Selected RD1 peptides for active tuberculosis diagnosis: comparison of a gamma interferon whole-blood enzyme-linked immunosorbent assay and an enzyme-linked immunospot assay

Abstract

We recently set up a gamma interferon (IFN-gamma) enzyme-linked immunospot assay (ELISPOT), using selected early secreted antigenic target 6 (ESAT-6) peptides, that appears specific for active tuberculosis (A-TB). However, ELISPOT is difficult to automate. Thus, the objective of this study was to determine if the same selected peptides may be used in a technique more suitable for routine work in clinical laboratories, such as whole-blood enzyme-linked immunosorbent assay (WBE). For this purpose, 27 patients with A-TB and 41 control patients were enrolled. Our WBE, using the already described selected peptides from ESAT-6 plus three new ones from culture filtrate protein 10, was performed, and data were compared with those obtained by ELISPOT. Using our selected peptides, IFN-gamma production, evaluated by both WBE and ELISPOT, was significantly higher in patients with A-TB than in controls (P < 0.0001). Statistical analysis showed a good correlation between the results obtained by WBE and ELISPOT (r = 0.80, P < 0.001). To substantiate our data, we compared our WBE results with those obtained by QuantiFERON-TB Gold, a whole-blood assay based on region of difference 1 (RD1) overlapping peptides approved for TB infection diagnosis. We observed a slightly higher sensitivity with QuantiFERON-TB Gold than with our WBE (89% versus 81%); however, our test provided a better specificity result (90% versus 68%). In conclusion, results obtained by WBE based on selected RD1 peptides significantly correlate with those generated by ELISPOT. Moreover, our assay appears more specific for A-TB diagnosis than QuantiFERON-TB Gold, and thus it may represent a complementary tool for A-TB diagnosis for routine use in clinical laboratories.

FIG.1.

IFN-γ response to selected RD1 peptides in A-TB patients and controls by WBE and ELISPOT. Detection of IFN-γ was performed by WBE (A), ELISPOT (B), and by both WBE and ELISPOT (C). The highest response to selected RD1 peptides in terms of IFN-γ is reported in IU/ml or SFC per million PBMC for each individual. Horizontal bars represent the mean IU/ml or SFC per million PBMC values for each group of patients. The P value denotes the difference between the responders in each group. Symbols: ○, active TB; ▾, TST-positive controls; ▵, TST-negative controls.

FIG. 2.

Correlation between WBE and QuantiFERON-TB Gold results. The highest IFN-γ-secreting T-cell response is reported in IU/ml for each individual. Symbols: ○, active TB; ▾, TST-positive controls; ▵, TST-negative controls.