To the Editors:
Tuberculosis (TB) remains a major global health problem. A major factor contributing to the current TB epidemic is the resistance of Mycobaterium tuberculosis to first-line anti-TB treatment. Therefore, there is a need for an appropriate and reliable tool to evaluate treatment efficacy.
Several studies using the T-SPOT®.TB assay (Oxford Immunotec Ltd, Oxford, UK) reported a decline in the number of spots after successful anti-TB treatment, particularly in the 6-kDa early secretory antigenic target (ESAT-6) panel [1–3]; however, to our knowledge, no data have been published on cured TB patients who contracted TB before isoniazid and rifampicin became available. Therefore, the naturally long-term range of ESAT-6- and culture filtrate protein (CFP)-10-specific T-cell responses in patients with TB who did not receive modern anti-TB treatment is not known. Because most patients in prior studies were born in high-burden, TB-endemic countries, the T-cell kinetics might be influenced by reinfection. Therefore, the aim of this report is to compare the ESAT-6- and CFP-10-specific T-cell responses in a low-burden country with a low risk of reinfection between patients with a history of active TB before 1955, and patients with recently treated active TB.
Venous blood for T-SPOT®.TB was collected from 24 outpatient subjects with a history of active TB before 1955 (group 1), obtained by clinical history-taking, patients’ medical records or documentation of a sanatorium admission. 16 of these patients had been admitted to a sanatorium, two patients underwent surgery and six patients were treated at home with para-aminosalicylic acid therapy or bedrest. Furthermore, 27 patients with recent active TB (group 2) were prospectively enrolled between March and May 2008. Active TB was defined as fulfilling one or more of the following criteria: 1) positive culture for M. tuberculosis (n=22); 2) positive PCR for M. tuberculosis (n=3); or 3) a high clinical suspicion of active TB and favourable outcome after receiving anti-TB treatment (n=2). Patients in group 2 had at least two T-SPOT®.TB samples: one before treatment (T0) and one after treatment (T1). The median follow-up time was 14 months (range 6–44 months).
T-SPOT®.TB was performed and results were interpreted according to the manufacturer’s instructions. The European cut-off value of 6 spot-forming cells (SFCs) was used for the binomial variables for ESAT-6 and CFP-10.
The Mann–Whitney U-test and Chi-squared test were used for between-group comparisons. Wilcoxon’s test and McNemar’s test were used for within-group analyses. All analyses were performed using SPSS for Windows, version 17.0 (IBM, Amsterdam, the Netherlands).
Patient characteristics for all subjects are shown in table 1. The median time between reported recovery from TB and current T-SPOT®.TB result was 59 yrs. In group 1, 16 (67%) subjects had positive T-SPOT®.TB results, seven (29%) had negative results and one (4%) had an indeterminate result. 13 (57%) patients were ESAT-6 positive and 13 (57%) patients were CFP-10 positive. The median (range) number of spots for ESAT-6 and CFP-10 was 7 (0–17) and 18 (0–50) SFCs, respectively (table 2).
In group 2, at T0, 26 (96%) subjects had positive T-SPOT®.TB results, none of the subjects had a negative result and one (4%) subject had an indeterminate result. At T1, 23 (85%) subjects had positive T-SPOT®.TB results, four (15%) had a negative result and none of the subjects had an indeterminate result. The number of subjects with positive T-SPOT®.TB results did not significantly decrease after treatment (nonsignificant).
There was a significant decrease in the number of subjects with positive ESAT-6 results from 25 (96%) at T0 to 17 (63%) at T1 (p=0.008). There was no significant difference in the number of subjects with positive CFP-10 results at T0 (n=25; 96%) and T1 (n=22; 82%) (p=0.250).
There was a significant decline in the median number of spots from 21 (14–42) to 9 (3–32) SFCs for ESAT-6 and from 41 (23–76) to 25 (9–59) SFCs for CFP-10 (p=0.003 and p=0.020, respectively).
T-SPOT®.TB results from group 1 were compared with those from group 2 at T0 and T1. There was a significant difference in the number of subjects with positive T-SPOT®.TB (p=0.003), ESAT-6 (p=0.001) or CFP-10 (p=0.001) results between group 1 and group 2 at T0. There was no significant difference in the number of positive T-SPOT®.TB (p=0.184), ESAT-6 (p=0.643) or CFP-10 (p=0.055) results between patients in groups 1 and 2 at T1.
The median number of spots in the ESAT-6 and CFP-10 panels were significantly different between patients in group 1 and patients in group 2 at T0 (p=0.001 and p=0.015, respectively). No differences in median numbers of spots for CFP-10 and ESAT-6 were observed between groups 1 and 2 at T1.
The correlation between the number of SFCs, and the length of time between treatment completion and blood collection for all patients in groups 1 and 2 was assessed. There was no association between the length of time after treatment completion and the number of spots for ESAT-6 (p=0.268) or CFP-10 (p=0.637).
This study is the first to compare T-cell kinetics in the T-SPOT®.TB in TB patients recently treated with anti-TB treatment, with results in patients with a history of active TB before the introduction of modern anti-TB treatment. Most importantly, patients with a history of “spontaneous” cure (group 1) had comparable T-SPOT®.TB results with patients with recent anti-TB treatment for active TB (group 2 at T1). In contrast, two prior studies showed that therapy resulted in a significant decline in the number of spots, whereas after treatment completion the number of spots did not decline further [4, 5].
In patients with cured TB before 1955, T-SPOT®.TB results were positive in 66.7% after a median survival of 59 yrs. These results demonstrate that positive T-SPOT®.TB results can persist over time in patients with a history of active TB who did not receive modern anti-TB treatment. This might be the result of a persistent in vivo antigen load in recovered TB patients [6].
Our data show that T-SPOT®.TB results in patients with TB before 1955 were significantly lower than the results in patients with recent TB before treatment (T0). One could hypothesise that after successful treatment, the number of SFCs will decline with time. However, our study and the study of Wu-Hsieh et al. [7] did not report an association between the time after recovery and the number of spots.
Moreover, we found that the number of positive T-SPOT®.TB results before (T0) and after (T1) treatment in treated and cured patients with recent active TB were not significantly different. Only 12% of the individuals with a positive T-SPOT®.TB before treatment converted to negative after treatment. However, we found a significant decline in the number of ESAT-6-positive patients after treatment. Consistent with our results, several studies reported a significant decline in the number of spots in the ESAT-6 panel during or after anti-TB treatment [2, 8]. The studies by Aiken et al. [1] in Gambia and Dheda et al. [9] in South Africa reported not only a significant decline in the number of spots after treatment, but also a significant decline in the amount of positive results for both ESAT-6 and CFP-10, despite high re-infection rates in Gambia and South Africa. One can only hypothesise that these conflicting observations between these studies in high- and low-burden endemic countries might be due to differences in exposure, M. tuberculosis strain, genetics, infectious burden, treatment regime and the impact of comorbidity on the immune response to M. tuberculosis [10].
ELISPOT results from patients with active disease were significant higher than those of patients with TB before 1955. This might be due to an acquired state of latent infection with naturally declining kinetics of the T-SPOT®.TB in the last group. After effective anti-TB treatment, the number of spots declined, which might be due to the eradication of M. tuberculosis because of effective therapy. Combining these results, it might be suggested that anti-TB treatment accelerates the naturally declining kinetics of the T-SPOT®.TB [6].
This study has some limitations: 1) the limited number of patients; 2) because the T-SPOT®.TB assay was not available in 1955, we could not compare pre- and post-treatment results of these patients; and 3) selection bias could have been introduced, as patients with TB before 1955 who did not recover or even died were not included.
In conclusion, our report is the first to describe the naturally long-term kinetics of the ESAT-6 and CFP-10 panels of the T-SPOT®.TB in patients with a history of TB before the introduction of modern anti-TB treatment. T-SPOT®.TB results do not differ between recently treated TB patients and patient with TB before 1955 in a low-burden, endemic setting. Although the number of spots decline after treatment, the number of patients with T-SPOT®.TB conversion is disappointing, indicating that the T-SPOT®.TB might not be an appropriate tool for evaluating treatment outcome and efficacy.
Footnotes
Statement of Interest
A statement of interest for A.W.J. Bossink can be found at www.erj.ersjournals.com/site/misc/statement.xhtml
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