Measurement of IFN-γ and IL-2 for the assessment of the cellular immunity against SARS-CoV-2.

The study of specific T-cell responses against SARS-CoV-2 is important for understanding long-term immunity and infection management. The aim of this study was to assess the dual IFN-γ and IL-2 detection, using a SARS-CoV-2 specific fluorescence ELISPOT, in patients undergoing acute disease, during convalescence, and after vaccination. We also evaluated humoral response and compared with T-cells with the aim of correlating both types of responses, and increase the number of specific response detection. Blood samples were drawn from acute COVID-19 patients and convalescent individuals classified according to disease severity; and from unvaccinated and vaccinated uninfected individuals. IgGs against Spike and nucleocapsid, IgMs against nucleocapsid, and neutralizing antibodies were also analyzed. Our results show that IFN-γ in combination with IL-2 increases response detection in acute and convalescent individuals (p = 0.023). In addition, IFN-γ detection can be a useful biomarker for monitoring severe acute patients, as our results indicate that those individuals with a poor outcome have lower levels of this cytokine. In some cases, the lack of cellular immunity is compensated by antibodies, confirming the role of both types of immune responses in infection, and confirming that their dual detection can increase the number of specific response detections. In summary, IFN-γ/IL-2 dual detection is promising for characterizing and assessing the immunization status, and helping in the patient management.

Development and Evaluation of an NTM-IGRA to Guide Pediatric Lymphadenitis Diagnosis.

BACKGROUND: Diagnosis of nontuberculous mycobacteria (NTM) infections remains a challenge. In this study, we describe the evaluation of an immunological NTM-interferon (IFN)-γ release assay (IGRA) that we developed using glycopeptidolipids (GPLs) as NTM-specific antigens. METHODS: We tested the NTM-IGRA in 99 samples from pediatric patients. Seventy-five were patients with lymphadenitis: 25 were NTM confirmed, 45 were of unknown etiology but compatible with mycobacterial infection and 5 had lymphadenitis caused by an etiologic agent other than NTM. The remaining 24 samples were from control individuals without lymphadenitis (latently infected with M. tuberculosis , uninfected controls and active tuberculosis patients). Peripheral blood mononuclear cells were stimulated overnight with GPLs. Detection of IFN-γ producing cells was evaluated by enzyme-linked immunospot assay. RESULTS: NTM culture-confirmed lymphadenitis patient samples had a significantly higher response to GPLs than the patients with lymphadenitis of unknown etiology but compatible with mycobacterial infection ( P < 0.001) and lymphadenitis not caused by NTM ( P < 0.01). We analyzed the response against GPLs in samples from unknown etiology lymphadenitis but compatible with mycobacterial infection cases according to the tuberculin skin test (TST) response, and although not statistically significant, those with a TST ≥5 mm had a higher response to GPLs when compared with the TST <5 mm group. CONCLUSIONS: Stimulation with GPLs yielded promising results in detecting NTM infection in pediatric patients with lymphadenitis. Our results indicate that the test could be useful to guide the diagnosis of pediatric lymphadenitis. This new NTM-IGRA could improve the clinical handling of NTM-infected patients and avoid unnecessary misdiagnosis and treatments.

Tuberculosis: current challenges and beyond.

Despite being a preventable and curable disease, tuberculosis (TB) is still a major global health threat and the second leading cause of death due to an infectious agent worldwide. All the efforts invested to end TB have resulted overall in rather slow decreases in TB incidence and mortality rates, which have been further negatively affected by the ongoing coronavirus disease 2019 (COVID-19) pandemic. While the majority of targets of the End TB Strategy remain off track, and we have not yet overcome the disruptions caused by the COVID-19 pandemic, recent conflicts such as the ongoing war in Ukraine are threatening the decrease of the burden of TB even further. To get back on track and get closer to ending TB, we need urgent, global, well-structured and committed multi-sectoral actions that go beyond national and global TB programmes with the support of deep investments in research and facilitation of equitable and rapid implementation of innovation worldwide.

Measuring T-Cell Responses against SARS-CoV-2 Is of Utility for Disease and Vaccination Management.

The measurement of specific T-cell responses can be a useful tool for COVID-19 diagnostics and clinical management. In this study, we evaluated the IFN-γ T-cell response against the main SARS-CoV-2 antigens (spike, nucleocapsid and membrane) in acute and convalescent individuals classified according to severity, and in vaccinated and unvaccinated controls. IgG against spike and nucleocapsid were also measured. Spike antigen triggered the highest number of T-cell responses. Acute patients showed a low percentage of positive responses when compared to convalescent (71.6% vs. 91.7%, respectively), but increased during hospitalization and with severity. Some convalescent patients showed an IFN-γ T-cell response more than 200 days after diagnosis. Only half of the vaccinated individuals displayed an IFN-γ T-cell response after the second dose. IgG response was found in a higher percentage of individuals compared to IFN-γ T-cell responses, and moderate correlations between both responses were seen. However, in some acute COVID-19 patients specific T-cell response was detected, but not IgG production. We found that the chances of an IFN-γ T-cell response against SARS-CoV-2 is low during acute phase, but may increase over time, and that only half of the vaccinated individuals had an IFN-γ T-cell response after the second dose.

Study of CD27, CD38, HLA-DR and Ki-67 immune profiles for the characterization of active tuberculosis, latent infection and end of treatment.

Background: Current blood-based diagnostic tools for TB are insufficient to properly characterize the distinct stages of TB, from the latent infection (LTBI) to its active form (aTB); nor can they assess treatment efficacy. Several immune cell biomarkers have been proposed as potential candidates for the development of improved diagnostic tools. Objective: To compare the capacity of CD27, HLA-DR, CD38 and Ki-67 markers to characterize LTBI, active TB and patients who ended treatment and resolved TB. Methods: Blood was collected from 45 patients defined according to clinical and microbiological criteria as: LTBI, aTB with less than 1 month of treatment and aTB after completing treatment. Peripheral blood mononuclear cells were stimulated with ESAT-6/CFP-10 or PPD antigens and acquired for flow cytometry after labelling with conjugated antibodies against CD3, CD4, CD8, CD27, IFN-γ, TNF-α, CD38, HLA-DR, and Ki-67. Conventional and multiparametric analyses were done with FlowJo and OMIQ, respectively. Results: The expression of CD27, CD38, HLA-DR and Ki-67 markers was analyzed in CD4+ T-cells producing IFN-γ and/or TNF-α cytokines after ESAT-6/CFP-10 or PPD stimulation. Within antigen-responsive CD4+ T-cells, CD27- and CD38+ (ESAT-6/CFP-10-specific), and HLA-DR+ and Ki-67+ (PPD- and ESAT-6/CFP-10-specific) populations were significantly increased in aTB compared to LTBI. Ki-67 demonstrated the best discriminative performance as evaluated by ROC analyses (AUC > 0.9 after PPD stimulation). Data also points to a significant change in the expression of CD38 (ESAT-6/CFP-10-specific) and Ki-67 (PPD- and ESAT-6/CFP-10-specific) after ending the anti-TB treatment regimen. Furthermore, ratio based on the CD27 median fluorescence intensity in CD4+ T-cells over Mtb-specific CD4+ T-cells showed a positive association with aTB over LTBI (ESAT-6/CFP-10-specific). Additionally, multiparametric FlowSOM analyses revealed an increase in CD27 cell clusters and a decrease in HLA-DR cell clusters within Mtb-specific populations after the end of treatment. Conclusion: Our study independently confirms that CD27-, CD38+, HLA-DR+ and Ki-67+ populations on Mtb-specific CD4+ T-cells are increased during active TB disease. Multiparametric analyses unbiasedly identify clusters based on CD27 or HLA-DR whose abundance can be related to treatment efficacy. Further studies are necessary to pinpoint the convergence between conventional and multiparametric approaches.

Tobacco Smoking and Second-Hand Smoke Exposure Impact on Tuberculosis in Children.

Little is known about whether second-hand smoke (SHS) exposure affects tuberculosis (TB). Here, we investigate the association of cigarette smoke exposure with active TB and latent TB infection (LTBI) in children, analyzing Interferon-Gamma Release Assays' (IGRAs) performance and cytokine immune responses. A total of 616 children from contact-tracing studies were included and classified regarding their smoking habits [unexposed, SHS, or smokers]. Risk factors for positive IGRAs, LTBI, and active TB were defined. GM-CSF, IFN-γ, IL-2, IL-5, IL-10, IL-13, IL-22, IL-17, TNF-α, IL-1RA and IP-10 cytokines were detected in a subgroup of patients. Being SHS exposed was associated with a positive IGRA [aOR (95% CI): 8.7 (5.9-12.8)] and was a main factor related with LTBI [aOR (95% CI): 7.57 (4.79-11.94)] and active TB [aOR (95% CI): 3.40 (1.45-7.98)]. Moreover, IGRAs' sensitivity was reduced in active TB patients exposed to tobacco. IL-22, GM-CSF, IL-5, TNF-α, IP-10, and IL-13 were less secreted in LTBI children exposed to SHS. In conclusion, SHS is associated with LTBI and active TB in children. In addition, false-negative IGRAs obtained on active TB patients exposed to SHS, together with the decrease of specific cytokines released, suggest that tobacco may alter the immune response.

Molecular and Cellular Mechanisms of M. tuberculosis and SARS-CoV-2 Infections-Unexpected Similarities of Pathogenesis and What to Expect from Co-Infection.

Tuberculosis is still an important medical and social problem. In recent years, great strides have been made in the fight against M. tuberculosis, especially in the Russian Federation. However, the emergence of a new coronavirus infection (COVID-19) has led to the long-term isolation of the population on the one hand and to the relevance of using personal protective equipment on the other. Our knowledge regarding SARS-CoV-2-induced inflammation and tissue destruction is rapidly expanding, while our understanding of the pathology of human pulmonary tuberculosis gained through more the 100 years of research is still limited. This paper reviews the main molecular and cellular differences and similarities caused by M. tuberculosis and SARS-CoV-2 infections, as well as their critical immunological and pathomorphological features. Immune suppression caused by the SARS-CoV-2 virus may result in certain difficulties in the diagnosis and treatment of tuberculosis. Furthermore, long-term lymphopenia, hyperinflammation, lung tissue injury and imbalance in CD4+ T cell subsets associated with COVID-19 could propagate M. tuberculosis infection and disease progression.

Diagnostic Performance of the Fujifilm SILVAMP TB-LAM in Children with Presumptive Tuberculosis.

Current diagnostics for tuberculosis (TB) only manage to confirm a small proportion of children with TB and require respiratory samples, which are difficult to obtain. There is a need for non-invasive biomarker-based tests as an alternative to sputum testing. Fujifilm SILVAMP TB lipoarabinomannan (FujiLAM), a lateral-flow test to detect lipoarabinomannan in urine, is a novel non-sputum-based point-of-care diagnostic reported to have increased sensitivity for the diagnosis of TB among human immunodeficiency virus (HIV)-infected adults. We evaluate the performance of FujiLAM in children with presumptive TB. Fifty-nine children attending a paediatric hospital in Haiti with compatible signs and symptoms of TB were examined using Xpert MTB/RIF, smear microscopy and X-rays, and classified according to the certainty of diagnosis into bacteriologically confirmed TB (n = 5), unconfirmed TB (bacteriologically negative, n = 50) and unlikely TB (n = 4). Healthy children (n = 20) were enrolled as controls. FujiLAM sensitivity and specificity were 60% and 95% among children with confirmed TB. FujiLAM's high specificity and its characteristics as a point-of-care indicate the test has a good potential for the diagnosis of TB in children.

Discovery and validation of an NMR-based metabolomic profile in urine as TB biomarker.

Despite efforts to improve tuberculosis (TB) detection, limitations in access, quality and timeliness of diagnostic services in low- and middle-income countries are challenging for current TB diagnostics. This study aimed to identify and characterise a metabolic profile of TB in urine by high-field nuclear magnetic resonance (NMR) spectrometry and assess whether the TB metabolic profile is also detected by a low-field benchtop NMR spectrometer. We included 189 patients with tuberculosis, 42 patients with pneumococcal pneumonia, 61 individuals infected with latent tuberculosis and 40 uninfected individuals. We acquired the urine spectra from high and low-field NMR. We characterised a TB metabolic fingerprint from the Principal Component Analysis. We developed a classification model from the Partial Least Squares-Discriminant Analysis and evaluated its performance. We identified a metabolic fingerprint of 31 chemical shift regions assigned to eight metabolites (aminoadipic acid, citrate, creatine, creatinine, glucose, mannitol, phenylalanine, and hippurate). The model developed using low-field NMR urine spectra correctly classified 87.32%, 85.21% and 100% of the TB patients compared to pneumococcal pneumonia patients, LTBI and uninfected individuals, respectively. The model validation correctly classified 84.10% of the TB patients. We have identified and characterised a metabolic profile of TB in urine from a high-field NMR spectrometer and have also detected it using a low-field benchtop NMR spectrometer. The models developed from the metabolic profile of TB identified by both NMR technologies were able to discriminate TB patients from the rest of the study groups and the results were not influenced by anti-TB treatment or TB location. This provides a new approach in the search for possible biomarkers for the diagnosis of TB.

Diagnostic Accuracy of Interferon Gamma-Induced Protein 10 mRNA Release Assay for Tuberculosis.

Interferon gamma (IFN-γ) release assays (IGRAs) are increasingly used to test for latent tuberculosis (TB) infection. Although highly specific, IGRAs have a relatively high false-negative rate in active TB patients. A more sensitive assay is needed. IFN-γ-induced protein 10 (IP-10) is an alternative biomarker with a 100-fold-higher expression level than IFN-γ, allowing for different analysis platforms, including molecular detection. The PCR technique is already an integrated tool in most TB laboratories and, thus, an obvious platform to turn to. In this case-control study, we investigated the diagnostic sensitivity and specificity of a molecular assay detecting IP-10 mRNA expression following antigen stimulation of a blood sample. We included 89 TB patients and 99 healthy controls. Blood was drawn in QuantiFeron-TB gold in-tube (QFT) assay tubes. Eight hours poststimulation, IP-10 mRNA expression was analyzed, and 20 h poststimulation, IP-10 and IFN-γ protein plasma levels were analyzed using an in-house IP-10 enzyme-linked immunosorbent assay (ELISA) and the official QFT ELISA, respectively. The IP-10 mRNA assay provided high specificity (98%), sensitivity (80%), and area under the concentration-time curve (AUC) (0.97); however, the QFT assay provided a higher overall diagnostic potential, with specificity of 100%, sensitivity of 90%, and AUC of 0.99. The IP-10 protein assay performed on par with the QFT assay, with specificity of 98%, sensitivity of 87%, and AUC of 0.98. We have provided proof of high technical performance of a molecular assay detecting IP-10 mRNA expression. As a diagnostic tool, this assay would gain from further optimization, especially on the kinetics of IP-10 mRNA expression.

Serial testing of health care workers for tuberculosis infection: A prospective cohort study.

Health Care Workers (HCW) may have an occupational risk of latent tuberculosis infection (LTBI) and TB disease. The objective of this study was to evaluate the performance of the 2-step strategy: tuberculin skin test (TST) followed by confirmation with Interferon (IFN)-γ- release assays (IGRAs) in HCW. A secondary objective was to determine the factors related to conversions and reversions. HCW at risk of occupational exposure who attended the Occupational Department of the Hospital Germans Trias i Pujol were included during the study period (2013-2016). All professionals testing negative for LTBI were included in a cohort study. These workers were followed up with the administration of a TST and an IGRA quantification at least one year after inclusion in the study. Workers with positive TST, regardless of the results of the IGRA tests, were followed-up with an IGRA. 255 workers were enrolled in the study and 108 workers from the same cohort were followed up. During the follow-up period, seven workers presented TST test conversion. One of these conversions was also confirmed by an IGRA test. There were 2 conversions of cases only testing positive with the IGRA. There have been only 2 reversions of cases testing negative with the IGRA. In this study, not all TST conversions were confirmed when using the IGRA test, which highlights the importance of the 2-step strategy. We have detected a low number of conversions and reversions. Our conclusions should be confirmed in studies with a longer follow-up time.

Cell-Mediated Immune Responses to in vivo-Expressed and Stage-Specific Mycobacterium tuberculosis Antigens in Latent and Active Tuberculosis Across Different Age Groups.

A quarter of the global human population is estimated to be latently infected by Mycobacterium tuberculosis (Mtb), the causative agent of tuberculosis (TB). TB remains the global leading cause of death by a single pathogen and ranks among the top-10 causes of overall global mortality. Current immunodiagnostic tests cannot discriminate between latent, active and past TB, nor predict progression of latent infection to active disease. The only registered TB vaccine, Bacillus Calmette-Guérin (BCG), does not adequately prevent pulmonary TB in adolescents and adults, thus permitting continued TB-transmission. Several Mtb proteins, mostly discovered through IFN-γ centered approaches, have been proposed as targets for new TB-diagnostic tests or -vaccines. Recently, however, we identified novel Mtb antigens capable of eliciting multiple cytokines, including antigens that did not induce IFN-γ but several other cytokines. These antigens had been selected based on high Mtb gene-expression in the lung in vivo, and have been termed in vivo expressed (IVE-TB) antigens. Here, we extend and validate our previous findings in an independent Southern European cohort, consisting of adults and adolescents with either LTBI or TB. Our results confirm that responses to IVE-TB antigens, and also DosR-regulon and Rpf stage-specific Mtb antigens are marked by multiple cytokines, including strong responses, such as for TNF-α, in the absence of detectable IFN-γ production. Except for TNF-α, the magnitude of those responses were significantly higher in LTBI subjects. Additional unbiased analyses of high dimensional flow-cytometry data revealed that TNF-α+ cells responding to Mtb antigens comprised 17 highly heterogeneous cell types. Among these 17 TNF-α+ cells clusters identified, those with CD8+TEMRA or CD8+CD4+ phenotypes, defined by the expression of multiple intracellular markers, were the most prominent in adult LTBI, while CD14+ TNF-α+ myeloid-like clusters were mostly abundant in adolescent LTBI. Our findings, although limited to a small cohort, stress the importance of assessing broader immune responses than IFN-γ alone in Mtb antigen discovery as well as the importance of screening individuals of different age groups. In addition, our results provide proof of concept showing how unbiased multidimensional multiparametric cell subset analysis can identify unanticipated blood cell subsets that could play a role in the immune response against Mtb.

E-cigarettes: Effects in phagocytosis and cytokines response against Mycobacterium tuberculosis.

Cigarette smoking and tuberculosis are a significant cause of death worldwide. Several epidemiological studies have demonstrated cigarette smoking is a risk factor for tuberculosis. Electronic cigarettes have recently appeared as a healthier alternative to conventional smoking, although their impact in tuberculosis is not well understood. The aim of this study was to explore the effect of electronic cigarettes in phagocytosis of Mycobacterium tuberculosis and cytokines production. In vitro infection was carried out by exposing THP-1 macrophages to four electronic vapor extracts and the intracellular burden of M. tuberculosis was determined. The percentage of infection was evaluated by confocal microscopy and the cytokine production by Luminex. A reduction of intracellular M. tuberculosis burden in THP-1 macrophages was found after its exposure to electronic vapor extract; the same trend was observed by confocal microscopy when Mycobacterium bovis BCG-GFP strain was used. Electronic cigarettes stimulate a pro-inflammatory cytokine response. We conclude that electronic cigarettes impair the phagocytic function and the cytokine response to M. tuberculosis.

A Model Based on the Combination of IFN-γ, IP-10, Ferritin and 25-Hydroxyvitamin D for Discriminating Latent From Active Tuberculosis in Children.

In recent years, pediatric research on tuberculosis (TB) has focused on addressing new biomarkers with the potential to be used as immunological non-sputum-based methods for the diagnosis of TB in children. The aim of this study was to characterize a set of cytokines and a series of individual factors (ferritin, 25-hydroxyvitamin D [25(OH)D], parasite infections, and nutritional status) to assess different patterns for discriminating between active TB and latent TB infection (LTBI) in children. The levels of 13 cytokines in QuantiFERON-TB Gold In-Tube (QFT-GIT) supernatants were analyzed in 166 children: 74 with active TB, 37 with LTBI, and 55 uninfected controls. All cytokines were quantified using Luminex or ELISA. Ferritin and 25(OH)D were also evaluated using CLIA, and Toxocara canis Ig-G antibodies were detected with a commercial ELISA kit. The combination of IP-10, IFN-γ, ferritin, and 25(OH)D achieved the best diagnostic performance to discriminate between active TB and LTBI cases in children in relation to the area under receiver operating characteristic (ROC) curve 0.955 (confidence interval 95%: 0.91-1.00), achieving optimal sensitivity and specificity for the development of a new test (93.2 and 90.0%, respectively). Children with TB showed higher ferritin levels and an inverse correlation between 25(OH)D and IFN-γ levels. The model proposed includes a combination of biomarkers for discriminating between active TB and LTBI in children to improve the accuracy of TB diagnosis in children. This combination of biomarkers might have potential for identifying the onset of primary TB in children.

Impact of Host Genetics and Biological Response Modifiers on Respiratory Tract Infections.

Host susceptibility to respiratory tract infections (RTI) is dependent on both genetic and acquired risk factors. Repeated bacterial and viral RTI, such as pneumonia from encapsulated microorganisms, respiratory tract infections related to respiratory syncytial virus or influenza, and even the development of bronchiectasis and asthma, are often reported as the first symptom of primary immunodeficiencies. In the same way, neutropenia is a well-known risk factor for invasive aspergillosis, as well as lymphopenia for Pneumocystis, and mycobacterial infections. However, in the last decades a better knowledge of immune signaling networks and the introduction of next generation sequencing have increased the number and diversity of known inborn errors of immunity. On the other hand, the use of monoclonal antibodies targeting cytokines, such as tumor necrosis factor alpha has revealed new risk groups for infections, such as tuberculosis. The use of biological response modifiers has spread to almost all medical specialties, including inflammatory diseases and neoplasia, and are being used to target different signaling networks that may mirror some of the known immune deficiencies. From a clinical perspective, the individual contribution of genetics, and/or targeted treatments, to immune dysregulation is difficult to assess. The aim of this article is to review the known and newly described mechanisms of impaired immune signaling that predispose to RTI, including new insights into host genetics and the impact of biological response modifiers, and to summarize clinical recommendations regarding vaccines and prophylactic treatments in order to prevent infections.

Study of CD27 and CCR4 Markers on Specific CD4+ T-Cells as Immune Tools for Active and Latent Tuberculosis Management.

The immunological characterization of different cell markers has opened the possibility of considering them as immune tools for tuberculosis (TB) management, as they could correlate with TB latency/disease status and outcome. CD4+ T-cells producing IFN-γ+ with a low expression of CD27 have been described as an active TB marker. In addition, there are unknown homing receptors related to TB, such as CCR4, which might be useful for understanding TB pathogenesis. The aim of our study is focused on the assessment of several T-cell subsets to understand immune-mechanisms in TB. This phenotypic immune characterization is based on the study of the specific immune responses of T-cells expressing CD27 and/or CCR4 homing markers. Subjects enrolled in the study were: (i) 22 adult patients with active TB, and (ii) 26 individuals with latent TB infection (LTBI). Blood samples were drawn from each patient. The expression of CD27 and/or CCR4 markers were analyzed within CD4+ T-cells producing: (i) IFN-γ+, (ii) TNF-α+, (iii) TNF-α+IFN-γ+, and (iv) IFN-γ+ and/or TNF-α+. The percentage of CD27- within all CD4+ T-cell populations analyzed was significantly higher on active TB compared to LTBI after PPD or ESAT-6/CFP-10 stimulation. As previously reported, a ratio based on the CD27 median fluorescence intensity (MFI) was also explored (MFI of CD27 in CD4+ T-cells over MFI of CD27 in IFN-γ+CD4+ T-cells), being significantly increased during disease (p < 0.0001 after PPD or ESAT-6/CFP-10 stimulation). This ratio was also assessed on the other CD4+ T-cells functional profiles after specific stimulation, being significantly associated with active TB. Highest diagnostic accuracies for active TB (AUC ≥ 0.91) were achieved for: (i) CD27 within IFN-γ+TNF-α+CD4+ T-cells in response to ESAT-6/CFP-10, (ii) CD27 and CCR4 markers together within IFN-γ+CD4+ T-cells in response to PPD, and (iii) CD27 MFI ratio performed on IFN-γ+TNF-α+CD4+ T-cells after ESAT-6/CFP-10 stimulation. The lowest diagnostic accuracy was observed when CCR4 marker was evaluated alone (AUC ≤ 0.77). CD27 and CCR4 expression detection could serve as a good method for immunodiagnosis. Moreover, the immunological characterization of markers/subset populations could be a promising tool for understanding the biological basis of the disease.

Immune-mediated inflammatory diseases differently affect IGRAs' accuracy for latent tuberculosis infection diagnosis in clinical practice.

BACKGROUND: Clinical accuracy of IGRAs remains unclear on patients with immune-mediated inflammatory diseases (IMIDs). Here, we assess the impact of immunosuppressants and IMIDs on QuantiFERON-TB Gold In-Tube (QFN-G-IT) and T-SPOT.TB accuracy. METHODS: Patients with IMIDs who required latent tuberculosis infection (LTBI) screening were enrolled and classified into: (i) 50 patients with inflammatory rheumatic diseases, (ii) 50 patients with psoriasis and (iii) 30 patients with Crohn's disease. A total of 44 healthy individuals without immunosuppression were also included as controls. Tuberculin skin test (TST), T-SPOT.TB and QFN-G-IT assays were performed. IGRAs were performed following manufacturer's instructions. RESULTS: Immunosuppressant's intake was more frequent on patients with Crohn's disease and psoriasis. Positive IGRAs and TST results were reduced in Crohn's disease patients, whereas rate of indeterminate T-SPOT.TB results was increased in this group with respect to the other IMIDs analysed and controls. When IFN-γ response was studied, the levels of this cytokine after mitogen stimulation were significantly lower in Crohn's and inflammatory rheumatic diseases than in psoriasis. Interestingly, psoriatic patients were the only ones not receiving corticosteroids. Furthermore, a negative correlation was observed between the IFN-γ secreted after mitogen stimulation and corticosteroids dose. CONCLUSIONS: IMIDs seem to negatively affect the clinical accuracy of IGRAs, being Crohn's disease patients the most affected individuals due to their concomitant drug-profile and impaired immune response.

Assessment of the influence of direct tobacco smoke on infection and active TB management.

BACKGROUND: Smoking is a risk factor for tuberculosis (TB) infection and disease progression. Tobacco smoking increases susceptibility to TB in a variety of ways, one of which is due to a reduction of the IFN-γ response. Consequently, an impaired immune response could affect performance of IFN-γ Release Assays (IGRAs). OBJECTIVE: In the present study, we assess the impact of direct tobacco smoking on radiological manifestations, sputum conversion and immune response to Mycobacterium tuberculosis, analyzing IFN-γ secretion by IGRAs. METHODS: A total of 525 participants were studied: (i) 175 active pulmonary TB patients and (ii) 350 individuals coming from contact tracing studies, 41 of whom were secondary TB cases. Clinical, radiological and microbiological data were collected. T-SPOT.TB and QFN-G-IT were processed according manufacturer's instructions. RESULTS: In smoking patients with active TB, QFN-G-IT (34.4%) and T-SPOT.TB (19.5%) had high frequencies of negative results. In addition, by means of an unconditional logistic regression, smoking was a main factor associated with IGRAs' false-negative results (aOR: 3.35; 95%CI:1.47-7.61; p<0.05). Smoking patients with active TB presented a high probability of having cavitary lesions (aOR: 1.88; 95%CI:1.02-3.46;p<0.05). Mean culture negativization (months) ± standard deviation (SD) was higher in smokers than in non-smokers (2.47±1.3 versus 1.69±1.4). Latent TB infection (LTBI) was favored in smoking contacts, being a risk factor associated with infection (aOR: 11.57; 95%CI:5.97-22.41; p<0.00005). The IFN-γ response was significantly higher in non-smokers than in smokers. Smoking quantity and IFN-γ response analyzed by IGRAs were dose-dependent related. CONCLUSIONS: Smoking had a negative effect on radiological manifestations, delaying time of sputum conversion. Our data establish a link between tobacco smoking and TB due to a weakened IFN-γ response caused by direct tobacco smoke.

Use of IFN-γ and IP-10 detection in the diagnosis of latent tuberculosis infection in patients with inflammatory rheumatic diseases.

OBJECTIVES: Biologic agents are used against rheumatic diseases, however, they increase the risk of developing severe infections and diseases such as tuberculosis. We aimed to determine the benefits of IP-10 detection to diagnose latent tuberculosis infection (LTBI) in patients with inflammatory rheumatic diseases on different immunosuppressive drug regimens, and compare these results with IFN-γ detection. MATERIALS AND METHODS: We included 64 patients with inflammatory rheumatic diseases. We used QuantiFERON Gold In-Tube (QFN-G-IT) and T-SPOT.TB to detect IFN-γ production, and an in-house ELISA for IP-10 detection from the previous QFN-G-IT stimulated samples. We assessed the combined use of IFN-γ release assays (IGRAs) and IP-10 test, and analyzed the influence of immunotherapy on the tests performance. RESULTS: We obtained 34.9% positive results by T-SPOT.TB, 25.0% by QFN-G-IT and 31.3% by IP-10 test. The combined use of IGRAs and IP-10 detection increased significantly the amount of positive results (p < 0.0001). Treatment intake had no significant effect on in vitro tests (p > 0.05). CONCLUSIONS: IP-10 and IFN-γ detection is comparable and their combined use could increase the number of positive results in the diagnosis of LTBI in rheumatic patients. The tested assays were not influenced by rheumatoid immunosuppressive therapy. Thus, IP-10 could be of use in the development of new and improved LTBI diagnostic tools.

Accessory gene regulator (Agr) functionality in Staphylococcus aureus derived from lower respiratory tract infections.

OBJECTIVE: Characterization of Staphylococcus aureus clinical isolates derived from lower respiratory tract infections (LRTIs), and correlation between the functionality of the accessory gene regulator (Agr) and genotypic and phenotypic characteristics, clinical variables and clinical outcome. METHODS: S aureus isolates derived from LRTIs and control groups (nasal carriage and bacteraemia) were genotyped using StaphyType DNA microarray. Agr activity was evaluated using the CAMP synergistic haemolysis assay and the Vesicle Lysis Test (VLT). Discordant strains were analysed by quantitative reverse-transcriptase real-time PCR (qRT-PCR). RESULTS: Agr was functional in 79.7% and 84.5% of strains according to the CAMP and VLT assays respectively. Higher concordance with RNAIII expression measured by qRT-PCR was observed with the VLT assay (76.2%) compared with the CAMP assay (23.8%). No statistically significant differences were observed in Agr functionality between the study groups, nor the phenotypical/genotypical bacterial characteristics. No association between increased mortality/respiratory complications and Agr function was observed. CONCLUSIONS: Agr activity was high (82.2%) in isolates from LRTIs suggesting the importance of this global regulator in lower respiratory tract colonisation and infection. However, equally high Agr activity was observed in isolates derived from nasal carriage and bacteraemia, contradictory to previous observations. Agr functionality measured by the VLT assay was superior to CAMP assay.