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The aim of this study was to test the use of IP-10 detection in dried plasma from contact studies individuals (contacts of smear positive patients), by comparing it with IP-10 and IFN-γ detection in direct plasma, to establish IP-10 detection in DPS as a useful assay for LTBI diagnosis. Whole blood samples were collected from 80 subjects: 12 with active tuberculosis (TB), and 68 from contact studies. The amount of IFN-γ produced by sensitized T cells was determined in direct plasma by QuantiFERON Gold In-Tube test. IP-10 levels were determined in direct and dried plasma by an in-house ELISA. For dried plasma IP-10 determination, two 25 µl plasma drops were dried in Whatman903 filter paper and sent by mail to the laboratory. Regarding TB patients, 100.0%, 91.7% and 75.0% were positive for IFN-γ detection and IP-10 detection in direct and dried plasma, respectively. In contacts, 69.1%, 60.3% and 48.5% had positive results after IFN-γ and IP-10 in direct and dried plasma, respectively. The agreement among in vitro tests was substantial and IP-10 levels in direct and dried plasma were strongly correlated (r = 0.897). In conclusion, IP-10 detection in dried plasma is a simple and safe method that would help improve LTBI management.

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.

Interferon (IFN)-γ release assays (IGRAs) are used to diagnose latent tuberculosis (TB) infection (LTBI). To improve the accuracy of these tests, different approaches, such as alternative cytokine detection and using different antigens, are considered. Following this purpose, this study aims to evaluate the addition of EspC, EspF and Rv2348-B to those present in the QuantiFERON-TB Gold In-Tube (QFN-G-IT). We included 115 subjects: 74 active TB patients, 17 LTBI individuals and 24 healthy controls. Whole blood samples were collected in QFN-G-IT and in-house tubes containing different combinations of EspC, EspF and Rv2348-B, together with ESAT-6, CFP-10, and TB7.7. After overnight incubation at 37 ºC, plasma was harvested and IFN-γ quantified. IFN-γ levels in the QFN-G-IT and in-house tubes correlated very good (Spearman Rho(r) > 0.86). In-house antigen combinations distinguished healthy individuals from those with active TB and LTBI (specificities and sensitivities higher than 87.5% and 96.3%, respectively [AUC > 0.938]). Adding EspC, EspF and Rv2348-B, increased the sensitivity of the test, being the addition of EspC and Rv2348-B the combination that yielded a higher sensitivity with no specificity loss. Addition of these antigens could improve diagnosis in patients with impaired or immature immune response who are at high risk of developing TB.

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-Guerin (BCG), does not adequately prevent pulmonary TB in adolescents and adults, thus permitting continued TB-transmission. Several Mtb proteins, mostly discovered through IFN-gamma 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-gamma 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-alpha, in the absence of detectable IFN-gamma production. Except for TNF-alpha, the magnitude of those responses were significantly higher in LTBI subjects. Additional unbiased analyses of high dimensional flow-cytometry data revealed that TNF-alpha+ cells responding to Mtb antigens comprised 17 highly heterogeneous cell types. Among these 17 TNF-alpha+ 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-alpha+ 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-gamma 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.

Summary 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 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.

Diagnosing non-tuberculous mycobacteria (NTM) infection is important to discard tuberculosis (TB) infection when test results are discordant and could be useful to handle patients with pulmonary infectious diseases (PID) with NTM isolations of unclear clinical relevance. This study, describes an immunological NTM-Interferon-gamma (IFN-γ) Release Assay (IGRA) that we have developed using specific NTM antigens to detect NTM sensitization. We enrolled 342 patients: 90 latently TB infected (LTBI), 69 active TB, 60 NTM culture positive, 13 NTM infection suspicion (positive TST, negative IGRA and no BCG vaccination record), 53 HIV positive (4 also LTBI), 32 unknown etiology lymphadenopathies, 3 non-NTM lymphadenopathies, and 22 uninfected controls with no TB exposure record. Peripheral blood mononuclear cells were isolated from whole blood samples. Cells were then stimulated overnight with NTM specific antigens. Detection of IFN-γ producing cells was evaluated by ELISPOT. NTM positive and suspicion groups had a significantly higher count of INF-γ-producing cells after NTM-antigen sensitization. Using ROC curve analysis, a positivity cut-off of 6 spots was determined. NTM suspicion, NTM positive and non-culture confirmed lymphadenopathies groups, had a higher number of positive results than LTBI, HIV positive, and active TB patients (76.9%, 73.3% and 37.5%, 26.7%, 22.6%, 17.4%, respectively). Uninfected individuals and those with non-NTM lymphadenopathies were negative. NTM-IGRA could serve as an NTM infection diagnosis test aiding clinicians in the patient’s clinical evaluation and treatment decision-making. Ongoing studies are focused on characterizing the immune response against these antigens.

This research was supported by: (i) a grant from the Instituto de Salud Carlos III (PI 13/01546, PI16/01912, and PI18/00411), integrated in the Plan Nacional de I+D+I and cofunded by the ISCIII Subdirección General de Evaluación and the Fondo Europeo de Desarrollo Regional (FEDER); and (ii) a grant from the Sociedad Española de Neumología y Cirugía Torácica (project 25/2016; SEPAR; Barcelona, Spain). JD is a researcher from the Miguel Servet programme. 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.

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.

BACKGROUND Available data to assess the optimal diagnostic approach in infants and preschool children at risk of tuberculosis (TB) are limited. METHODS We conducted a prospective observational study in children younger than 5 years undergoing assessment with both tuberculin skin tests (TST) and QuantiFERON-TB Gold In-Tube (QFT-GIT) assays at 2 tertiary TB units in Barcelona, Spain. RESULTS A total of 383 children were included. One of 304 participants considered uninfected developed active TB during follow-up {median [interquartile range (IQR)]: 47 [30; 48] months}, compared with none of 40 participants with latent TB infection [follow-up since completion of anti-TB treatment: 42 (32; 45) months]. Overall test agreement between TST and QFT-GIT was moderate (κ = 0.551), but very good in children screened after TB contact (κ = 0.801) and in Bacillus Calmette-Guerin (BCG)-unvaccinated children (κ = 0.816). Discordant results (16.8%, all TST+/QFT-GIT-) were mainly observed in new-entrant screening and in BCG-vaccinated children. Children with indeterminate QFT-GIT results were on average younger than those with determinate results (median age: 12 vs. 30 months; P < 0.001). The sensitivity of TSTs and QFT-GIT assays in children with confirmed active TB was 100% (95% confidence interval: 79.4%-100%) and 93.7% (95% confidence interval: 69.8%-99.8%), respectively. In patients with latent TB infection or active TB, there was no correlation between age and antigen-stimulated interferon-γ responses (r = -0.044; P = 0.714). CONCLUSIONS In young BCG-unvaccinated children with recent TB contact, a dual testing strategy using TST and QFT-GIT in parallel may not be necessary. However, TST+/QFT-GIT- discordance is common, and it remains uncertain if this constellation indicates TB infection or not. In active TB, QFT-GIT assays do not perform better than TSTs.

Utility of the in-vitro immunodiagnostic methods, based on the detection of interferon-γ released by T-cells after specific Mycobacterium tuberculosis antigen stimulation (IGRA), has been an improvement in the accuracy of the latent tuberculosis infection diagnosis. IGRA have a well-known higher specificity than the tuberculin skin testing (TST). Moreover, they can obtain a larger number of positive results than the TST in immunocompromised patients. IGRA have shown a high correlation with M. tuberculosis exposure, but their positive and negative predictive value are similar than those obtained by TST. Nevertheless, given their high specificity, they allow reducing number of unnecessary preventive treatments. In addition, these in-vitro techniques are less affected than TST by the different immunosuppressing status. In this review is discussed up-to-date applicability of IGRA in different patient groups: contact studies, pediatric population, immunosuppressed patients, health care workers and active tuberculosis patients. Furthermore, it has been included possible future directions for latent tuberculosis infection and active tuberculosis diagnosis.

The identification of patients with latent tuberculosis infection, who are at higher risk to develop active disease, is an important component of disease control.We aim to compare the usefulness of the QuantiFERON-TB Gold in-tube assay and the tuberculin skin test to predict the development of active tuberculosis during follow-up, using positive and negative predictive values, positive likelihood ratios, and stratified level of risk.The study included contacts of tuberculosis cases diagnosed between 2007 and 2009. All contacts included were from the first circle of exposure. Tuberculin skin test and QuantiFERON test were performed and a chest radiograph was obtained during the contact's study.A total of 1,335 contacts were followed up for 4 years: a smear-positive index case was identified for 937 contacts, of whom 15 developed active tuberculosis and had initially presented with positive tuberculin skin test/QuantiFERON results, a normal chest radiograph, and no symptoms. The positive predictive value was 4% for QuantiFERON and 2% for the tuberculin skin test (when ≥5 mm). The probability of developing active disease was 2.36 times higher with a positive QuantiFERON, and 1.3 times higher with a positive tuberculin skin test. The positive predictive value was 17%, and the positive likelihood ratio was 7.53 for untreated contacts with a positive QuantiFERON. Stratifying according to initial QuantiFERON results showed a 6.36 times higher risk of developing active tuberculosis for patients with a QuantiFERON result greater than or equal to 10 IU/ml. Among bacillus Calmette-Guérin-vaccinated patients, a tuberculin skin test induration greater than or equal to 15 mm correlated better with a positive QuantiFERON.QuantiFERON results were more accurate than tuberculin skin test results in predicting tuberculosis. Although all contacts with QuantiFERON-positive results are at risk of developing tuberculosis, those with a tuberculin skin test induration greater than or equal to 15 mm and QuantiFERON greater than or equal to 10 IU/ml are at highest risk. This has important implications in the clinical management of tuberculosis contacts.

Background: Deregulation of microRNAs (miRNAs)has been associated with various human pathologies including bacterial infections.Aim: To investigate the role of miRNAs as blood biomarkers for the diagnosis of active pulmonary tuberculosis (TB).Materials and methods: We studied 50 patients, classified into 3 groups:(i) 17individuals with latent tuberculosis infection (LTBI) (ii) 17 active pulmonary TB patients (iii) 16 healthy individuals. Blood was collected in a PAXgene Blood RNA tubefor thelater isolation oftotal RNA. MiRNA quantification was performed with SurePrint G3 8x60K miRNA microarrays (Agilent). Real time-quantitative polymerase chain reaction (RT-qPCR)with the miScript PCR System (Qiagen) was used to validateselectedmiRNAexpression levels.Results: MiRNA expression patternswere comparedin whole blood from active TBpatients, LTBI individuals and healthy controls. Aset of miRNAs differentially deregulated during active pulmonary TB vs.LTBI and healthy controlswas identified. We then selected nine miRNAsfor validation in a larger patient cohort. The set of miRNAs consisting of miR-150, miR-21, miR-29cand miR-194 enabled to rapidly diagnose pulmonary TB with 91.21% sensitivity and 87.95% specificity.Conclusions:Theidentified novel miRNA signature opens the possibility of developinga simple, rapid and cheap TB diagnostic test from blood This work was supported by agrant from the Spanish Ministerio de Ciencia e Innovación SAF2010–21336. Andreas Meyerhans is a researcher founded from the Institucio Catalana de Recerca i Estudis Avancats (ICREA). Irene Latorre wasthe recipient of a joint ERS/SEPAR Long-Term fellowship (LTRF 82-2012)during this work

Advanced age was the only risk factor for false-negative IGRAs in this study of active tuberculosis http://ow.ly/Cvp5G

Detection and treatment of latent tuberculosis infection (LTBI) is an essential measure for tuberculosis (TB) control in low-incidence countries. However, such strategy is limited by the low predictive ability of the diagnostic tests for the development of active TB among infected people and the long-term and toxic treatment regimens. The in vitro interferon-gamma release assays are more specific and sensitive than the tuberculin skin test (TST), and enable a better selection of cases requiring treatment. Nonetheless, their capacity to predict development of TB is still poor. In addition, treatment regimens for LTBI are long, and compliance rates are low. This review discusses the use of the available diagnostic tests and the new approaches to the diagnosis of LTBI, as well as its management in different clinical scenarios. (C) 2017 Elsevier Espana, S.L.U. and Sociedad Espanola de Enfermedades Infecciosas y Microbiologia Clinica. All rights reserved.

Summary Objective In this study, we have performed a direct comparison between both T-cell based assays (QFN-G-IT and T-SPOT.TB) and TST in patients with psoriasis taking different immunosuppressant drug-regimens. Methods We have prospectively studied 103 patients with moderate-to-severe psoriasis who required latent tuberculosis infection (LTBI) screening before starting systemic immunosuppressive treatment or during its sustained use. Results Overall number of positive results was 16.5%, 17.5% and 8.7% using T-SPOT.TB, QFN-G-IT and TST, respectively. Differences in the percentage of positive results between TST with T-SPOT.TB and QFN-G-IT were significant ( p = 0.005 and p = 0.008, respectively). A total of 24.3% of the subjects enrolled were positive for at least one of the three tests performed. Sixteen patients with negative TST (17%) were positive for one of the two IGRAs. We obtained seven indeterminate results by T-SPOT.TB and two by QFN-G-IT. Seven patients with negative TST presented indeterminate results by either of two IFN-γ assays. Positive TST, T-SPOT.TB and QFN-G-IT results were not affected by clinical therapeutic profile. Conclusions Our results reveal that in vitro assays are useful methods for LTBI diagnosis in patients with psoriasis, suggesting that they might be less influenced by immunosuppression than TST.

The objective was to assess the annual risk of tuberculosis infection by means of tuberculin skin testing (TST) in children, evaluating whether QuantiFERON-TB Gold In-Tube (QFN-G-IT) could improve the accuracy. On the basis of the positive TST results, the global annual incidence was estimated at 0.78%, with an increase in the prevalence from 0.64% to 1.68% in 2 years. However, QFN-G-IT was only positive in 6 of the 25 children with positive TST. The confirmation of the positive TST results by QFN-G-IT provided more accurate annual incidence estimation.

Interferon (IFN)-γ release assays (IGRAs) are probably the most accurate tests for the detection of latent Mycobacterium tuberculosis infection, but IGRAs are labour intensive and the transport of samples over longer distances is difficult. IFN-γ-induced protein (IP)-10 is expressed at 100-fold higher levels than IFN-γ, and IP-10 release assays have comparable performance to IGRAs. The aim of this study was to explore the diagnostic potential of a novel IP-10 release assay based on dried plasma spots (DPS). The presence of IP-10 and IFN-γ was determined in plasma and in DPS by ELISA. Diagnostic algorithms for plasma and DPS tests for IP-10 were developed on a training cohort comprising 60 tuberculosis (TB) patients and 59 healthy controls. Diagnostic accuracy was assessed in a validation cohort comprising 78 TB patients and 98 healthy controls. Plasma was measured in Spain and DPS samples were sent to Denmark using the conventional postal service for analysis. IP-10 was readily detectable in both plasma and DPS, and correlation was excellent (r2 = 0.95). QuantiFERON-TB Gold In-Tube (QFT-TB) and IP-10 in DPS and plasma rendered comparable sensitivity (78%, 82% and 84%, respectively), specificity (100%, 97% and 97%, respectively) and indeterminate rates (p>0.55). The DPS-based IP-10 test has comparable diagnostic accuracy to the QFT-TB and samples can be sent via conventional mail over long distances for analysis without affecting the results.

Objectives: To investigate the immune response against M. tuberculosis (MTB) by Interferon Gamma Release Assays (IGRAs) in Tuberculosis (TB) and Latent TB Infected (LTBI) patients exposed to cigarette smoke. Methods: A cross sectional study from two clinics in Barcelona. Subjects included were: (i) 216 active pulmonary TB, (ii) 309 asymptomatic contact tracing individuals. At first visit blood was collected for IGRAs. The impact of cigarette smoke on MTB immune response was analyzed as the median of IFN-γ secreted (UI/mL) in QFN-G-IT and as the number (median) of T-cells producing IFN-γ [spot forming cells (SFC)] in T-SPOT.TB. Results: False-negative results in TB patients were higher in smokers (aOR:4.42; p Conclusions: Tobacco exposure was associated with IGRAs false-negative results in TB patients and was an independent risk factor for LTBI acquisition. A less IFN-γ response was observed in smokers than non-smokers. An increment on tobacco smoking produces a reduction on immune responses observed by both IGRAs. PII –Tabaquismo. 2013 SEPAR grant.

Summary Introduction Interferon-γ (IFN-γ) inducible protein 10kD (IP-10) and IFN-γ release assays (IGRAs) are immunodiagnostic tests aiming to identify the presence of specific cellular immune responses, interpreted as markers for latent infection with Mycobacterium tuberculosis. Incubation at higher temperatures could affect IFN-γ and IP-10 responsiveness in order to improve the performance of IP-10 release assays and IGRAs. Aim The aim of this study was to assess the robustness of whole blood based IP-10 release assay and IGRAs and the effect of hyper-thermic incubation (39 °C) on the diagnostic accuracy of IP-10 release assay and IGRAs. Results We included 65 patients with confirmed pulmonary tuberculosis and 160 healthy controls from 6 European centres collaborating in the TBnet. In patients, IP-10 responses increased 1.07 (IQR 0.90–1.36) fold and IFN-γ responses decreased 0.88 (IQR 0.57–1.02) fold, with 39 °C compared to 37 °C incubation temperature. At 37 °C IGRA sensitivity was 85% and IP-10 sensitivity was 82%, whereas specificity was 97% for both tests (p > 0.8). These minor changes observed as a result of hyper-thermic incubation were not sufficient to impact IGRA and IP-10 release assay test performance. Conclusion The performance of IGRA and IP-10 release assays is robust despite variations in the incubation temperature between 37 °C and 39 °C.

The aim of the present study was to compare the performance of the interferon (IFN)-γ tests (QuantiFERON®-TB Gold In-Tube (QFT-G-IT) and T-SPOT®.TB) with the tuberculin skin test (TST) in diagnosing tuberculosis (TB) infection in children, and to analyse discordant results. This was a prospective study including 98 children from contact-tracing studies and 68 children with TST indurations ≥ 5 mm recruited during public health screenings. Positive IFN-γ tests results were associated with risk of exposure (p

The aim of the present study was to determine the role of previous non-tuberculous mycobacteria sensitisation in children as a factor of discordant results between tuberculin skin test (TST) and an in vitro T-cell based assay (T-SPOT.TB; Oxford Immunotec, Oxford, UK). We enrolled 21 non-bacille Calmette-Guerin-vaccinated paediatric patients for suspicious of latent tuberculosis infection (LTBI). These patients yielded a positive TST and a negative T-SPOT.TB. Cells were stimulated with Mycobacterium avium sensitin (having cross-reaction with Mycobacterium intracellulare and Mycobacterium scrofulaceum ) and the presence of reactive T-cells was determined by an ex vivo ELISPOT. From the 21 patients, in 10 cases (47.6%), we obtained a positive ELISPOT result after stimulation with M. avium sensitin, in six (28.6%) cases, the result was negative and in the remaining five (23.8%) cases, the result was indeterminate. In conclusion, previous non-tuberculous mycobacteria sensitisation induces false-positive results in the TST for diagnosing LTBI and the use of γ-interferon tests could avoid unnecessary chemoprophylaxis treatment among a child population.

Background: Immunocompromised persons infected with Mycobacterium tuberculosis (MTB) have increased risk of tuberculosis (TB) reactivation, but their management is hampered by the occurrence of false-negative results of the tuberculin skin test (TST). The T-cell interferon (IFN)-γ release blood assays T-SPOT.TB (TS.TB) [Oxford Immunotec; Abingdon, UK] and QuantiFERON-TB Gold In-Tube (QFT-IT) [Cellestis Ltd; Carnegie, VIC, Australia] might improve diagnostic accuracy for latent TB infection (LTBI) in high-risk persons, although their performance in different groups of immunocompromised patients is largely unknown. Methods and results: Over a 1-year period, we prospectively enrolled patients in three different immunosuppressed groups, as follows: 120 liver transplantation candidates (LTCs); 116 chronically HIV-infected persons; and 95 patients with hematologic malignancies (HMs). TST, TS.TB, and QFT-IT were simultaneously performed, their results were compared, and intertest agreement was evaluated. Overall, TST provided fewer positive results (10.9%) than TS.TB (18.4%; p < 0.001) and QFT-IT (15.1%; p = 0.033). Significantly fewer HIV-infected individuals had at least one positive test (9.5%) compared with LTCs (35.8%; p < 0.001) and patients with HMs (29.5%; p < 0.001). Diagnostic agreement between tests was moderate (κ = 0.40 to 0.65) and decreased in the HIV-infected group when the results of the TS.TB were compared with either TST (κ = 0.16) or QFT-IT (κ = 0.19). Indeterminate blood test results due to low positive control values were significantly more frequent with QFT-IT (7.2%) than with TS.TB (0.6%; p < 0.001). Conclusions: Blood tests identified significantly more patients as being infected with MTB than TST, although diagnostic agreement varied across groups. Based on these results, we recommend tailoring application of the new blood IFN-γ assays for LTBI in different high-risk groups and advise caution in their current use in immunosuppressed patients.

SUMMARYIn vitro tests have been developed for the diagnosis of tuberculosis (TB) infection. The objective was to analyse latent TB infection in drug and alcohol abusers through two interferon-γ techniques. One hundred and thirty-nine patients were admitted between February 2006 and May 2007. Mean age was 39·8 years [31% HIV positive]. The enzyme immunoassay (EIA) and enzyme-linked immunospot (ELISPOT) interferon-γ assays were positive in 34% of patients with an agreement of 83% (κ=0·63). Tuberculin skin test (TST) was positive in 29% of patients and the agreement of TST with EIA and ELISPOT interferon-γ assays was 85% (κ=0·62) and 83% (κ=0·57), respectively. Almost 50% of patients with history of TB had a positive in vitro test. In conclusion, we observed a high prevalence of latent TB and good agreement between the new in vitro tests that otherwise may continue to be positive long after developing TB disease.

Chronic inflammatory diseases such as Crohn's disease or ulcerative colitis, are major autoimmune disorders with increasing incidence.1 Although conventional therapies based on corticosteroids and immunosuppressants play a major role in the management of these diseases, the corticoisteroid-associated adverse events and the existence of immunosuppressant-refractory patients have prompted the development of new disease-modifying therapies. Biological agents, especially the anti-Tumor Necrosis Factor (TNF)-α agents, have emerged as an effective treatment for these diseases.1 One of the primary concerns with anti-TNF-α agents is the possibility of collateral effects on host defence mechanisms. Patients undergoing TNF-α inhibition are at increased risk of developing severe infections. A temporal association between anti-TNF-α antibodies and reactivation of latent tuberculosis (TB) infection has been established.2,3 In fact, TNF-α is one of the key molecules involved in granuloma formation and maintenance. Therefore, previous to start TNF-α inhibition, appropriate screening of latent TB infection and early active TB diagnosis has become mandatory.4 The current guidelines for screening latent TB are based on the tuberculin skin test (TST) that has been used since the late 1800s for diagnosing latent TB infection. TST attempts to measure cell-mediated immunity as assessed by a delayed-type hypersensitivity response to its components.5,6 TST is produced by steaming cultures of Mycobacterium tuberculosis in a sterilizer and purifying the proteins (purified protein derivative [PPD]) by repeated precipitation with neutral ammonium sulphate.7 In the TST, 0.1 ml of PPD solution are injected intradermically in the forearm of the patient. After 48 h, the diameter of induration is measured. A TST induration > 5mm is considered positive, independently of the Mycobacterium bovis bacilli Calmette-Guerin (BCG) vaccination status. In case of induration < 5mm, it is necessary to repeat the TST between 1–2 weeks later (booster effect), also considering as positive …

We evaluated the T-SPOT.TB and Quantiferon-TB Gold In tube (QFN-G-IT) tests for diagnosing Mycobacterium tuberculosis infection. T-SPOT.TB was more sensitive than QFN-G-IT in diagnosing both active and latent infection. Both gamma interferon tests were unaffected by prior Mycobacterium bovis BCG vaccination. Among children who were not BCG vaccinated but had a positive tuberculin skin test, QFN-G-IT was negative in 53.3% of cases, and T-SPOT.TB was negative in 50% of cases.

Utility of the in-vitro immunodiagnostic methods, based on the detection of interferon-γ released by T-cells after specific Mycobacterium tuberculosis antigen stimulation (IGRA), has been an improvement in the accuracy of the latent tuberculosis infection diagnosis. IGRA have a well-known higher specificity than the tuberculin skin testing (TST). Moreover, they can obtain a larger number of positive results than the TST in immunocompromised patients. IGRA have shown a high correlation with M. tuberculosis exposure, but their positive and negative predictive value are similar than those obtained by TST. Nevertheless, given their high specificity, they allow reducing number of unnecessary preventive treatments. In addition, these in-vitro techniques are less affected than TST by the different immunosuppressing status. In this review is discussed up-to-date applicability of IGRA in different patient groups: contact studies, pediatric population, immunosuppressed patients, health care workers and active tuberculosis patients. Furthermore, it has been included possible future directions for latent tuberculosis infection and active tuberculosis diagnosis.

Latent infection with Mycobacterium tuberculosis is defined by a positive IFN-γ release assay (IGRA) result in the absence of active tuberculosis. Only few, mostly monocentric studies have evaluated the role of IGRAs to predict the development of tuberculosis in recent contacts in low-incidence countries of tuberculosis.To analyze IGRA results and the effect of preventive chemotherapy on tuberculosis progression rates among recent contacts.Results from contact investigations at 26 centers in 10 European countries including testing for latent infection with M. tuberculosis by the QuantiFERON-TB Gold In-Tube (QFT) test or the T-SPOT.TB (TSPOT) were prospectively collected and analyzed.Among 5,020 contacts of 1,023 index cases, 25 prevalent secondary cases were identified at screening. Twenty-four incident cases occurred among 4,513 contacts during 12,326 years of cumulative follow-up. In those with a positive IGRA result, tuberculosis incidence was 0.2 (QFT) and 0 (TSPOT) per 100 patient-years when contacts received preventive chemotherapy versus 1.2 (QFT) and 0.8 (TSPOT) per 100 patient-years in those not treated (38 and 37 patients needed to be treated to prevent one case, respectively). Positive and negative predictive values were 1.9% (95% confidence interval [CI], 1.1-3.0) and 99.9% (95% CI, 99.7-100) for the QFT and 0.7% (95% CI, 0.1-2.6) and 99.7% (95% CI, 99.1-99.9) for the TSPOT.Tuberculosis rarely developed among contacts, and preventive chemotherapy effectively reduced the tuberculosis risk among IGRA-positive contacts. Although the negative predictive value of IGRAs is high, the risk for the development of tuberculosis is poorly predicted by these assays.

Tuberculosis (TB) is an infectious disease of important global health concern. On 2016, TB caused 10.4 million new cases and 1.8 million deaths (1). Compared to adults, young children infected with Mycobacterium tuberculosis ( M.TB ) are at high risk of progression and have an increased risk of developing a severe TB outcome (2). TB screening recommendations in children and other risk groups are described in several international and national guidelines, as well as the recommended prophylaxis (3,4). However, in some cases, prophylaxis may turn out to be unnecessary and depending on the setting it may be impossible to access. In order to better characterize who would benefit from prophylaxis, there is a need to identify those at a higher risk of developing active TB.

The aim of our work here was to evaluate the immunogenicity of 60 mycobacterial antigens, some of which have not been previously assessed, notably a novel series of in vivo-expressed Mycobacterium tuberculosis (IVE-TB) antigens. We enrolled 505 subjects and separated them in individuals with and without latent tuberculosis infection (LTBI) vs. patients with active tuberculosis (TB). Following an overnight and 7 days stimulation of whole blood with purified recombinant M. tuberculosis antigens, interferon-γ (IFN-γ) levels were determined by ELISA. Several antigens could statistically significantly differentiate the groups of individuals. We obtained promising antigens from all studied antigen groups [dormancy survival regulon (DosR regulon) encoded antigens; resuscitation-promoting factors (Rpf) antigens; IVE-TB antigens; reactivation associated antigens]. Rv1733, which is a probable conserved transmembrane protein encoded in DosR regulon, turned out to be very immunogenic and able to discriminate between the three defined TB status, thus considered a candidate biomarker. Rv2389 and Rv2435n, belonging to Rpf family and IVE-TB group of antigens, respectively, also stood out as LTBI biomarkers. Although more studies are needed to support our findings, the combined use of these antigens would be an interesting approach to TB immunodiagnosis candidates.

Summary Objective Performance of IFN-γ assays in children is compromised. Therefore, we investigated the utility of IP-10 for the detection of active tuberculosis (TB) and latent tuberculosis infection (LTBI) diagnosis in children; comparing its positivity with QuantiFERON-TB Gold In-Tube (QFN-G-IT) and T-SPOT.TB. Methods We studied 230 children from three groups: active TB, screening (healthy children without known exposure to active TB patient screened at school or by their paediatrician) and contact-tracing studies. IFN-γ release was determined by QFN-G-IT and T-SPOT.TB. IP-10 was detected in QFN-G-IT supernatants by ELISA. Results When combining QFN-G-IT and IP-10 assays, positive results improved significantly from 38.3% in QFN-G-IT and 33.9% in IP-10 to 41.3%. Age and type of contact were significant risk factors associated with positive QFN-G-IT and IP-10 results. IP-10 levels after antigen-specific stimulation were significantly higher in comparison to IFN-γ levels. Correlation between the three assays was good ( κ = 0.717–0.783). Conclusions IP-10 cytokine is expressed in response to TB specific-antigens used in QFN-G-IT. In conclusion, the use of IFN-γ T-cell based assays in combination with an additional IP-10 assay detection could be useful for diagnosing active TB and LTBI in children.

Rationale: In the absence of active tuberculosis, a positive tuberculin skin test (TST) or interferon-gamma 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.

Recently, a selection of HLA class II-restricted epitopes of ESAT-6 and CFP-10 Mycobacterium tuberculosis proteins from the region of difference (RD) 1 have been described. We have evaluated the host interferon-gamma (IFN-γ) T cell response to these RD1 selected peptides at the beginning and during anti-tuberculosis therapy.We studied 29 pulmonary TB patients enrolled at the beginning of treatment and 24 enrolled during treatment. We performed T-SPOT.TB and ELISPOT with RD1 selected peptides.Patients included at the beginning of treatment responded producing IFN-γ after antigen stimulation in 89.7% by means of T-SPOT.TB and 79.3% by means of RD1 selected ELISPOT. In contrast, for patients included during treatment the percentages were 87.5% and 25%, respectively. Differences in sensitivities between patients evaluated at the beginning and during treatment were only significant for RD1 selected ELISPOT (p0.0001).The host immune response to RD1 selected peptides is lower than to T-SPOT.TB during therapy. Immunological assays based on RD1 selected peptides may be useful tools for studying the immune response during anti-tuberculosis therapy.

Biological agents, particularly anti-Tumour Necrosis Factor (TNF)-α agents, have emerged as an effective treatment in patients with chronic inflammatory diseases. An association between anti-TNF-α antibodies and reactivation of latent tuberculosis infection (LTBI) has been established. Appropriate screening for TB infection has become mandatory before starting a treatment based on TNF-α inhibition. The objective was to determine the usefulness of IFN-γ release assays in diagnosing LTBI in patients with inflammatory rheumatic diseases scheduled for anti-TNF-α treatment. The study included 53 individuals with inflammatory rheumatism. All patients had a TST, a chest radiograph, QuantiFERON Gold In-Tube (QFN-G-IT) and T-SPOT.TB. To investigate the influence of non-tuberculous mycobacteria (NTM) infections on non-BCG-vaccinated patients, with a positive TST result and both negative IFN-γ assays, we performed an ex vivo ELISPOT, stimulating the cells separately with NTM sensitins. TST was positive in 7 cases, T-SPOT.TB in 11 and QFN-G-IT in 9 cases. Agreement between TST and T-SPOT.TB and QFN-G-IT was 77.35% (κ = 0.33 and κ = 0.40, respectively), and between both in vitro tests, it was 83.01% (κ = 0.57). Of the three patients with positive TST and negative T-SPOT.TB and QFN-G-IT, one positive ELISPOT result was obtained after stimulation with NTM sensitins. Positive TST, T-SPOT.TB and QFN-G-IT results were not affected by the immunosuppressive therapies. IFN-γ release assays are useful methods for avoiding TST false-positive results, but in those patients with a high risk of developing active TB and in the absence of predictive value studies in this specific kind of population for knowing how safe is the use of IGRAs alone, the combined use of TST and IFN-γ tests should be recommended in order to increase the overall number of LTBI diagnoses.

Background Individuals infected with human immunodeficiency virus (HIV) have an increased risk of progression to active tuberculosis following Mycobacterium tuberculosis infection. The objective of the study was to determine IFN-γ responses for the detection of latent tuberculosis infection (LTBI) with QuantiFERON-TB GOLD In Tube (QFT-G-IT) and T-SPOT.TB in HIV patients, and evaluate the influence of CD4 cell count on tests performance. Methods We studied 75 HIV patients enrolled for ongoing studies of LTBI with T-SPOT.TB, QFN-G-IT and TST. Mean CD4 cell counts ± standard deviation was 461.29 ± 307.49 cells/μl. Eight patients had a BCG scar. Results T-SPOT.TB, QFN-G-IT and TST were positive in 7 (9.3%), 5 (6.7%) and 9 (12%) cases, respectively. Global agreement between QFN-G-IT and T-SPOT.TB was 89% (κ = 0.275). The overall agreement of T-SPOT.TB and QFN-G-IT with TST was 80.8% (κ = 0.019) and 89% (κ = 0.373), respectively. We have found negative IFN-γ assays results among 2 BCG-vaccinated HIV-infected individuals with a positive TST. In non BCG-vaccinated patients, QFN-G-IT and TST were positive in 5 cases (7.5%) and T-SPOT.TB in 7 (10.4%). In contrast, in BCG-vaccinated patients, only TST was positive in 4/8 (50%) of the cases. The differences obtained in the number of positive results between TST and both IFN-γ assays in BCG vaccinated patients were significant (95% CI 3-97%, p = 0.046), however, the confidence interval is very wide given the small number of patients. In patients with CD4< 200, we obtained only one (5%) positive result with T-SPOT.TB; however, QFN-G-IT and TST were negative in all cases. On the contrary, percentages of positive results in patients with CD4> 200 were 10.9% (6/55), 9.1% (5/55) and 16.4% (9/55) with T-SPOT.TB, QFN-G-IT and TST, respectively. Conclusions IFN-γ tests have the benefit over TST that are less influenced by BCG vaccination, consequently they are more specific than TST. Although our number of patients with advance immunosuppression is limited, our study suggests that IFN-γ assays are influenced with level of immunosuppression. The use of IFN-γ assays could be a helpful method for diagnosing LTBI in HIV population.

BACKGROUND:Health care workers (HCWs) are a group at risk of latent tuberculosis infection (LTBI). The aims of this study were to determine IFN-gamma response by QuantiFERON-TB GOLD In Tube (QFN-G-IT) and T-SPOT.TB in HCWs, comparing the results with tuberculin skin test (TST); and to analyze the capacity of IFN-gamma tests to detect recent versus remote LTBI with a prolonged stimulation test (PST). METHODOLOGY/PRINCIPAL FINDINGS:A total of 147 HCWs were enrolled; 23 of whom were BCG vaccinated. 95 HCWs (64.6%) had a previous positive TST and were not retested; and 52 HCWs had a previous negative TST or were tested for the first time. When we analysed individuals without previous positive TST, the number of positive results for T-SPOT.TB was 12/52 (23.1%); and for QFN-G-IT, 9/52 (17.3%). The global concordance (kappa) between T-SPOT.TB and QFN-G-IT with TST was 0.754 and 0.929 respectively. Of individuals with previous positive TST, T-SPOT.TB and QFN-G-IT were negative in 51.6% (49/95) and 62.1% (59/95) respectively, decreasing the concordance to 0.321 and 0.288, respectively. In non-BCG vaccinated HCWs with previous positive TST a positive IFN-gamma test was associated with degree of exposure and diameter of TST. PST was performed in 24 HCW with previous positive TST and negative IFN-gamma tests. PST was developed in 3 cell cultures stimulated with medium alone, ESAT-6 and CFP-10, respectively. In the third and sixth day of incubation period, part of the supernatants were replaced with complete medium supplemented with (rIL)-2. On day 9, ELISPOT assay was performed. In 14 samples PST was not valid due to not having enough cells. In 8 cases, the response was negative, and in 2 cases positive, suggesting that these patients were infected with Mycobacterium tuberculosis in some point in the past. CONCLUSIONS:Both IFN-gamma tests showed a similar number of positive results, and concordance between the tests was excellent. None of the tests was affected by prior BCG vaccination. IFN-gamma tests are a useful tool for detecting recent infection in HCW population.

We have evaluated the quantitative T-cell response after specific Mycobacterium tuberculosis antigen stimulation in active tuberculosis (TB) and latent TB infection (LTBI) patients. In adults, the median number of T cells after RD1 antigen stimulation was significantly higher in active TB patients than in LTBI patients. In children, the number of responder T cells against the specific antigens was higher in active TB than in LTBI patients, although the differences were not significant. In summary, in patients with suspected clinical TB, although there is overlapping in the number of responder T cells between both groups, a T-cell count above the described threshold could suggest active TB, especially in patients with a high probability of having active TB and low probability of having LTBI. In addition, the results are consistent with the current evidence that T-cell response may indicate mycobacterial burden and disease activity.

The objectives of the study were to assess the performance of the QuantiFERON-TB Gold In-Tube (QFN-G-IT) and the T-SPOT.TB tests in the immunodiagnosis of active tuberculosis (TB) in adult patients, and to study the T-cell interferon gamma (IFN-gamma) responses during treatment and in patients who have recovered after curative treatment and self-healed TB patients. When only analyzing patients included at the beginning of treatment, the sensitivity was 83.3% for T-SPOT.TB and 69.4% for QFN-G-IT. In contrast, when evaluating patients during treatment, the sensitivity of the T-SPOT.TB and QFN-G-IT decreased to 69.8% and 48.8%, respectively. The response to the specific antigens increased after finishing the treatment compared with the values during the treatment. The T-SPOT.TB was more sensitive in diagnosing active TB than the QFN-G-IT. The IFN-gamma tests could be used as a complementary method in the diagnosis of active TB.

From the authors: We do appreciate the comments by M. Tebruegge and co-workers about our recently published manuscript in the European Respiratory Journal 1. The aim of this reply is to clarify some points in order to interpret better the results of the study, given that we think there were some misunderstandings. In vitro assays for measuring interferon (IFN)-γ released by the T-cells after specific Mycobacterium tuberculosis stimulation have demonstrated promising results in adults and also in children for diagnosing tuberculosis (TB) infection 2, 3. However, there are discordant results between IFN-γ based assays and the tuberculin skin test (TST) that require clarification in order to assess the real utility of the in vitro tests in the management of patients 2, 4. In our study we determined the potential role of non-tuberculous mycobacteria (NTM) sensitisation in children as a factor of discordant results between TST and an in vitro T-cell based assay (T.SPOT. TB ; Oxford Immunotec, Oxford, UK). We enrolled 21 non-bacille Calmette–Guerin vaccinated paediatric …

We read with interest the article by Dheda et al 1 who followed our approach for a rapid diagnosis of smear-negative tuberculosis by bronchoalveolar lavage (BAL) enzyme-linked immunospot (ELISpot)2 in a country of high tuberculosis incidence, including individuals with HIV-1 infection. The authors report a sensitivity of 88.9% and specificity of 94.7% of the BAL ELISpot (T-SPOT.TB test) for the diagnosis of tuberculosis in suspects with scarce or negative acid-fast bacilli (AFB) sputum smears. This observation is important, as it confirms the findings of other …