that there are no actual or potential con icts of interest in relation to this article.   Figure 1