Risk assessment of tuberculosis in immunocompromised patients. A TBNET study.

RATIONALE: In the absence of active tuberculosis, a positive tuberculin skin test (TST) or interferon-γ release assay (IGRA) result defines latent infection with Mycobacterium tuberculosis, although test results may vary depending on immunodeficiency. OBJECTIVES: This study compared the performance of TST and IGRAs in five different groups of immunocompromised patients, and evaluated their ability to identify those at risk for development of tuberculosis. METHODS: Immunocompromised patients with HIV infection, chronic renal failure, rheumatoid arthritis, solid-organ or stem-cell transplantation, and healthy control subjects were evaluated head-to-head by the TST, QuantiFERON-TB-Gold in-tube test (ELISA), and T-SPOT.TB test (enzyme-linked immunospot) at 17 centers in 11 European countries. Development of tuberculosis was assessed during follow-up. MEASUREMENTS AND MAIN RESULTS: Frequencies of positive test results varied from 8.7 to 15.9% in HIV infection (n = 768), 25.3 to 30.6% in chronic renal failure (n = 270), 25.0% to 37.2% in rheumatoid arthritis (n = 199), 9.0 to 20.0% in solid-organ transplant recipients (n = 197), 0% to 5.8% in stem-cell transplant recipients (n = 103), and 11.2 to 15.2% in immunocompetent control subjects (n = 211). Eleven patients (10 with HIV infection and one solid-organ transplant recipient) developed tuberculosis during a median follow-up of 1.8 (interquartile range, 0.2-3.0) years. Six of the 11 patients had a negative or indeterminate test result in all three tests at the time of screening. Tuberculosis incidence was generally low, but higher in HIV-infected individuals with a positive TST (3.25 cases per 100 person-years) than with a positive ELISA (1.31 cases per 100 person-years) or enzyme-linked immunospot result (1.78 cases per 100 person-years). No cases of tuberculosis occurred in patients who received preventive chemotherapy. CONCLUSIONS: Among immunocompromised patients evaluated in this study, progression toward tuberculosis was highest in HIV-infected individuals and was poorly predicted by TST or IGRAs. Clinical trial registered with www.clinicaltrials.gov (NCT 00707317).

Antigen-specific CD4- and CD8-positive signatures in different phases of Mycobacterium tuberculosis infection.

Current diagnostic standards for Mycobacterium tuberculosis (MTB) infection do not distinguish between active and latent tuberculosis (TB). To identify specific biomarkers characterizing the different forms of TB infection, we investigated in parallel with the QuantiFERON -TB Gold In-Tube (QFT-IT) the use of flow cytometry measuring CD4 and CD8 MTB-specific immune response in 17 active-TB patients, 21 health care workers (HCW), 14 recent contacts of TB patients (RC-TB), and 10 bacille Calmette Guerin (BCG)-vaccinated healthy controls (BCG-HC). A correlation (r = 0.4526, P = 0.0002) was found only between the amount of IFN-γ measured by QFT-IT and the frequency of CD4+/CD69+/IFN-γ+ T cells. The frequency of CD4+/CD69+/IFNγ+ responding T cells was higher in active-TB patients (0.254 ± 0.336%, P < 0.01) compared to the other groups. The response of QFT-IT antigen-specific CD8+/CD69+/IFNγ+ T cells was significantly higher in RC-TB (0.245 ± 0.305%, P < 0.05) compared to the other study groups.

Association of age with the level of response in the QuantiFERON-TB Gold In-Tube assay for children with active tuberculosis.

QuantiFERON-TB data from 50 children with tuberculosis were analysed to evaluate age related effects. Significantly higher IFN-? responses to TB-specific antigens were associated with younger age, but no difference was found with Mitogen responses. Extrapolating IGRA responses to a Mitogen does not reflect those induced by an antigen-specific stimulus. QFT-IT responses to TB-specific antigens are not compromised with young age.

Rational use of immunodiagnostic tools for tuberculosis infection: guidelines and cost effectiveness studies.

Tuberculosis (TB) remains a public health challenge and its control requires the use efficient diagnostic tools. Mycobacterium tuberculosis (MTB) elicits a strong immune response upon infection, a phenomenon measured by the old tuberculin skin test (TST). However, this test has many limitations and a high rate of positivity in BCG-vaccinated subjects. Recent studies have identified several MTB-antigens for diagnostic use, including the ESAT-6 and CFP-10 proteins. Based on these antigens, one of the most significant developments in the diagnostic armamentarium for TB has been the assays based on IFN- determination (IGRAs). The assays stem from the principle that T-cells of infected individuals produce IFN-gamma when they re-encounter the MTB antigens in vitro and this can be measured by a conventional ELISA test. The evaluation of IGRAs in different clinical settings showed many advantages over TST. The worldwide diffusion of IGRAs has increased the knowledge on their clinical use and a number of guidelines have been devised for their application. The two-step approach (first using TST followed by IGRA for confirmation) is the most favored strategy for IGRA-use in the general population, while the use of IGRAs alone is suggested in particular clinical settings and/or patient groups. Even if these tests are still costly there are a number of cost effective advantages in the "targeted" use of IGRAs over the TST. The work we present summarises all these aspects.

Design of immunogenic peptides from Mycobacterium tuberculosis genes expressed during macrophage infection.

In vitro diagnosis of MTB-infection uses MTB-proteins coded for by genes of the region of differentiation 1 (RD1) of the MTB genome. This study wants to test if proteins preferentially expressed during MTB-intracellular growth might provide new targets for the diagnosis of MTB-infection. To this end seventy-five multiepitopic HLA-promiscuous MTB-peptides were designed by quantitative implemented peptide-binding motif analysis from 3 MTB-protein genes expressed in activated human macrophages (MA), 4 genes expressed during growth in non-activated human macrophages (MN-A), 12 housekeeping genes (HKG) and 6 genes of the RD1 region (RD1) as control. ELISpot for IFN-was performed to measure the responses of PBMCs deriving from 45 patients affected by active tuberculosis and 34 controls. In active-TB patients, the mean response to RD1-derived peptides was higher than that to either MA (p<0.01), MN-A (p<0.008) or HKG (p<0.01) derived peptides. In TST-positive subjects all selected peptides elicited significant IFN-T-cell responses (p<0.02 compared to TST-negatives), but without differences between the subgroups. Further, T-cell responses to RD1 peptides were lower in the 23 active-TB treated patients than in the untreated ones (p<0.01). The response to MA peptides in treated active-TB was higher than when untreated (p<0.01). These results demonstrate that the use of in vitro models of MTB-intracellular infection to select MTB gene products for further in silico and in vitro assessment of their immunogenicity have the potential to identify novel antigens amenable to the design of new tools for diagnosis and monitoring of tuberculosis.

Accuracy of immunodiagnostic tests for active tuberculosis using single and combined results: a multicenter TBNET-Study.

BACKGROUND: The clinical application of IFN-gamma 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.

Cellular and humoral systemic and mucosal immune responses stimulated in volunteers by an oral polybacterial immunomodulator "Dentavax".

The oral polybacterial immunomodulator Dentavax (D), composed of killed cells from Klebsiella pneumoniae, Streptococcus pyogenes, Staphylococcus aureus, Candida albicans and Lactobacillus acidophilus and their lysates was created for immunoprophylaxis and therapy of oral mucosa and parodont inflammations. The stimulating effect of the preparation was evaluated in twelve volunteers immunized for 10 consecutive days. On days 7, 14, 21, 28 and 49 after the last immunization peripheral blood (PB) lymphocyte subsets, T lymphocyte activation and PB phagocytic activity, were studied by flow cytometry. PB lymphocyte proliferative responses to PHA, rIL-2, LPS and D were evaluated radiometrically. The production of TNF-alpha in supernatants of in vitro stimulated lymphocytes and specific IgA, IgM and IgG antibodies in serum and saliva was determined by ELISA. Ultrastructural morphologic changes in T and B lymphocyte populations were also investigated. Although no significant changes in the levels of basic lymphocyte subsets were detected, the early/late (CD57+/CD57-) CD8 T effectors ratio was increased at the end of the studied period, as were the percentage of PHA-responding (CD69+) T cells and PB phagocytizing cells. The most prominent lymphoprolipherative responses were measured upon costimulation with LPS+D and PHA+D on day 21. Electron-microscopic studies demonstrated a significant effect of D on both T and B cell activity. TNF-alpha concentration increased progressively from day 7 till the end of the investigation. Maximal concentrations were observed after stimulation with D and LPS. An increased level of specific salivary and serum antibodies against the components of D was found, with highest levels between days 7 and 21. Specific secretory IgA predominated in saliva as compared to IgM and IgG. Our results demonstrate the stimulating effect of Dentavax on PB lymphocyte functional activity and the specific humoral systemic and mucosal immunity.