Santin et al. compared 18 studies to estimate the diagnostic performance of TSPOT and estimated a pooled rate of indeterminate results that was 5.9 % in a total of 2239 individuals. However, the number of indeterminate tests in low and intermediate burden countries was only 3.5 %. [8] Our rate of indeterminate results was 6.1 % out of 914 tests and thus higher than comparators for a low burden country. Lee et al. found a similar rate of indeterminate TSPOT (8.7 %) in their study population of patients with suspected extra-pulmonary tuberculosis and no correlation between indeterminate result and immunosuppression, however they did not analyse the different immunosuppressive medications. [9]
In a number of studies, indeterminate test results have been found more frequently in patients with malignant diseases, patients receiving immunosuppressive treatment and patients with consumptive (under nutrition) conditions for both T.Spot-TB and QFT . [10]
HIV and indeterminate test results
Several studies have evaluated the performance of both TSPOT and QFT-GIT in HIV positive individuals. The sensitivity of TSPOT in HIV positive individuals has been found to be lower in comparison to HIV negative individuals. [8, 11]
In our study, out of 88 HIV infected individuals, 11 (11.7 %) had indeterminate results, whereas among the HIV uninfected individuals there were only 5.5 % of indeterminate results. This outcome is consistent with the findings of other reports, as the number of indeterminate test results both in QFT-GIT and TSPOT has been known to be higher in HIV-positive than in HIV-negative individuals. Of all of the immunmodulating factors, HIV-positivity was the only one that reached statistical significance on TSPOT result validity.
CD4+ and indeterminate test results
The sensitivity of TSPOT has been equally high in patients with normal CD4+ T-cell count as in patients with low CD4+ T-cell count in two studies. [12, 13] Also, it could not be confirmed that the level of circulating CD4+ T-cells was in any way related to the number of indeterminate test results. [11, 14] According to these studies, the level of CD4+ T-cell depletion in HIV-positive individuals does not interfere with the functionality of TSPOT. However, various studies have confirmed that QFT-GIT and TST are both influenced by the number of circulating CD4+ T cells, more accurately, that the QFT-GIT result inversely correlated with CD4+ lymphocyte count. [14, 15]
This finding may indicate that the diagnostic performance of TSPOT may be superior to QFT-GIT in patients that are HIV positive and have a low CD4+ T-cell count. In our study, we did not statistically analyse the influence of the CD4+ T-cell count on the performance of TSPOT as most of the HIV positive patients were undergoing antiretroviral treatment and a current CD4+ T-cell count was not available for most of our patients. It would thus be reasonable to assume that the majority of patients on ART would have an adequate CD4+ count, however this remains speculative.
Immunosuppressive medication and indeterminate test results
Previous studies assessing the effect of immunosuppressive treatment on IGRA performance have been inconsistent. While some studies detected an effect on IGRA performance, especially on QFT-GIT performance, others, similar to our analysis, found no effect for TSPOT. [6, 16]
In 2017, Edwards et al. conducted a study that used an ex vivo model to determine the influence of corticosteroids and infliximab on the performance of QFT-GIT assays. [17] Both corticosteroids and infliximab were added after the blood was taken from the patient, the results of these assays were compared with the test results of patients’ blood without added drugs. Both drugs impaired the QFT-GIT performance significantly. Unfortunately, there is to date no such study for TSPOT.
Cortisone and indeterminate test results
Corticosteroids have been known to influence and suppress the cytokine and chemokine production of T-cells, including IFN-g. [18, 19] This effect explains why the performance of QFT-GIT in patients receiving corticosteroids is impaired compared to the performance of patients not taking any corticosteroids. A significant increase in indeterminate tests due to reduced IFN-g release in the control after mitogen stimulation (PHA) was reported by Latorre et al. in patients receiving steroids. [20] In a QuantiFERON Gold in-tube assay they were able to show that this effect was dose related with a sharp decrease in IFN-g release at doses greater than 20 mg/day.
Belard et al. demonstrated that oral prednisolone suppresses both QFT-GIT and TST performance and was associated with a greater risk of indeterminate QFT-GIT results (adjusted odds ratio, 16.1) whereas long-acting corticosteroids and other immunosuppressants do not have a similar effect. [6] This effect was dose related with doses of > 10 mg prednisolone per day associated with a 27 % risk of an indeterminate result. A recent study reported a significant risk of indeterminate results in patients receiving steroids using the QuantiFERON Gold in-tube assay but were not able to demonstrate a dose-related effect. [21] Another study conducted by Arias-Guillén in 2014 underlined these findings; all patients with indeterminate test results in QFT-GIT were also receiving corticosteroid treatment. [22] In this study T-cell sub-popultions were analysed by flow cytometry and the authors were able to demonstrate that in 9 of 12 indeterminate TSPOT assays the T-cell sub-population counts for CD3+, CD4+ and CD8+ cells were normal. None of these studies found any statistically significant evidence that prednisolone or corticosteroids in general influence the functionality of TSPOT. While in our study the number of indeterminate test results among patients taking corticosteroids was higher than in the control group, the statistical analysis did not support the hypothesis that corticosteroids impair TSPOT validity.
CIBD, Psoriasis vulgaris and indeterminate test results
Neither CIBD nor Psoriasis vulgaris were associated with indeterminate test results. Interestingly, patients with psoriasis vulgaris had a lower rate of indeterminate test results than patients who do not have psoriasis vulgaris.
There are several limitations to our study. We were unable to distinguish between different groups of immunosuppressive agents and doses and also did not account for duration of treatment or disease activity. All of these factors could have an effect on indeterminate results. In addition, the sub-group analysis only contained relatively small numbers of patients, which may impact on the ability to detect significant differences. HIV was associated with a higher risk of indeterminate result and it would be logical to assume that the CD4+ cell count would influence this observation; we were unable to determine with accuracy the current cell count as most patients were on a stable treatment regime. That the CD4+ cell count was likely to be high is however speculative. Other determinants for an indeterminate result, such as hypoalbuminaemia or anaemia [23] were not measured as this study was primarily aimed at the effect of steroid use on TSPOT performance. Furthermore, the study was performed retrospectively and thus not all datasets could be included as the medical records were not always complete.
In conclusion, the study demonstrated that steroid therapy did not influence the validity
of the T.Spot-TB IGRA and secondarily that, of the conditions investigated in this study, only HIV was associated with an increased rate of indeterminate tests.