Boosting effect of IL-7 in interferon gamma release assays to diagnose Mycobacterium tuberculosis infection

A quarter of the world's population is estimated to be infected with Myobacterium tuberculosis (Mtb). Infection is detected by immune response to M. tuberculosis antigens using either tuberculin skin test (TST) and interferon gamma release (IGRA's), tests which have low sensitivity in immunocompromised. IL-7 is an important cytokine for T-cell function with potential to augment cytokine release in in-vitro assays. This study aimed to determine whether the addition of IL-7 in interferon-gamma release assays (IGRAs) improves its diagnostic performance of Mtb infection.; 44 cases with confirmed TB and 45 household contacts without TB were recruited and 1ml of blood was stimulated in two separate IGRA's tube set: one set of standard Quantiferon TB gold tubes mitogen, TB antigen and TB Nil; one set of customized Quantiferon TB gold tubes with added IL-7. Following IFN-γ and IP-10 release was determined using ELISA.; We found that the addition of IL-7 led to significantly higher release of IFN-γ in individuals with active TB from 4.2IU/ml (IQR 1.4-6.9IU/ml) to 5.1IU/ml (IQR 1.5-8.1IU/ml, p = 0.0057), and we found an indication of a lower release of both IFN-γ and IP-10 in participants with negative tests.; In TB cases addition of IL-7 in IGRA tubes augments IFN-γ but not IP-10 release, and seems to lower the response in controls. Whether IL-7 boosted IGRA holds potential over standard IGRA needs to be confirmed in larger studies in high and low TB incidence countries

First description of agonist and antagonist IP-10 in urine of patients with active TB

Objectives: Biomarkers for tuberculosis (TB) diagnosis and clinical management are needed to defeat TB. In chronic hepatitis, patients not responding to interferon/ribavirin treatment had high levels of an antagonist form of IP-10. Recently, antagonist IP-10 has been shown to be involved also in TB pathogenesis. Here, we investigated IP-10 agonist/antagonist forms as potential inflammatory biomarkers to support TB diagnosis and monitoring. Methods: Total IP-10 and its agonist/antagonist forms were measured by SIMOA digital ELISA in urine obtained from patients with active TB at baseline and after treatment. Healthy donors (HD) and patients with pneumonia were enrolled as controls. Results: Patients with active TB had significantly higher levels of total and agonist IP-10 at baseline compared to HD; conversely, no differences were observed between IP-10 levels in active TB vs pneumonia. Moreover, in active TB a decline of total urine IP-10 was observed at therapy completion; agonist/antagonist forms reflected this decline although their differences were not statistically significant. Conclusions: We showed for the first time that agonist/antagonist IP-10 forms are measurable in urine. IP-10 levels associate with TB and pneumonia disease, suggesting their association with acute inflammation. Further studies are needed to assess their role to monitor TB treatment efficacy. Keywords: Tuberculosis, Biomarker, IP-10 antagonism, Urine, Treatment monitorin

Development of a One-Step Probe Based Molecular Assay for Rapid Immunodiagnosis of Infection with <i>M. tuberculosis</i> Using Dried Blood Spots

<div><p>Background</p><p>Antigen specific release of IP-10 is the most promising alternative marker to IFN-γ for infection with <i>M. tuberculosis</i>. Compared to Interferon-γ release assays (IGRA), IP-10 is released in high levels enabling novel approaches such as field friendly dried blood spots (DBS) and molecular detection.</p><p>Aim</p><p>To develop a robust IP-10 based molecular assay for the diagnosis of infection with <i>M. tubercuolsis</i> from whole blood and DBS.</p><p>Method</p><p>We developed a one-step probe based multiplex RT-qPCR assay for detecting IP-10 and IFN-γ mRNA expression from whole blood and DBS samples. The assay was validated and applied for the diagnosis of <i>M. tuberculosis</i> infection in DBS samples from 43 patients with confirmed TB, 13 patients with latent TB and 96 presumed uninfected controls. In parallel, IP-10 and INF-γ levels were measured in Quantiferon (QFT-TB) plasma supernatants.</p><p>Results</p><p>IP-10 mRNA upregulation was detectable at 4 hours after stimulation (6 fold upregulation) peaking at 8 hours (108 fold upregulation). IFN-γ expression occurred in concert but levels were lower (peak 6.7 fold upregulation). IP-10 gene expression level was significantly higher in patients with tuberculosis (median 31.2, IQR 10.7–67.0) and persons with latent tuberculosis infection (LTBI) (41.2, IQR 9.8–64.9) compared to healthy controls (1.6, IQR 1.1–2.4; p<0.0001). The IP-10 mRNA and protein based tests had comparable diagnostic accuracy to QFT-TB, sensitivity (85% and 88% vs 85%) and specificity (96% and 96% vs 97%, p = ns.).</p><p>Conclusion</p><p>We developed a rapid, robust and accurate molecular immunodiagnostic test for <i>M. tuberculosis</i> infection. By combining DBS based sample acquisition, mail or currier based sample transport with centralized molecular detection, this immunodiagnostic test concept can reduce the local technological requirements everywhere and make it possible to offer highly accurate immunodiagnostic tests in low resource settings.</p></div

Interplay of DDP4 and IP-10 as a Potential Mechanism for Cell Recruitment to Tuberculosis Lesions

International audienceMycobacterium tuberculosis is one of the world's most successful pathogens equipped to establish itself within the human host as a subclinical infection without overt disease. Unable to eradicate the bacteria, the immune system contains the infection in a granuloma structure. Th1 cells that are essential for infection control are recruited to the site of infection directed by chemokines, predominantly CXCL10. It has previously been shown that CXCL10 in the plasma of patients chronically infected with hepatitis C virus is present primarily in an antagonist form. This is due to N-terminal truncation by the enzyme DPP4, which results in the antagonist form that is capable of binding its receptor CXCR3, but does not induce signaling. We aimed to explore whether such CXCL10 antagonism may have an impact on the pathogenesis of tuberculosis (TB)

Comparison of mRNA extraction from whole blood (WB) and dried blood spots (DBS).

<p>Whole blood from 12 TB patients and 8 LTBI persons was incubated in QFT-TB tubes for 8 hours at 37°C. mRNA was extracted directly from WB and DBS samples were prepared for later mRNA extraction. IP-10 gene expression was determined using our RT-qPCR assay. The difference was analysed using a Wilcoxon matched pairs test <i>p</i> = 0.003.</p

IP-10 mRNA expression and IP-10 and IFN-γ protein release.

<p>Whole blood from 96 healthy controls, 43 TB patients and 13 LTBI persons was stimulated with ESAT-6, CFP-10 and TB7.7. IP-10 gene expression was analysed from DBS after 8 hours stimulation (A) and IP-10 and IFN-γ protein levels were analysed from plasma after 20 hours stimulation (B and C respectively). A Kruskal Wallis test was performed to analyse the differences between the groups. IFN-γ mRNA gene expression was not measured in this experiment.</p

Baseline.

<p>Baseline.</p