Conventional diagnostic methods for tuberculosis are not satisfying in clinical practice as acid-fast bacilli (AFB) smear lacks sensitivity and mycobacterial culture is time consuming . Although Xpert MTB/RIF can detect MTB and rifampin-resistance mutations in several hours with high accuracy, it is estimated that 30–50% patients failed to provide appropriate samples for the test . Our previous study showed that less than 30% of patients diagnosed with ATB could be confirmed by etiology . Thus, clinicians urgently need new methods to help diagnose TB, especially for patients who cannot obtain evidence of the pathogens.
T-cell immunity plays a critical role in controlling MTB infection . IGRA works by detecting the secretion of Th1 cytokine IFN-γ after MTB-specific antigen stimulation to diagnose TB infection . In addition to IFN-γ, TNF-α is also involved in immunity against MTB infection. Secreted by activated dendritic cells, γδT cells , macrophages, CD4+ as well as CD8+ T cells, TNF-α attaches to endothelin cells and improves vascular permeability so that neutrophils and monocytes in circulation recruited by inflammation cytokines can enter infection sites and further form granuloma. TNF-α maintains granuloma integrity through transforming inactivated macrophage to M1 subtype, a process synergizing with IFN-γ . Clinical studies observed higher reactivation and incidence of TB in patients with anti-TNF-α therapy [18,19,20]. Therefore, whether MTB-specific TNF-α secretion can contribute to the diagnosis of ATB is worthy of further study.
Our study showed that the frequencies of MTB specific IFN-γ-secreting T cells in the ATB group were significantly higher than those in the LTBI and HC groups. These findings were consistent with our previous research . The frequencies of TNF-α-secreting T cells was comparable to the results of other studies using ELISA or flow cytometry (FCM) [22,23,24]. The FluoroSpot assay based on the ELISPOT is more accurate compared to ELISA or FCM. The Fluorospot assay may be preferable for studies requiring the detection of low-level responses or qualitative results . In addition, compared to FCM, FluoroSpot assay enables single-cell measurements of the actual secretion of bioactive molecules rather than intracellular analytes, which can be critical for understanding functional properties of T cells, as it may help in better identification of infection status .
Interestingly, ESAT-6 seemed to activate T cells more effectively than CFP-10. The frequencies of TNF-α-secreting T cells stimulated by ESAT-6 were significantly higher than those stimulated by CFP-10, though no difference was detected in the IFN-γ secretion under the stimulation of these two antigens. The immune mechanism is still unclear and remains to be further studied.
In this study, the frequencies of ESAT-6-specific total TNF-α-secreting T cells might be a better potential marker for IFN-γ/TNF-α FluoroSpot assay to differentiate ATB and LTBI, compared with IFN-γ-secreting T cells. Wang’s study showed that the detection of. of MTB-specific TNF-α secretion by ELISA assay could distinguish ATB from LTBI, with a sensitivity of 72% and a specificity of 90.91% . Another study evaluated the diagnostic accuracy of differentiating ATB from LTBI by combining the IGRA and the TNF-α-release assay (TARA). Compared with IGRA only, the combination of IGRA and TARA improved the specificity without compromising the sensitivity . IFN-γ/TNF-α FluoroSpot assay was a new method to diagnose ATB, and only one study showed that IFN-γ/TNF-α dual release assay had the best accuracy to differentiate ATB and LTBI, with a sensitivity of 84% and a specificity of 94%. This was slightly different from our results, which might be due to the different inclusion criteria of the ATB patients. In addition to the microbiologically confirmed cases, the study also included clinically diagnosed TB cases, who possibly displayed different cytokine secretion patterns. It should also be noted that the patients’ medication was not mentioned in the study, which may affect the results .
Furthermore, this study showed that double-testing the ESAT-6 with the CFP-10 might not increase the differential diagnostic accuracy. The result was in consistency with a study of North India population which also observed significantly higher IFN-γ and TNF-α response to ESAT-6 stimulation in ATB group compared to healthy household contacts (considered as LTBI), while no significant difference was observed with CFP-10 stimulation . Clifford’s study showed that the level of TNF-α stimulated by ESAT-6 declined significantly over the course of therapy in ATB cases, but the decrease was not significant under CFP-10 stimulation . Therefore, using ESAT-6 antigen alone might reduce the cost of testing without compromising diagnostic accuracy.
Medication may disturb the cytokine secretion. Several studies of IGRAs indicated that anti-tuberculosis drugs, corticosteroids and immunosuppressants may interfere T-cell responses towards MTB antigens [13, 32,33,34]. Therefore, immunosuppressive therapy may also influence the efficacy of the FluoroSpot assay. In our study, one patient with rheumatoid arthritis (RA) were taking prednisone (at 15 mg/d) when tested for both T-SPOT.TB and FluoroSpot. Both results of the RA patients were positive and similar for overall study population. Although it is highly possible that immunosuppressive therapy did not interfere the results of the study, further research on the potential influence of the immunosuppressive therapy is needed.
Both our study and previous research found that a number of iSFCs secreting TNF-α in the nil control of some samples were detected, but no iSFCs secreting IFN-γ were found . A possible reason was that these spots formed without antigen stimulation may be caused by TNF-producing monocytes or dendritic cells instead of lymphocytes. TNF-α, a monokine involved in innate immune system, was mainly secreted by activated monocytes and macrophages, yet activated dendritic cells, γδT cells, CD4 + T cells and CD8 + T cells can also produce TNF-α . In addition to lymphocytes, the PBMC for FluoroSpot testing also included monocytes and DC cells, and the innate immune response in which they participated also produced TNF-α, causing background spots in the nil control without MTB specific antigen stimulation.
However, this study has some limitations. First, we excluded patients with malignancy or undergoing TNF-α antagonist therapy, whose TNF-α secretion may be disturbed. Thus, whether IFN-γ/TNF-α FluoroSpot can be applied to these population required further research. Second, the use of T-SPOT.TB as a diagnostic criterion for LTBI may lead to selection bias . However, IGRA is one of the recognized methods for detecting LTBI with satisfying specificity, it is reasonable to reduce the risk of misdiagnosis in the control group. Third, the overlapping in cytokines responses still exist, which may affect the correct distinction between groups. The high background in the nil control when detecting TNF-α should be concerned, which may affect the results. Forth, this was only a preliminary study of diagnostic test with case-control design, making overestimation of the diagnostic accuracy highly possible, and the results need to be further verified by cross-sectional or prospective cohort studies.