Use of IP-10 detection in dried plasma spots for latent tuberculosis infection diagnosis in contacts via mail

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

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

EP27-355-23 Characterization of the metabolic urine profile using nuclear magnetic resonance (NMR) spectroscopy to monitor treatment of tuberculosis

The emergence of drug-resistant tuberculosis (TB) challenges the control of TB with an increase in treatment failure. Surveillance during treatment is essential to monitor adherence and treatment response. Metabolomics can be used to obtain a "fingerprint" of the metabolites presents in a biological sample, allowing the study of sets of metabolites affected by host-pathogen interactions and the identification of therapeutic markers to improve the evaluation of the TB outcome. In this proof of concept, we aimed to characterize the urinary metabolic profile of patients receiving anti-TB treatment and its association to TB treatment outcome

Study of CD27 and CCR4 markers on specific CD4+ T-cells as immune tools for active and latent tuberculosis management

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

Use of IP-10 detection in dried plasma spots for latent tuberculosis infection diagnosis in contacts via mail

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

Diagnostic benefits of adding EspC, EspF and Rv2348-B to the QuantiFERON Gold In-tube antigen combination

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 T