An IFN-γ and TNF-α dual release fluorospot assay for diagnosing active tuberculosis

Abstract

Objectives: Currently available interferon (IFN)-g-release assays (IGRA) cannot discriminate active tuberculosis (TB) from latent TB infection (LTBI), and so have limited clinical utility for diagnosing active TB. Since numbers of tumour necrosis factor (TNF)-a-producing T cells are highly correlated with active TB, we hypothesized that detecting IFN-g-and/or TNF-a-producing T cells would overcome this limitation of IGRA. This study evaluated the diagnostic performances of the IFN-g and TNF-a dual release fluorospot assay for active TB. Methods: Adult patients with suspected TB including recent TB exposers were prospectively enrolled over a 28-month period. In addition to the conventional IGRA test (i.e. QuantiFERON-In-Tube), a fluorospot assay for detecting IFN-g-and TNF-a-producing T cells was performed. The final diagnoses were classified by clinical category. Patients with confirmed or probable TB were regarded as active TB, and patients with not active TB were further classified as having not active TB with and without LTBI, based on the QuantiFERON-In-Tube results. Results: A total of 153 patients including 45 with active TB and 108 with not active TB (38 LTBI vs. 70 not LTBI) were finally analysed. The sensitivity and specificity of the QuantiFERON-In-Tube assay for active TB were 84% (95% confidence interval (CI), 70e93) and 70% (95% CI 61e79), respectively. The IFN-g/TNF-a dual release assay by fluorospot had substantially higher diagnostic specificity (94%) for diagnosing active TB than the IFN-g single release assay (72%, p < 0.001), without compromising sensitivity (84% vs. 89%, p 0.79). Conclusions: The fluorospot-based IFN-g/TNF-a dual release assay appears to be a simple and useful test for diagnosing active TB.

Combined plasma cell-free DNA detection and IFN-γ/TNF-α dual fluorospot assays for diagnosing active tuberculosis

“…The uorospot assay can detect simultaneously cells which secret different cytokines using uorescencebased enzyme-linked immuno-spot technology [9,22]. In our previous study, the uorospot-based IFN-γ /TNF-α dual release assay showed 84% sensitivity and 94% speci city for active TB [9]. However, this sensitivity was still suboptimal for ruling out active TB.…”

“…All patients were classi ed by a physician investigator (SY Lim) blinded to the uorospot results, based on clinical, histopathological, radiological, and microbiological information collected over at least 3 months of follow-up. The clinical categories of patients with suspected TB have been described previously [9,11] and are presented in Supplementary Table 1. Patients with possible TB were excluded from the nal analysis.…”

“…The uorospot assay was performed according to the modi ed procedure described previously [9]. Brie y, aliquots of 100 µl of peripheral blood mononuclear cells (PBMC, 2 x 10 5 ) were seeded in the wells of 96well plates precoated with anti-IFN-γ, and anti-TNF-α capture antibodies (Mabtech AB, Nacka Strand, Sweden), and 50 µl of TB stimulators, 2 µg/ml each of ESAT-6, CFP-10, and Ag85B peptide pools (JPT Peptide Technologies Inc., Berlin, Germany) were added.…”

“…Following overnight drying of the plates, two-color uoro-spots were counted with a FluoroSpot Reader (AID, Strassberg, Germany) and con rmed manually by an investigator (JY Kim). The results of the uorospot assay were classi ed as previously described [9], and the results of the combination of cfDNA detection and the IFN-γ /TNF-α dual release assay were considered to be positive if the cfDNA detection or the dual release assay yielded positive results…”

Challenges and the Way forward in Diagnosis and Treatment of Tuberculosis Infection

“…Although a combination of IFN-γ and TNF-α levels had lower AUC (0.900), the specificity of the test increased to 97.1% from single TNF-α (94.3%) and IFN-γ (77.1%) [ 164 ]. A study by Kim et al (2020) had also shown that the diagnostic specificity of the ESAT-6 and CFP-10-stimulated in an IFN-γ/TNF-α dual release assay (94%) by fluorospot was greater than the IFN-γ single release assay alone (72%) [ 165 ].…”