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Goletti D.,Lazzaro Spallanzani National Institute for Infectious Diseases INMI | Raja A.,Tuberculosis Research Center | Kabeer B.S.A.,Tuberculosis Research Center | Rodrigues C.,Pd Hinduja National Hospital And Research Center | And 9 more authors.
PLoS ONE | Year: 2010

Background: The suboptimal sensitivity of Interferon (IFN)-γ-based in-vitro assays, especially in immunocompromised individuals, emphasizes the need for alternative markers for diagnosing tuberculosis (TB). The objective of this study was to evaluate whether interferon-inducible protein (IP)-10, monocyte chemotactic protein (MCP)-2 and interleukin (IL)-2 can be useful biomarkers for evaluating a specific response to RD1 antigens associated to active TB disease in HIV-infected individuals. Methodology/Principal Findings: The study was carried out in India, the country with the highest TB burden in the world. Sixty-six HIV-infected individuals were prospectively enrolled, 28 with active-pulmonary-TB and 38 without. The whole blood assay based on RD1-selected peptides (experimental test) and QuantiFERON-TB Gold In tube (QFT-IT) was performed. Plasma was harvested at day-1-post-culture and soluble factors were evaluated by ELISA. The results indicate that by detecting IP-10, the sensitivity of the experimental test and QFT-antigen (75% and 85.7% respectively) for active TB was higher compared to the same assays based on IFN-γ (42.9% and 60.7% respectively) and was not influenced by the ability to respond to the mitogen. By detecting IP-10, the specificity of the experimental test and QFT-antigen (57.9% and 13.2% respectively) for active TB was lower than what was reported for the same assays using IFN-γ-detection (78.9% and 68.4% respectively). On the other side, in vitro IL-2 and MCP-2 responses were not significantly associated with active TB. Conclusions: HIV infection does not impair RD1-specific response detected by IP-10, while it significantly decreases IFN-γ-mediated responses. At the moment it is unclear whether higher detection is related to higher sensitivity or lower specificity of the assay. Further studies in high and low TB endemic countries are needed to elucidate this. © 2010 Goletti et al.


Kabeer B.S.A.,Tuberculosis Research Center | Raja A.,Tuberculosis Research Center | Raman B.,Tuberculosis Research Center | Thangaraj S.,bioMerieux | And 5 more authors.
BMC Infectious Diseases | Year: 2011

Background: There is an urgent need of prognosis markers for tuberculosis (TB) to improve treatment strategies. The results of several studies show that the Interferon (IFN)-γ-specific response to the TB antigens of the QuantiFERON TB Gold (QFT-IT antigens) decreases after successful TB therapy. The objective of this study was to evaluate whether there are factors other than IFN-γ [such as IFN-γ inducible protein (IP)-10 which has also been associated with TB] in response to QFT-IT antigens that can be used as biomarkers for monitoring TB treatment.Methods: In this exploratory study we assessed the changes in IP-10 secretion in response to QFT-IT antigens and RD1 peptides selected by computational analysis in 17 patients with active TB at the time of diagnosis and after 6 months of treatment. The IFN-γ response to QFT-IT antigens and RD1 selected peptides was evaluated as a control. A non-parametric Wilcoxon signed-rank test for paired comparisons was used to compare the continuous variables at the time of diagnosis and at therapy completion. A Chi-square test was used to compare proportions.Results: We did not observe significant IP-10 changes in whole blood from either NIL or QFT-IT antigen tubes, after 1-day stimulation, between baseline and therapy completion (p = 0.08 and p = 0.7 respectively). Conversely, the level of IP-10 release to RD1 selected peptides was significantly different (p = 0.006). Similar results were obtained when we detected the IFN-γ in response to the QFT-IT antigens (p = 0.06) and RD1 selected peptides (p = 0.0003). The proportion of the IP-10 responders to the QFT-IT antigens did not significantly change between baseline and therapy completion (p = 0.6), whereas it significantly changed in response to RD1 selected peptides (p = 0.002). The proportion of IFN-γ responders between baseline and therapy completion was not significant for QFT-IT antigens (p = 0.2), whereas it was significant for the RD1 selected peptides (p = 0.002), confirming previous observations.Conclusions: Our preliminary study provides an interesting hypothesis: IP-10 response to RD1 selected peptides (similar to IFN-γ) might be a useful biomarker for monitoring therapy efficacy in patients with active TB. However, further studies in larger cohorts are needed to confirm the consistency of these study results. © 2011 Kabeer et al; licensee BioMed Central Ltd.


PubMed | National Institute for Infectious Diseases L Spallanzani INMI, INMI, Scientific Direction and San Raffaele Scientific Institute
Type: Journal Article | Journal: The Journal of infection | Year: 2016

QuantiFERONWe enrolled 27 active tuberculosis (TB) patients and 30 LTBI individuals. Following stimulation with TB1 and TB2, antigen-specific T-cells were characterized by flow cytometry. Data were also correlated with the grade of TB severity.TB1 mainly elicited a CD4 T-cell response while TB2 induced both CD4 and CD8 responses. Moreover, the TB2-specific CD4 response was detected for both active TB and LTBI patients, whereas the TB2-specific CD8 response was primarily associated with active TB (p=0.01).To our knowledge, we report the first characterization of the CD4 and CD8 T-cell response to QFT-Plus. CD8 T-cell response is mainly due to TB2 stimulation which is largely associated to active TB. These results provide a better knowledge on the use of this assay.


Ammassari A.,INMI | Cicconi P.,Institute of Tropical Medicine | Ladisa N.,University of Bari | Di Sora F.,Hospital San Giovanni Addolorata | And 6 more authors.
HIV Medicine | Year: 2013

Objectives: The aim of the study was to investigate whether HIV diagnosis affected reproductive planning over time and to assess independent predictors of abortion overall and following HIV diagnosis. Methods: Donne con Infezione da HIV (DIDI) is an Italian multicentre study based on a questionnaire survey carried out in 585 HIV-positive women between November 2010 and February 2011. The incidence and predictors of abortion were measured by person-years analysis and Poisson regression. Results: The crude incidence rate of abortion was 18.8 [95% confidence interval (CI) 16.5-21.4] per 1000 person-years of follow-up (PYFU). Compared with women who terminated their pregnancy before HIV diagnosis, women who terminated their pregnancy after HIV diagnosis but before 1990 showed a 2.56-fold (95% CI 1.41-4.65) higher risk. During 1990-1999 and 2000-2010, HIV diagnosis was not significantly associated with outcome [adjusted rate ratio (ARR) 0.93 (95% CI 0.55-1.59) and ARR 0.69 (95% CI 0.32-1.48), respectively]. Age [ARR 0.96 (95% CI 0.94-0.99) per 1 year older] and injecting drug use [ARR 1.38 (95% CI 0.98-1.94)] were found to be predictors of abortion overall. After HIV diagnosis, being on combination antiretroviral therapy [ARR 0.54 (95% CI 0.28-1.02)], monthly income<€800 [ARR 1.76 (95% CI 0.99-3.12)], younger age [ARR 0.95 (95% CI 0.91-1.00) per 1 year older] and fear of vertical transmission [ARR 1.95 (95% CI 1.04-3.67)] were found to be independently associated with abortion. Conclusions: We observed a higher incidence of abortion compared with data available for the general Italian population. Awareness of HIV diagnosis was predictive of abortion only in the 1980s. Women with HIV infection are still worried about vertical HIV transmission. Interventions promoting HIV screening among women who plan to have an abortion and informative counselling on motherhood planning in the setting of HIV care are needed. © 2012 British HIV Association.


Vanini V.,L Spallanzani National Institute For Infectious Diseases Inmi | Petruccioli E.,L Spallanzani National Institute For Infectious Diseases Inmi | Gioia C.,Laboratory of Cellular Immunology | Cuzzi G.,L Spallanzani National Institute For Infectious Diseases Inmi | And 9 more authors.
Journal of Infection | Year: 2012

Objective: In Indian HIV-infected patients, IP-10 response to QuantiFERON-TB Gold In tube (QFT-IT) antigens has been associated to tuberculosis (TB). However, specificity for active TB was lower than that reported by QFT-IT, making accuracy for TB detection questionable. To investigate this uncertainty, likely due to India being highly endemic for TB, and to better identify TB correlates, we evaluated the IP-10-based assay in HIV-infected subjects in Italy, a low-TB endemic country. Methods: 195 individuals were prospectively enrolled; 118 were HIV-infected (21 with active TB, 97 without active TB, and distinguished as high/low-TB-risk). QFT-IT was performed and IP-10 was evaluated by ELISA. Results: Among the HIV-infected individuals, sensitivity for active TB was 66.7% by IP-10-based test and 52.4% (p = 1) by QFT-IT. IP-10-based assay showed a lower dependence on mitogen-response and CD4 counts than QFT-IT. Among subjects without active TB, a higher proportion of IP-10 responders was shown in high-TB-risk subjects than low-TB-risk subjects (40.0% vs 12.9%), similar to QFT-IT (37.1% vs 4.8%). Low-TB risk subjects showed 87.1% specificity for active TB by IP-10-based test vs 95.2% by QFT-IT. Conclusions: In a low-TB endemic country, besides IFN-γ, IP-10 response to QFT-IT is associated with active TB and TB risk factors in HIV-infected patients with lower dependence on mitogen-response and CD4 counts. © 2012 The British Infection Association.


Chiacchio T.,L Spallanzani National Institute For Infectious Diseases Inmi | Petruccioli E.,L Spallanzani National Institute For Infectious Diseases Inmi | Vanini V.,L Spallanzani National Institute For Infectious Diseases Inmi | Cuzzi G.,L Spallanzani National Institute For Infectious Diseases Inmi | And 5 more authors.
Journal of Infection | Year: 2014

Objectives: Polyfunctional T-cells associate with chronic viral infection control while their involvement in tuberculosis (TB) is unclear. We evaluated TB-specific polyfunctional T-cell response and memory status in antiretroviral treatment (ART)-naïve HIV-infected patients from a low TB-endemic country. Methods: We prospectively enrolled HIV-infected patients, 12 with active TB (HIV-TB) and 15 with latent tuberculosis infection (LTBI). Peripheral blood cells were stimulated with TB antigens (RD1 proteins/peptides), HIV antigens, cytomegalovirus (CMV) and staphylococcal enterotoxin B (SEB) and analyzed by cytometry. Results: The HIV-TB showed a higher frequency of polyfunctional CD4+ T-cells in response to RD1 antigens than HIV-LTBI (p=0.007). Among the CD8+ T-cells, both groups showed a significantly higher frequency of RD1-specific monofunctional cells than polyfunctional cells(p=0.03). Analyzing the cytokine profile, IFNγ+ TNFα+ CD4+ T-cells associated with HIV-TB (p≤0.02) whereas IL2+ TNFα+ associated with HIV-LTBI (p=0.009). CD4+ T-cell response presented an effector-memory status in HIV-TB (p=0.007) and an effector-memory terminally-differentiated phenotype in HIV-LTBI (p=0.03). CD8+ T-cell response presented an effector status in HIV-LTBI (p=0.02). No significant cytokine profile pattern associated with responses to the other stimuli tested. Conclusions: In HIV-infection, polyfunctional CD4+ T-cell-response associates with active TB, characterized by a high proportion of IFNγ+ TNFα+ and an effector-memory phenotype. © 2014 The British Infection Association.


Petruccioli E.,L Spallanzani National Institute For Infectious Diseases Inmi Irccs | Petrone L.,L Spallanzani National Institute For Infectious Diseases Inmi Irccs | Vanini V.,L Spallanzani National Institute For Infectious Diseases Inmi Irccs | Cuzzi G.,L Spallanzani National Institute For Infectious Diseases Inmi Irccs | And 5 more authors.
Journal of Infection | Year: 2015

There are still no reliable tests to distinguish active tuberculosis (TB) from latent TB infection (LTBI). Assessment of CD27 modulation on CD4+ T-cells has been suggested as a tool to diagnose different TB stages. Objectives: To use several cytometric approaches to evaluate CD27 expression on Mycobacterium tuberculosis (Mtb)-specific CD4+ T-cells to differentiate TB stages. Methods: 55 HIV-uninfected subjects were enrolled: 13 active TB; 12 cured TB; 30 LTBI. Whole blood was stimulated with RD1-proteins or Cytomegalovirus-lysate (CMV). Interferon (IFN)-γ response was evaluated by cytometry. The proportion of CD27-/+ within the IFN-γ+ CD4+ T-cells or RATIO of the CD27-median fluorescence intensity (MFI) of CD4+ T-cells over the CD27 MFI of IFN-γ+ CD4+ T-cells was evaluated. Results: The greatest diagnostic accuracy in discriminating active TB vs. LTBI or cured TB was reached by evaluating the CD27+ CD45RA- cells within the IFN-γ+ CD4+ T-cell subset (76.92 sensitivity for both, and 90% and 91.67% specificity, respectively), although the use of the CD27 MFI RATIO allows for stricter data analysis, independent of the operator. Conclusions: the study of CD27 expression using different approaches, whether it involves evaluation of CD45RA expression or not, is a robust biomarker for discriminating TB stages. © 2015 The British Infection Association.


Chiacchio T.,L Spallanzani National Institute For Infectious Diseases Inmi | Petruccioli E.,L Spallanzani National Institute For Infectious Diseases Inmi | Vanini V.,L Spallanzani National Institute For Infectious Diseases Inmi | Butera O.,L Spallanzani National Institute For Infectious Diseases Inmi | And 8 more authors.
PLoS ONE | Year: 2011

Rationale: Due to the invasive nature of the procedures involved, most studies of Mycobacterium tuberculosis (Mtb)-specific immunity in humans have focused on the periphery rather than the site of active infection, the lung. Recently, antigens associated with Mtb-latency and -dormancy have been described using peripheral blood (PB) cells; however their response in the lung is unknown. The objective of this report was to evaluate, in patients prospectively enrolled with suspected active tuberculosis (TB), whether the latency antigen Rv2628 induces local-specific immune response in bronchoalveolar lavage (BAL) cells compared to PB cells. Material/Methods: Among the 41 subjects enrolled, 20 resulted with active TB. Among the 21 without active disease, 9 were defined as subjects with latent TB-infection (LTBI) [Quantiferon TB Gold In-tube positive]. Cytokine responses to Rv2628 were evaluated by enzyme linked immunospot (ELISPOT) assay and flow cytometric (FACS) analysis. RD1-secreted antigen stimulation was used as control. Results: There was a significantly higher frequency of Rv2628- and RD1-specific CD4 + T-cells in the BAL of active TB patients than in PB. However the trend of the response to Rv2628 in subjects with LTBI was higher than in active TB in both PB and BAL, although this difference was not significant. In active TB, Rv2628 and RD1 induced a cytokine-response profile mainly consisting of interferon (IFN)-γ-single-positive over double-IFN-γ/interleukin (IL)-2 T-cells in both PB and BAL. Finally, BAL-specific CD4 + T-cells were mostly effector memory (EM), while peripheral T-cell phenotypes were distributed among naïve, central memory and terminally differentiated effector memory T-cells. Conclusions: In this observational study, we show that there is a high frequency of specific T-cells for Mtb-latency and RD1-secreted antigens (mostly IFN-γ-single-positive specific T-cells with an EM phenotype) in the BAL of active TB patients. These data may be important for better understanding the pathogenesis of TB in the lung. © 2011 Chiacchio et al.


Petruccioli E.,L Spallanzani National Institute For Infectious Diseases Inmi | Petrone L.,L Spallanzani National Institute For Infectious Diseases Inmi | Vanini V.,L Spallanzani National Institute For Infectious Diseases Inmi | Sampaolesi A.,INMI | And 4 more authors.
Journal of Infection | Year: 2013

Objectives: Controversial results were reported on the role of polyfunctional T-cells in tuberculosis (TB). Our aim was to simultaneously characterize the Mycobacterium tuberculosis (Mtb)-specific immune response as cytokine production and memory phenotype by flow cytometry after in vitro stimulation with region of difference 1 (" RD1" ) recombinant proteins (ESAT-6 and CFP-10) in patients at different TB stage in a low TB endemic country. To assess the specificity of these findings, we evaluated the response to cytomegalovirus (CMV), an unrelated antigen. Methods: We enrolled subjects with active TB, cured TB, latent TB infection (LTBI). Cytokine and phenotype profiles of T-cells from whole blood stimulated with " RD1" proteins and CMV were characterized by multi-parametric flow cytometry. Results: Bifunctional IFNγ+ TNFα+ CD4+ T-cells and effector memory phenotype significantly associated with active TB compared to the LTBI group (p = 0.008, at least p ≤ 0.009 respectively) whereas " RD1" -T-cell response in cured TB and LTBI was characterized by a central memory phenotype (at least p ≤ 0.013 and p ≤ 0.004 respectively vs active TB). In contrast, response to CMV antigen was not associated with a TB-specific status. Conclusion: We identified qualitative associations between Mtb-specific T-cell and TB status in terms of functional capacity and memory status. These immune correlates may be helpful to trace natural history of TB. © 2013 The British Infection Association.


Delogu G.,Catholic University of the Sacred Heart | Chiacchio T.,L Spallanzani National Institute For Infectious Diseases Inmi | Vanini V.,L Spallanzani National Institute For Infectious Diseases Inmi | Butera O.,L Spallanzani National Institute For Infectious Diseases Inmi | And 10 more authors.
PLoS ONE | Year: 2011

Background: A challenge in tuberculosis (TB) research is to develop a new immunological test that can help distinguish, among subjects responsive to QuantiFERON TB Gold In tube (QFT-IT), those who are able to control Mtb replication (remote LTBI, recent infection and past TB) from those who cannot (active TB disease). IFN-γ response to the Heparin-binding-hemagglutinin (HBHA) of Mtb has been associated with LTBI, but the cumbersome procedures of purifying the methylated and immunological active form of the protein from Mtb or M. bovis Bacillus Calmette et Guerin (BCG) have prevented its implementation in a diagnostic test. Therefore, the aim of the present study was to evaluate the IFN-γ response to methylated HBHA of Mtb produced in M. smegmatis (rHBHAms) in individuals at different stages of TB who scored positive to QFT-IT. Methodology/Principal Findings: 87 individuals at different stages of TB who scored positive to QFT-IT were selected. IFN-γ response to in vitro whole blood stimulation with rHBHAms was evaluated by short-term and long-term tests and detected by ELISA or flow cytometry. We demonstrated that the IFN-γ response to rHBHAms is mediated by CD4 + T-cells with an effector-memory phenotype. This response, evaluated by short-term-tests, is significantly lower in active TB than in remote LTBI (p = 0.0010) and past TB (p = 0.0152). These results were confirmed by long-term tests. The qualitative data confirmed that IFN-γ responses higher than the cut-off point identified by ROC analysis are associated with the status of non-active disease. Conclusions: In this study we show that the T-cell response to a recombinant and methylated HBHA of Mtb produced in M. smegmatis is useful to discriminate between active and non-active TB disease among those responsive to QFT-IT in a whole blood system. Further studies are needed to improve the accuracy of the assay. © 2011 Delogu et al.

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