Ilaria Sauzullo

Accuracy of Immunodiagnostic Tests for Active Tuberculosis Using Single and Combined Results: A Multicenter TBNET-Study

Background The clinical application of IFN-γ release assays (IGRAs) has recently improved the diagnosis of latent tuberculosis infection. In a multicenter study of the Tuberculosis Network European Trialsgroup (TBNET) we aimed to ascertain in routine clinical practice the accuracy of a novel assay using selected peptides encoded in the mycobacterial genomic region of difference (RD) 1 for the diagnosis of active tuberculosis in comparison with tuberculin skin test (TST), QuantiFERON-TB GOLD In-Tube (Cellestis Ltd., Carnegie, Australia) and T-SPOT.TB (Oxfordimmunotec, Abingdon, UK). Principal Findings 425 individuals from 6 different European centres were prospectively enrolled. We found that sensitivity of the novel test, TST, QuantiFERON-TB GOLD In-Tube and T-SPOT.TB was respectively 73.1%, 85.3%, 78.1%, and 85.2%; specificity was respectively 70.6%, 48.0%, 61.9% and 44.3%; positive likelihood ratios were respectively 2.48, 1.64, 2.05, and 1.53; negative likelihood ratios were respectively 0.38, 0.31, 0.35, 0.33. Sensitivity of TST combined with the novel test, QuantiFERON-TB GOLD In-Tube and T-SPOT.TB increased up to 92.4%, 97.7% and 97.1%, respectively. The likelihood ratios of combined negative results of TST with, respectively, the novel test, QuantiFERON-TB GOLD In-Tube and T-SPOT.TB were 0.19, 0.07 and 0.10. Conclusions The assay based on RD1 selected peptides has similar accuracy for active tuberculosis compared with TST and commercial IGRAs. Then, independently of the spectrum of antigens used in the assays to elicit mycobacterial specific immune responses, the novel test, IGRAs, and the TST do not allow an accurate identification of active tuberculosis in clinical practice. However, the combined use of the novel assay or commercial IGRAs with TST may allow exclusion of tuberculosis.

In Vivo and In Vitro Effects of Antituberculosis Treatment on Mycobacterial Interferon-γ T Cell Response

Background In recent years, the impact of antituberculous treatment on interferon (IFN)-γ response to Mycobacterium tuberculosis antigens has been widely investigated, but the results have been controversial. The objective of the present study was: i) to evaluate longitudinal changes of IFN-γ response to M. tuberculosis-specific antigens in TB patients during antituberculous treatment by using the QuantiFERON-TB Gold (QFT-G) assay; ii) to compare the differences in T-cell response after a short or prolonged period of stimulation with mycobacterial antigens; iii) to assess the CD4+ and CD8+ T cells with effector/memory and central/memory phenotype; iv) to investigate the direct in vitro effects of antituberculous drugs on the secretion of IFN-γ. Principal Findings 38 TB patients was evaluated at baseline and at month 2 and 4 of treatment and at month 6 (treatment completion). 27 (71%) patients had a QFT-G reversion (positive to negative) at the end of therapy, while 11 (29%) TB patients remained QFT-G positive at the end of therapy. Among the 11 patients with persistent positive QFT-G results, six had a complete response to the treatment, while the remaining 5 patients did not have a resolution of the disease. All 27 patients who became QFT-G negative had a complete clinical and microbiological recovery of the TB disease. In these patients the release of IFN-γ is absent even after a prolonged 6-day incubation with both ESAT-6 and CFP-10 antigens and the percentage of effector/memory T-cells phenotype was markedly lower than subjects with persistent positive QFT-G results. The in vitro study showed that antituberculous drugs did not exert any inhibitory effect on IFN-γ production within the range of therapeutically achievable concentrations. Conclusions The present study suggests that the decrease in the M. tuberculosis-specific T cells responses following successful anti-TB therapy may have a clinical value as a supplemental tool for the monitoring of the efficacy of pharmacologic intervention for active TB. In addition, the antituberculous drugs do not have any direct down-regulatory effect on the specific IFN-γ response.

Influence of previous tuberculin skin test on serial IFN-γ release assays

Tuberculin skin test (TST) and interferon-γ release assays (IGRAs) have been proposed for serial testing in tuberculosis. In the present study, we assessed the effect of TST on subsequent QuantiFERON-TB Gold In-Tube (QFT-GIT) results by monitoring the evolution of responses during a follow-up period of 6 weeks. One hundred and two subjects were initially tested with QFT-GIT and subsequently with TST; then the QFT-GIT was performed serially 1, 2, 4, and 6 weeks after the TST. A subgroup of 40 subjects was also assessed by older version of the QuantiFERON-TB Gold (QFT-G) assay. The results showed no significant variation in IFN-γ response over time in the tested patients, although two TST-positive subjects showed evidence of possible boosting effect. In addition, a direct comparison between the QFT-G and QFT-GIT test showed no significant differences at any time point with excellent agreement between two tests. No significant differences were seen in IFN-γ responses between BCG-unvaccinated and BCG-vaccinated patients at each time point. In conclusion, our findings indicate that TST does not influence the outcome of subsequent IGRAs testing in individuals with negative TST results, but it can boost the IFN-γ response in subjects sensitized to TB antigens and not detected by IGRA.

Mycobacterial Interferon-γ Release Variations During Longterm Treatment with Tumor Necrosis Factor Blockers: Lack of Correlation with Clinical Outcome

Objective. To assess the performance of serial QuantiFeron-TB Gold In-Tube (QFT-GIT) tests in patients with rheumatic diseases during longterm systemic treatment with biologic therapy, evaluating conversions and reversions in relation to the clinical outcome. Methods. We conducted a prospective study on patients awaiting biologic agents. At baseline, they had chest radiographs, QFT-GIT tests, and tuberculin skin tests (TST); QFT-GIT was repeated at 3, 6, 12, and 18 months after onset of biologic therapy. In patients with no evidence of latent tuberculosis infection (LTBI) at baseline, TST was repeated at 12 months of biologic treatment. Results. Among patients (n = 102; women 65.7%; median age 47 yrs, range 20–82), 14 (13.7%) were considered as having LTBI because of a minimum of 1 abnormal screening test. The agreement between QFT-GIT and TST was 88% (κ = 0.14). During biologic treatment, both patients with (n = 14) and those without (n = 88) evidence of LTBI at baseline showed conversions and reversions in QFT-GIT results at different timepoints. These fluctuations were not paralleled by significant clinical changes. The TST repeated at 12 months in patients with no evidence of LTBI at baseline continued to be negative. The median baseline interferon-γ (IFN-γ) concentration was not significantly different from that observed at each subsequent timepoint. Conclusion. Dynamic changes occur with serial IFN-γ release assay testing in patients treated with biologic therapy that do not correlate with clinical outcome. A careful and integrated evaluation of the patient, including clinical information, should guide the treatment decision. This study was underpowered for definite conclusions and further studies are needed to determine the significance of these findings.

Plasmacytoid Dendritic Cells Count in Antiretroviral-Treated Patients is Predictive of HIV Load Control Independent of CD4+ T-Cell Count

The depletion in circulating dendritic cells (DCs) and inverse correlation with viral load have been described in human immunodeficiency virus (HIV)-infected patients. The aim of this study was to investigate whether the DC blood count in antiretroviral-treated patients might be predictive of viral load control independent of CD4+ T cell count. Plasmacytoid DCs (pDCs) and myeloid DCs (mDCs) were enumerated using a newly developed flow cytometric assay based on TruCOUNT. A significant reduction of circulating pDCs and mDCs was detected both in untreated and -treated subjects. The probability of experiencing viral load increase according to pDC, and CD4 count at baseline was evaluated in 39 treated patients. Individuals with lower baseline pDCs were more likely to have an increase of HIV-RNA during the 30 month follow-up in comparison with patients with high pDCs (p <0.001). In particular, the pDC measurement may be useful in the context of a high CD4 count, to distinguish the patients who have virological failure despite high CD4 counts. These findings suggest that in treated patients the enumeration of circulating DCs, especially pDC count, can augment the predictive value of CD4 measurement in predicting virologic failure.

Severe and Persistent Depletion of Circulating Plasmacytoid Dendritic Cells in Patients with 2009 Pandemic H1N1 Infection

Background Dysregulation of host immune responses plays a critical role in the pathogenesis of severe 2009 pandemic H1N1 infection. Whether H1N1 virus could escape innate immune defense in vivo remains to be investigated. The aim of this study was to evaluate the pattern of innate immune response during human 2009 H1N1 infection. We performed the enumeration of circulating myeloid dendritic cells (mDC) and plasmacytoid DC (pDC) in blood from patients with H1N1 pneumonia shortly after the onset of symptoms and during follow-up at different intervals of time. The analysis of CD4 and CD8 count, CD38 T-cell activation marker and serum cytokine/chemokine plasma levels was also done. Methodology/Principal Findings Blood samples were collected from 13 hospitalized patients with confirmed H1N1-related pneumonia at time of admission and at weeks 1, 4, and 16 of follow-up. 13 healthy donors were enrolled as controls. In the acute phase of the disease, H1N1-infected patients exhibited a significant depletion in both circulating pDC and mDC in conjunction with a decrease of CD4 and CD8 T cell count. In addition, we found plasmatic hyperproduction of IP-10 and RANTES, whereas increase in T-cell immune activation was found at all time points. When we assessed the changes in DC count over time, we observed a progressive normalization of mDC number. On the contrary, H1N1-infected patients did not achieve a complete recovery of pDC count as values remained lower than healthy controls even after 16 weeks of follow-up. Conclusions H1N1 disease is associated with a profound depletion of DC subsets. The persistence of pDC deficit for several weeks after disease recovery could be due to H1N1 virus itself or to a preexisting impairment of innate immunity.

HIV protease inhibitor therapy reverses neutrophil apoptosis in AIDS patients by direct calpain inhibition

The reduction of neutrophils apoptosis is one of the main non-virological effects of protease inhibitor (PI) therapy. We explore here whether this may be due to the cross-inhibition of calpain, an important non-virological target of PI in vitro. We found that the high basal level of neutrophils apoptosis in AIDS patients is strictly related to an increased intracellular calpain activity. Both alterations disappear after PI treatment, with apoptosis and calpain going back to normal levels after 3 months of PI therapy, independently of a proficient antiviral effect. PI drugs exerted a similar antiapoptotic and anticalpain effects on neutrophils in ex vivo experiments: strikingly, the effects were mimicked by commercially available calpain inhibitors. This study shows, for the first time, that apoptosis of neutrophils in AIDS patients is mediated by calpain, and that neutrophil survival in PI treated AIDS patients is a non virological effect due to calpain inhibition.

Multifunctional Analysis of CD4+ T-Cell Response as Immune-Based Model for Tuberculosis Detection

Mono- and multifunctional specific CD4+ and CD8+ T-cell responses were evaluated to improve the immune-based detection of active tuberculosis (TB) and latent infection (LTBI). We applied flow cytometry to investigate cytokines profile (IFN-γ, TNF-α, and IL-2) of T cells after stimulation with TB antigens in 28 TB-infected subjects (18 active TB and 10 LTBI) and 10 uninfected controls. Cytokines production by CD4+ T cells at single-cell levels was higher in TB-infected subjects than uninfected controls (P < 0.0001). Assigning to activated CD4+ T cells, producing any of the three cytokines, a cut-off >0.45%, it was possible to differentiate TB-infected (>0.45%) by uninfected subjects (<0.45%). Among TB-infected subjects, the frequencies of multifunctional CD4+ T cells, simultaneously producing all 3 cytokines, are lower in active TB than LTBI subjects (P = 0.003). Thus, assigning to triple-positive CD4+ T cells a cut-off <0.182%, TB-infected individuals could be classified as active TB subjects (<0.182%) or LTBI subjects (>0.182%). The magnitude of CD8+ T-cell responses showed no differences between active TB and LTBI. Multifunctional CD4+ T-cell responses could have the potential to identify at single time point subjects without TB infection and patients having active or latent TB.

QuantiFERON-TB Gold and selected region of difference 1 peptide-based assays for the detection of Mycobacterium tuberculosis infection in a cohort of patients enrolled with suspected tuberculosis

Recently, a simple whole blood test, QuantiFERON-TB Gold (QFT-Gold), has been adopted for immunologic diagnosis of Mycobacterium tuberculosis infection. A total of 369 individuals with suspected tuberculosis (TB) were tested with the QFT-Gold, whereas a subgroup of 62 patients was tested simultaneously with QFT-Gold test and an assay based on selected peptides from early secreted antigenic target 6 and culture filtrate protein 10 proteins. The sensitivity of the QFT-Gold assay for active TB was 72.9%, and the agreement between this test and tuberculin skin test was 75%. Using selected peptides, we found a positive response in the majority of active TB patients, with a sensitivity of 84% and a specificity of 89%. Our study suggests the usefulness of QFT-Gold in routine clinical practice on unselected patients with suspected TB. Moreover, the assay based on selected peptides is a useful tool to discriminate between active and latent TB and shows a high predictive value.

Long-term IFN-γ and IL-2 response for detection of latent tuberculosis infection in healthcare workers with discordant immunologic results

Discordant results between the interferon-gamma release assays (IGRAs) and tuberculin skin test (TST) are common in latent tuberculosis infection (LTBI). We evaluated whether the measurement of IFN-γ and interleukin (IL)-2T-cell responses, after prolonged Mycobacterium tuberculosis-specific antigen stimulation, can be used as adjunctive biomarker for LTBI detection in subjects with discordant results between TST and QuantiFERON-Gold In-Tube (QFT). 196 healthcare workers were screened for LTBI and in 90 of those participants, the QFT was repeated after 18 h, and IFN-γ/IL-2 immune response was measured after 72 h long-term stimulation. Of the 196 patients, 34 had positive, 155 negative, and 7 indeterminate QFT results. Discordant TST+/QFT- results were found in 29 (14.7%) patients, of whom 6 (20.6%) were Bacillus Calmette-Guerin (BCG) vaccinated. None of 23 non-BCG vaccinated subjects showed a specific IFN-γ immune response after 18 h nor 72 h of incubation, whereas 3/23 (13.04%) discordant subjects produced a specific long-term IL-2 response, which might reflect a LTBI status. In LTBI group (TST+/QFT+) both cytokine levels were increased after long-term in comparison to short-term stimulation. No significant long-term IFN-γ/IL-2 secretion was detected in control group (TST-/QFT-). Taken together, our data showed that the 87% of discordant patients who did not respond to the long-term assay, as controls subjects, were judged LTBI negative. The use of classic QFT and long-term IL-2 response may have a potential role to clarify the LTBI status in individuals in whom the diagnosis of LTBI is uncertain due to the discordance of the available diagnostic tests, such as TST and IGRA.