PE_PGRS33 is a surface-exposed protein of
(
) which exerts its role in macrophages entry and immunomodulation. In this study, we aimed to investigate the polymorphisms in the
gene of
clinical ...isolates and evaluate their impact on protein functions. We sequenced
in a collection of 135 clinical strains, genotyped by 15-loci MIRU-VNTR and spoligotyping and belonging to the
complex (MTBC). Overall, an association between
alleles and MTBC genotypes was observed and a dN/dS ratio of 0.64 was obtained, suggesting that a purifying selective pressure is acting on
against deleterious SNPs. Among a total of 19
alleles identified in this study, 5 were cloned and used to complement the
knock-out mutant strain of
H37Rv (
Δ33) to assess the functional impact of the respective polymorphisms in
infections of primary macrophages. In human monocyte-derived macrophages (MDMs) infection, large in-frame and frameshift mutations were unable to restore the phenotype of
H37Rv, impairing the cell entry capacity of
, but neither its intracellular replication rate nor its immunomodulatory properties.
studies performed in the murine model of tuberculosis (TB) demonstrated that the
Δ33 mutant strain was not impaired in the ability to infect and replicate in the lung tissue compared to the parental strain. Interestingly,
Δ33 showed an enhanced virulence during the chronic steps of infection compared to
H37Rv. Similarly, the complementation of
Δ33 with a frameshift allele also resulted in a
strain capable of causing a surprisingly enhanced tissue damage in murine lungs, during the chronic steps of infection. Together, these results further support the role of PE_PGRS33 in the pathogenesis and virulence of
.
Abstract Objective/background QuantiFERON-TB Gold In-Tube (QFT-GIT, Qiagen, Hilden, Germany) is an interferon-γ (IFN-γ) release assay designed to detect latent tuberculosis infection (LTBI). Although ...QFT-GIT has several advantages (mainly that it is not affected by the Bacille Calmette–Guérin vaccination), it has a poor sensitivity in immune-compromised individuals as it involves an immune response-based detection. Recently, QuantiFERON-TB Gold Plus (QFT-Plus) assay has been proposed as a new generation of QFT-GIT. QFT-Plus includes two tubes, TB1 and TB2 with Mycobacterium tuberculosis antigens to elicit a specific immune response. TB1 contains peptides derived from the antigens ESAT-6 and CFP-10 (TB-7.7, present in QFT-GIT, has been removed), and it is designed to induce a specific CD4 T-cell response. TB2 contains newly designed peptides stimulating IFN-γ production by both CD4 and CD8 T cells. The additional peptides for eliciting CD8 T-cell responses have been included to increase the sensitivity of the test for LTBI detection. The aim of the study was to evaluate specific CD4 and CD8 T-cell responses to the M. tuberculosis antigens contained within the QFT-Plus test by flow cytometry in individuals with active TB and LTBI. Methods We enrolled 23 individuals with active TB and 30 individuals with LTBI. QFT-Plus assay and intracellular staining were performed. 1 × 106 PBMC in 1 mL of medium were dispensed in QFT-Plus tubes. Following 16–24 h stimulation, antigen-specific T cells were characterized by flow cytometry evaluating CD4, CD8, CD3 markers, and IFN-γ production. For statistical analysis, nonparametric tests were performed. Results We found that CD4 T-cell responses were induced by both TB1 and TB2. Differently, the CD8 T-cell response was mainly induced by TB2 and was significantly higher than that induced by TB1 ( p = 0.01). The frequency observed in individuals with active TB was significantly higher than in those with LTBI ( p = 0.04). Finally, TB2-specific CD8 T-cell responses in individuals with active TB were associated with high radiological severity of lung lesions and microbiological diagnosis (based on M. tuberculosis isolation in sputum culture). Conclusion This is the first characterization of CD4 and CD8 T-cell responses to QFT-Plus TB1 and TB2 tubes in individuals with active TB and LTBI enrolled in a low TB-endemic country such as Italy. We demonstrated that the increased sensitivity is a consequence of the ability of TB2 to induce a CD8 T-cell response which is mainly associated with active TB. This assay has the potential to be very useful in conditions of immune depression due to CD4 T-cell impairments.
Abstract Objective/Background Interferon (IFN)-γ-release assays (IGRAs) are designed for the diagnosis of tuberculosis (TB) infection. The new IGRA called QuantiFERON-TB Plus (QFT-Plus) is based on ...enzyme-linked immunosorbent assay (ELISA) detection of IFN-γ following Myobacterium tuberculosis -antigen stimulation with TB1 and TB2 antigens. TB1 elicits a cell-mediated immune response by CD4 T cells and TB2 elicits a response from both CD4 and CD8 T cells. Here, we characterized variations IFN-γ release detected by ELISA to QFT-IT and QFT-Plus in patients with active TB and latent TB infection (LTBI) at baseline and during or after specific treatment (follow-up). Methods We studied seven patients with active TB and 10 patients with LTBI at baseline and during treatment either for active disease or preventive therapy. IFN-γ release detected by ELISA to QFT-IT and QFT-Plus was concomitantly evaluated over time. Statistical analysis was performed using a nonparametrical test for a paired dataset (Wilcoxon test). Results All participants responded to the mitogen, with all active-TB patients responding to QFT-IT or QFT-Plus at baseline. The responses did not change over time either qualitatively (number of responders) or quantitatively (IFN-γ release evaluated as IU/mL). Among the LTBI group, although all participants responded to both QFT-IT and QFT-Plus and the responses did not change over time, the quantitative responses to QFT-Plus showed a different trend. Specifically, response to TB2 was significantly lower at follow-up as compared with that observed at baseline ( p = .004), whereas the response to TB1 was not significantly different ( p = .16). Conclusion To our knowledge, this is the first report characterizing IFN-γ responses to QFT-Plus antigens in participants with active TB and LTBI over time. The data need to be confirmed in larger settings; however, we showed that monitoring IFN-γ release in response to TB2 can be used to evaluate preventive therapy immune changes. This can be useful also as a tool for public health control strategies in settings where preventive treatment is recommended.
Abstract Objective/Background Interferon (IFN)-γ release assays (IGRA) are designed for diagnosing tuberculosis (TB) infection. The new IGRA, QuantiFERON-TB Plus (QFT-Plus), is based on the ...enzyme-linked immunosorbent assay detection of IFN-γ after stimulation with Mycobacterium tuberculosis TB1 and TB2 antigens. TB1 elicits a cellular-mediated immune (CMI) response by CD4 T cells, and TB2 contains peptides recognized by both CD4 and CD8 T cells. The aim of the study is to characterize the CMI to QFT-Plus peptides in active TB and latent TB infection (LTBI) at baseline and during or after specific treatment (follow-up). Methods We enrolled 7 individuals with active TB and 11 individuals with LTBI at baseline and followed them during the treatment, either for active diseases or preventive therapy. Peripheral blood mononuclear cells were stimulated with QFT-Plus antigens (TB1, TB2, and mitogen). Cytokine profile (IFNγ, tumor necrosis factor-α, interleukin-2) and phenotype (CD45RA, CD27) of CD4 and CD8 T cells were characterized by flow cytometry. Results All the individuals responded to mitogen. CD4 T-cell responses to TB1 and TB2 were similar in both individuals with active TB and those with LTBI evaluated over time. Differently, we found a higher number of TB2-associated CD8 T-cell responders in individuals with active TB than in those with LTBI. For individuals with active TB, there was no change in the specific response overtime. Differently, in individuals with LTBI, the number of CD8 responders to QFT-Plus antigens increased during preventive treatment (TB1 = 5/11 45%, TB2 = 5/11 45%) compared with that at the time of enrolment (TB1 = 1/11 9%, TB2 = 1/11 9%). Moreover, we analyzed the effector memory profile of T cells responding to QFT-Plus antigens. The largest component of antigen-specific CD4 T cells (65%) had a central memory (CD45RA-CD27+) phenotype at enrolment and during follow-up. In contrast, specific CD8 T cells, which were analyzed only at follow-up because they were almost absent at baseline, were characterized by a large component with naïve (CD45RA+CD27+) phenotype (40%) and a minor component with central memory (25%) features. Conclusion To our knowledge, this is the first report characterizing CD4 and CD8 T-cell responses of individuals with active TB and with LTBI, followed overtime, to QFT-Plus antigens by flow cytometry. The results, although preliminary, may help in identifying better tools for monitoring therapy, especially in those with LTBI undergoing preventive treatment.
To examine whether specific T-cell-responses to SARS-CoV-2 peptides can be detected in COVID-19 using a whole-blood experimental setting, which may be further explored as a potential diagnostic tool.
...We evaluated interferon (IFN)-γ levels after stimulating whole-blood with spike and remainder-antigens peptides megapools (MP) derived from SARS-CoV-2 sequences; interleukin (IL)-1β, IL-1RA, IL-2, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-10, IL-12p70, IL-13, IL-15, IL-17A, eotaxin, basic fibroblast growth factor (FGF), granulocyte-colony stimulating factor (G-CSF), granulocyte-macrophage colony-stimulating factor (GM-CSF), IFN-γ, Interferon gamma-induced protein 10 (IP-10), monocyte chemoattractant protein-1 (MCP-1), macrophage inflammatory protein (MIP)-1α, MIP-1β, Platelet-derived growth factor (PDGF), RANTES (regulated on activation, normal T cell expressed and secreted), tumour necrosis factor-alpha (TNF-α), vascular endothelial growth factor (VEGF) were also evaluated.
IFN-γ-response to spike and remainder-antigens MPs was significantly increased in 35 COVID-19 patients compared with 29 ‘no COVID-19’ individuals (medians spike-MP: 0.26 vs 0, p = 0.0002; medians remainder-antigens-MP: 0.07 vs 0.02; p = 0.02).
This response was detected independently of patients' clinical parameters. IFN-γ-response to SARS-CoV-2-unrelated antigens cytomegalovirus (CMV) and Staphylococcal Enterotoxin B (SEB) was similar in COVID-19 compared with ‘no COVID-19’ individuals (median CMV: 3.46 vs 5.28, p = 0.16; median SEB: 12.68 vs 15.05; p = 0.1). In response to spike-MPs in COVID-19- compared with ‘no COVID-19’ -individuals, we found significant higher median of IL-2 (50.08 vs 0, p = 0.0018), IFN-γ (90.16 vs 0, p = 0.01), IL-4 (0.52 vs 0, p = 0.03), IL-13 (0.84 vs 0, p = 0.007) and MCP-1 (4602 vs 359.2, p = 0.05).
Immune response to SARS-CoV-2 peptides in a whole-blood assay is associated with COVID-19 and it is characterized by both Th1 and Th2 profile. This experimental approach may be useful for developing new T-cell based diagnostic tests for disease and vaccine settings.
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GEOZS, IJS, IMTLJ, KILJ, KISLJ, NLZOH, NUK, OILJ, PNG, SAZU, SBCE, SBJE, UILJ, UL, UM, UPCLJ, UPUK, ZAGLJ, ZRSKP
To evaluate the immune-specific response after full severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) vaccination of patients with multiple sclerosis (MS) treated with different ...disease-modifying drugs by the detection of both serologic and T-cell responses.
Healthcare workers (HCWs) and patients with MS, having completed the 2-dose schedule of an mRNA-based vaccine against SARS-CoV-2 in the past 2-4 weeks, were enrolled from 2 parallel prospective studies conducted in Rome, Italy, at the National Institute for Infectious diseases Spallanzani-IRCSS and San Camillo Forlanini Hospital. Serologic response was evaluated by quantifying the region-binding domain (RBD) and neutralizing antibodies. Cell-mediated response was analyzed by a whole-blood test quantifying interferon (IFN)-γ response to spike peptides. Cells responding to spike stimulation were identified by fluorescence-activated cell sorting analysis.
We prospectively enrolled 186 vaccinated individuals: 78 HCWs and 108 patients with MS. Twenty-eight patients with MS were treated with IFN-β, 35 with fingolimod, 20 with cladribine, and 25 with ocrelizumab. A lower anti-RBD antibody response rate was found in patients treated with ocrelizumab (40%,
< 0.0001) and fingolimod (85.7%,
= 0.0023) compared to HCWs and patients treated with cladribine or IFN-β. Anti-RBD antibody median titer was lower in patients treated with ocrelizumab (
< 0.0001), fingolimod (
< 0.0001), and cladribine (
= 0.010) compared to HCWs and IFN-β-treated patients. Serum neutralizing activity was present in all the HCWs tested and in only a minority of the fingolimod-treated patients (16.6%). T-cell-specific response was detected in the majority of patients with MS (62%), albeit with significantly lower IFN-γ levels compared to HCWs. The lowest frequency of T-cell response was found in fingolimod-treated patients (14.3%). T-cell-specific response correlated with lymphocyte count and anti-RBD antibody titer (ρ = 0.554,
< 0.0001 and ρ = 0.255,
= 0.0078 respectively). IFN-γ T-cell response was mediated by both CD4
and CD8
T cells.
mRNA vaccines induce both humoral and cell-mediated specific immune responses against spike peptides in all HCWs and in the majority of patients with MS. These results carry relevant implications for managing vaccinations, suggesting promoting vaccination in all treated patients with MS.
This study provides Class III data that SARS-CoV-2 mRNA vaccination induces both humoral and cell-mediated specific immune responses against viral spike proteins in a majority of patients with MS.
Abstract Introduction QuantiFERON®-TB Gold Plus (QFT-Plus) is the new generation of QuantiFERON-TB Gold In-Tube test to identify latent tuberculosis infection (LTBI). QFT-Plus includes TB1 and TB2 ...tubes which contain selected Mycobacterium tuberculosis (Mtb) peptides designed to stimulate both CD4 and CD8 T-cells. Aim of this study is the flow cytometric characterization of the specific CD4 and CD8 T-cell responses to Mtb antigens contained within QFT-Plus. Methods: we 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. Results 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). Conclusions 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.
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GEOZS, IJS, IMTLJ, KILJ, KISLJ, NUK, OILJ, PNG, SAZU, SBCE, SBJE, UL, UM, UPCLJ, UPUK
Summary 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.
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GEOZS, IJS, IMTLJ, KILJ, KISLJ, NUK, OILJ, PNG, SAZU, SBCE, SBJE, UL, UM, UPCLJ, UPUK
29.
Update on the diagnosis of tuberculosis Kontsevaya, Irina; Cabibbe, Andrea Maurizio; Cirillo, Daniela Maria ...
Clinical microbiology and infection,
2023-Jul-23
Journal Article
Peer reviewed
Tuberculosis remains a global public health threat, and the development of rapid and precise diagnostic tools is the key to enabling the early start of treatment, monitoring response to treatment, ...and preventing the spread of the disease.
An overview of recent progress in host- and pathogen-based tuberculosis diagnostics.
We conducted a PubMed search of recent relevant articles and guidelines on tuberculosis screening and diagnosis.
An overview of currently used methods and perspectives in the following areas of tuberculosis diagnostics is provided: immune-based diagnostics, X-ray, clinical symptoms and scores, cough detection, culture of Mycobacterium tuberculosis and identifying its resistance profile using phenotypic and genotypic methods, including next generation sequencing, sputum- and non-sputum-based molecular diagnosis of tuberculosis and monitoring of response to treatment.
A brief overview of the most relevant advances and changes in international guidelines regarding screening and diagnosing tuberculosis is provided in this review. It aims at reviewing all relevant areas of diagnostics, including both pathogen- and host-based methods.
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FZAB, GEOZS, GIS, IJS, IMTLJ, KILJ, KISLJ, NLZOH, NUK, OILJ, PNG, SAZU, SBCE, SBJE, SBMB, UILJ, UL, UM, UPCLJ, UPUK, ZAGLJ, ZRSKP
•QFT-Plus has similar sensitivity for active TB detection independently of HIV infection.•CD4 count does not influence IFNγ values of QFT-Plus in HIV-TB patients.•HIV-LTBI have high proportion of ...IFNγ values in the “uncertain range” of QFT-Plus.•Impairment of CD4 response to QFT-Plus antigens in the HIV-infected subjects.•Similar CD8 response to QFT-Plus antigens in HIV-infected and –uninfected subjects.
HIV-infection increases the risk to progress to active-tuberculosis (TB). Detection of latent TB infection (LTBI) is needed to eventually propose preventive-therapy and reduce TB reservoir. QuantiFERON-TB Plus (QFT-Plus)-test identifies LTBI. Currently, only two studies on QFT-Plus accuracy in HIV-infected-population are available in high TB-endemic-countries. Therefore we aimed to evaluate the effect of HIV-infection on QFT-Plus accuracy to detect LTBI in a low TB-endemic-country.
We enrolled 465 participants, among the 167 HIV-infected-persons: 32 with active-TB (HIV-TB), 45 remote-LTBI (HIV-LTBI) and 90 at low M. tuberculosis (Mtb)-infection risk. Among the 298 HIV-uninfected-persons: 170 with active-TB, 76 recent-LTBI, 34 remote-LTBI and 18 with low Mtb-infection risk.
QFT-Plus sensitivity was similar in TB regardless of HIV-status. CD4-count did not influence the distribution of IFN-γ values in HIV-TB and HIV-LTBI. Moreover HIV-LTBI and HIV-uninfected remote LTBI had a similar proportion of results in the uncertain range (IFNγ ≥0.2 ≤ 0.7 IU/ml) differently from those LTBI-persons reporting recent-exposure (p = 0.016). Cytometry results demonstrated that CD8-response was similar in HIV-infected- and -uninfected-persons whereas CD4-response was impaired in HIV-infected-persons (p = 0.011).
HIV-infection does not affect QFT-Plus response in active-TB, whereas the time of exposure influences the proportion of uncertain-results in LTBI.
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GEOZS, IJS, IMTLJ, KILJ, KISLJ, NLZOH, NUK, OILJ, PNG, SAZU, SBCE, SBJE, UILJ, UL, UM, UPCLJ, UPUK, ZAGLJ, ZRSKP
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