This study evaluating an interferon-gamma release assay, T-SPOT.TB, in 64 HIV-infected adults who all developed culture-confirmed TB after enrolment in the Swiss HIV Cohort Study indicates that T-SPOT.TB has a similar sensitivity to TST to detect latent TB in HIV-infected individuals. However, there was poor agreement between T-SPOT.TB and TST results. In contrast to TST, sensitivity of T-SPOT.TB was independent of the level of immunodeficiency, although a trend towards higher number of spot forming units with increasing CD4 cell count was observed in patients with a positive T-SPOT.TB. Importantly, combination of TST and T-SPOT.TB with at least one test positive resulted in improved sensitivity. This is likely to be useful in clinical practice to better identify HIV-infected individuals with latent TB who qualify for preventive treatment.
Sensitivity of T-SPOT.TB of 39-58% in our study was lower than in published studies [10, 11, 23, 26–28]. In the absence of a gold standard to diagnose latent or culture negative TB, assessments of accuracy of tests for TB are difficult, especially in the context of immunodeficiency. So far, sensitivity of IGRA for latent TB has been mainly estimated by comparison with TST in cross-sectional studies, or by assessing the number of positive IGRA results in patients with culture-confirmed TB. Because TST and IGRA are indirect tests that measure immunologic responses and do not detect the causative organism, assessment of sensitivity among persons with positive TST or active TB might not reliably estimate sensitivity for latent TB. In our study, T-SPOT.TB was performed in a highly selected population, namely HIV-infected individuals shortly before they developed culture-confirmed TB, providing a reliable approximation of patients with latent TB in an area of low TB transmission, since reinfection with M. tuberculosis was unlikely. The quite low sensitivity of T-SPOT.TB in our population was independent of socio-demographic characteristics, including ethnicity and country of origin, level of immunodeficiency and antiretroviral treatment, suggesting that immunologic differences implied in the progression of latent infection to active disease might negatively affect IGRA results. The suboptimal sensitivity of Interferon-based in-vitro assays, especially in immunocompromised individuals, emphasizes the need for alternative markers for diagnosing tuberculosis, such as interferon-inducible protein (IP-10), which appeared less influenced by HIV infection. Further studies are needed to test the clinical impact of these findings [15, 29].
The major concern in using T-cell based assays in the setting of HIV infection is the influence of immunodeficiency on sensitivity. While some studies suggested that IGRA were less influenced by HIV infection [15, 18, 26–28, 30, 31] other studies reported loss of sensitivity with severe immunodeficiency, in particular when CD4 cell counts were less than 100 cells/μl [15, 28, 30, 32–35]. We observed a clear association between CD4 cell count and TST but not between CD4 cell count and T-SPOT.TB, although a trend towards higher number of spot forming cells with increasing CD4 cell count was noted in patients with positive T-SPOT.TB result. As both tests rely on a T-cell mediated immune response, this difference might also result from the cut-off chosen for scoring a positive T-SPOT.TB. A recent publication suggests that accuracy in HIV-infected patients improves when the number of spot forming cells from the T-SPOT.TB test is related to CD4 count .
In line with prior studies [18, 28, 37–39], we observed poor agreement between TST and T-SPOT.TB. However, combination of TST and T-SPOT.TB with at least one test positive was shown to enhance sensitivity to detect latent TB in HIV co-infection. This is clinically relevant, since easier and more accurate diagnosis of latent TB implies start of preventive treatment, which was demonstrated to be highly effective . In multivariate analysis, older age was the only risk factor of scoring both tests negative, indicating a limitation of this strategy.
The high number of indeterminate test results in our study was not associated with lower CD4 cell counts in contrast to other reports [37, 38, 40]. As no correlation between time of lymphocyte sampling and performance of T-SPOT.TB was noted, it is also unlikely that the quality of cell samples was impaired.
Limitations and strengths
We acknowledge some limitations. The use of frozen stored lymphocytes to perform T-SPOT.TB might have impaired the ability of producing gamma-interferon, leading to an underestimation of sensitivity. However, viability of cells was meticulously checked, validated in several studies of the Swiss HIV Cohort Study [23–25], and T-SPOT.TB was always performed together with mitogens and nil controls, as described in the method section. As TB negative subjects were not included in our study, we could not estimate specificity of T-SPOT.TB and TST. However, the focus of this study was on sensitivity, because improvement of sensitivity is the main diagnostic need in latent TB in HIV-infected individuals. Due to the retrospective character of this study, results of TST were not available for all patients, and information on previous BCG vaccination was not collected in the SHCS database. Due to the small amount of viable peripheral blood mononuclear cells routinely stored in the Swiss HIV Cohort Study (3 aliquots of 1.5 million cells) we were not able to perform additional analysis such as the measurement of T regulatory cells. Moreover, we were not able to repeat T-SPOT.TB at later time points before culture-confirmed active tuberculosis was diagnosed, because cells samples were collected only once a year after enrolment in the Swiss HIV Cohort Study according to the protocol.
Strengths of this study were the inclusion of HIV-infected individuals with latent TB who all developed culture-confirmed TB in a low endemic area, and evaluation of combined testing with TST to enhance sensitivity of T-SPOT.TB. This is clinically relevant, since easier and more accurate diagnosis of latent TB implies start of preventive chemotherapy, that was shown to be highly effective in HIV-infected individuals .