Reversion of the ELISPOT test after treatment in Gambian tuberculosis cases

Abstract

Background: New tools are required to improve tuberculosis (TB) diagnosis and treatment, including enhanced ability to compare new treatment strategies. The ELISPOT assay uses Mycobacterium tuberculosis-specific antigens to produce a precise quantitative readout of the immune response to pathogen. We hypothesized that TB patients in The Gambia would have reduced ELISPOT counts after successful treatment.

Methods: We recruited Gambian adults with sputum smear and culture positive tuberculosis for ELISPOT assay and HIV test, and followed them up one year later to repeat testing and document treatment outcome. We used ESAT-6, CFP-10 and Purified Protein Derivative (PPD) as stimulatory antigens. We confirmed the reliability of our assay in 23 volunteers through 2 tests one week apart, comparing within and between subject variation.

Results: We performed an ELISPOT test at diagnosis and 12 months later in 89 patients. At recruitment, 70/85 HIV-negative patients (82%) were ESAT-6 or CFP-10 (EC) ELISPOT positive, 77 (90%) were PPD ELISPOT positive. Eighty-two cases (96%) successfully completed treatment: 44 (55%; p < 0.001) were EC ELISPOT negative at 12 months, 17 (21%; p = 0.051) were PPD ELISPOT negative. Sixty (73%) cured cases had a CFP-10 ELISPOT count decrease, 64 (78%) had an ESAT-6 ELISPOT count decrease, 58 (70%) had a PPD ELISPOT count decrease. There was a mean decline of 25, 44 and 47 SFU/2 x 105 cells for CFP-10, ESAT-6 and PPD respectively (p < 0.001 for all). Three of 4 HIV positive patients were cured, all 3 underwent ELISPOT reversion; all 4 not cured subjects (3 HIV-negative, 1 HIV positive) were ESAT-6, CFP-10 and PPD ELISPOT positive at 12 months.

Conclusion: Successful tuberculosis treatment is accompanied by a significant reduction in the M. tuberculosis-specific antigen ELISPOT count. The ELISPOT has potential as a proxy measure of TB treatment outcome. Further investigation into the decay kinetics of T-cells with treatment is warranted.

Figure 1

The proportions of cured sputum positive TB cases that have positive ESAT-6, CFP-10 or PPD ELISPOT test at recruitment (diagnosis) and 12 months later (n = 82). P values shown in figure are from tests of proportions.

Figure 2

Paired recruitment and 12 months quantitative counts of Spot Forming Units (SFU)/2 × 10⁵ cells in ELISPOT tests using CFP-10 (Fig. 2a), E-SAT6 (Fig. 2b) and PPD (Fig. 2c) as stimulatory antigens for cured, HIV-negative patients (n = 82). Filled black squares (■) represent the SFU count at recruitment; white squares (□) represent SFU counts 12 months later, after successful completion of TB treatment. The change in counts between the two timepoints is represented by the linking vertical bar. With all three stimulatory antigens, the average within-subject decline in SFU counts is highly significantly (p < 0.001).