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.

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.

Urine NMR-based TB metabolic fingerprinting for the diagnosis of TB in children.

Tuberculosis (TB) is a major cause of morbidity and mortality in children, and early diagnosis and treatment are crucial to reduce long-term morbidity and mortality. In this study, we explore whether urine nuclear magnetic resonance (NMR)-based metabolomics could be used to identify differences in the metabolic response of children with different diagnostic certainty of TB. We included 62 children with signs and symptoms of TB and 55 apparently healthy children. Six of the children with presumptive TB had bacteriologically confirmed TB, 52 children with unconfirmed TB, and 4 children with unlikely TB. Urine metabolic fingerprints were identified using high- and low-field proton NMR platforms and assessed with pattern recognition techniques such as principal components analysis and partial least squares discriminant analysis. We observed differences in the metabolic fingerprint of children with bacteriologically confirmed and unconfirmed TB compared to children with unlikely TB (p = 0.041 and p = 0.013, respectively). Moreover, children with unconfirmed TB with X-rays compatible with TB showed differences in the metabolic fingerprint compared to children with non-pathological X-rays (p = 0.009). Differences in the metabolic fingerprint in children with different diagnostic certainty of TB could contribute to a more accurate characterisation of TB in the paediatric population. The use of metabolomics could be useful to improve the prediction of TB progression and diagnosis 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.

Novel intracellular antibiotic delivery system against Staphylococcus aureus: cloxacillin-loaded poly(d,l-lactide-co-glycolide) acid nanoparticles.

Aim: First, to compare in vitro minimum inhibitory concentrations (MIC) of free cloxacillin and cloxacillin-containing nanoparticles (NP) against methicillin-susceptible (MSSA) and resistant Staphylococcus aureus (MRSA) and second, to assess NP antimicrobial activity against intracellular S. aureus. Methods: Poly(d,l-lactide-co-glycolide) acid (PLGA)-NP were loaded with cloxacillin and physico-chemically characterized. MICs were determined for reference strains Newman-(MSSA) and USA300-(MRSA). Murine alveolar macrophages were infected, and bacterial intracellular survival was assessed after incubating with free-cloxacillin or PLGA-cloxacillin-NP. Results & conclusion: For both isolates, MICs for antibiotic-loaded-NP were lower than those obtained with free cloxacillin, indicating that the drug encapsulation improves antimicrobial activity. A sustained antibiotic release was demonstrated when using the PLGA-cloxacillin-NP. When considering the lowest concentrations, the use of drug-loaded NP enabled a higher reduction of intracellular bacterial load.

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.

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.

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.