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.

Diagnostic Accuracy of Early Secretory Antigenic Target-6-Free Interferon-gamma Release Assay Compared to QuantiFERON-TB Gold In-tube.

BACKGROUND: Early secretory antigenic target-6 (ESAT-6) is an immunodominant Mycobacterium tuberculosis (M.tb) antigen included in novel vaccines against tuberculosis (TB) and in interferon-gamma (IFN-γ) release assays (IGRAs). Therefore, the availability of an ESAT-6-free IGRA is essential to determine M.tb infection status following vaccination with ESAT-6-containing vaccines. We aimed to qualify a recently developed ESAT-6-free IGRA and to assess its diagnostic performance in comparison to QuantiFERON-TB Gold In-tube (QFT). METHODS: Participants with different levels of M.tb exposure and TB disease were enrolled to determine the ESAT-6-free IGRA cutoff, test assay performance in independent cohorts compared to standard QFT, and perform a technical qualification of antigen-coated blood collection tubes. RESULTS: ESAT-6-free IGRA antigen recognition was evaluated in QFT-positive and QFT-negative South African adolescents. The ESAT-6-free IGRA cutoff was established at 0.61 IU/mL, based on receiver operating characteristic analysis in M.tb-unexposed controls and microbiologically confirmed pulmonary TB patients. In an independent cohort of healthy adolescents, levels of IFN-γ released in QFT and ESAT-6-free IGRA were highly correlated (P < .0001, r = 0.83) and yielded comparable positivity rates, 41.5% and 43.5%, respectively, with 91% concordance between the tests (kappa = 0.82; 95% confidence interval, 0.74-0.90; McNemar test P = .48). ESAT-6-free IGRA blood collection tubes had acceptable lot-to-lot variability, precision, and stability. CONCLUSIONS: The novel ESAT-6-free IGRA had diagnostic accuracy comparable to QFT and is suitable for use in clinical trials to assess efficacy of candidate TB vaccines to prevent established M.tb infection.

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.

A Suction Blister Protocol to Study Human T-cell Recall Responses In Vivo.

Cutaneous antigen-recall models allow for studies of human memory responses in vivo. When combined with skin suction blister (SB) induction, this model offers accessibility to rare populations of antigen-specific T-cells representative of the cellular memory response as well as the cytokine microenvironment in situ. This report describes the practical procedure of a cutaneous recall, an SB induction, and a harvest of antigen-specific T-cells. To exemplify the method, the tuberculin skin test is used for antigenic recall in individuals who, prior to this study, underwent a Bacillus Calmette-Guérin vaccination against an infection with Mycobacterium tuberculosis. Finally, examples of multiplex and flow cytometric analyses of SB specimens are provided, illustrating high fractions of antigen-specific polyfunctional CD4+ T-cells available by this sampling method compared with cells isolated from the blood. The method described here is safe and minimally invasive, provides a unique opportunity to study both innate and adaptive immune responses in vivo, and may be beneficial to a broad community of researchers working with cell-mediated immunity and human memory responses, in the context of vaccine development.

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

BACKGROUND: 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. METHODS: 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. RESULTS: 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. CONCLUSIONS: 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.

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

INTRODUCTION: Mycobacterium 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). RESULTS: We measured plasma levels of agonist and antagonist CXCL10 by Simoa digital ELISA, as well as DPP4 enzyme activity in the plasma of 20 patients with active TB infection, 10 patients with pneumonia infection, and a group of 10 healthy controls. We found higher levels of total and antagonist CXCL10 and reduced DPP4 enzyme activity in the plasma of TB patients compared to controls. We traced the source of CXCL10 secretion using immunohistochemical and confocal analysis to multinucleated giant cells in the TB lesions, and variable expression by macrophages. Interestingly, these cells were associated with DPP4-positive T cells. Moreover, the analysis of lymphocytes at the site of TB infection (bronchoalveolar lavage) showed a reduced frequency of CXCR3+ T cells. INTERPRETATION: Our data suggests that CXCL10 antagonism may be an important regulatory mechanism occurring at the site of TB pathology. CXCL10 can be inactivated shortly after secretion by membrane bound DPP4 (CD26), therefore, reducing its chemotactic potential. Given the importance of Th1 cell functions and IFN-γ-mediated effects in TB, our data suggest a possible unappreciated regulatory role of DPP4 in TB. PERSPECTIVES: DPP4 is the target for a class of enzyme inhibitors used in the treatment of diabetes, and the results from this study suggest that these drugs could be repurposed as an adjunct immunotherapy of patients with TB and MDR-TB.

Optimization and Interpretation of Serial QuantiFERON Testing to Measure Acquisition of Mycobacterium tuberculosis Infection.

RATIONALE: Conversion from a negative to positive QuantiFERON-TB test is indicative of Mycobacterium tuberculosis (Mtb) infection, which predisposes individuals to tuberculosis disease. Interpretation of serial tests is confounded by immunological and technical variability. OBJECTIVES: To improve the consistency of serial QuantiFERON-TB testing algorithms and provide a data-driven definition of conversion. METHODS: Sources of QuantiFERON-TB variability were assessed, and optimal procedures were identified. Distributions of IFN-γ response levels were analyzed in healthy adolescents, Mtb-unexposed control subjects, and patients with pulmonary tuberculosis. MEASUREMENTS AND MAIN RESULTS: Individuals with no known Mtb exposure had IFN-γ values less than 0.2 IU/ml. Among individuals with IFN-γ values less than 0.2 IU/ml, 0.2-0.34 IU/ml, 0.35-0.7 IU/ml, and greater than 0.7 IU/ml, tuberculin skin test positivity results were 15%, 53%, 66%, and 91% (P < 0.005), respectively. Together, these findings suggest that values less than 0.2 IU/ml were true negatives. In short-term serial testing, "uncertain" conversions, with at least one value within the uncertainty zone (0.2-0.7 IU/ml), were partly explained by technical assay variability. Individuals who had a change in QuantiFERON-TB IFN-γ values from less than 0.2 to greater than 0.7 IU/ml had 10-fold higher tuberculosis incidence rates than those who maintained values less than 0.2 IU/ml over 2 years (P = 0.0003). By contrast, "uncertain" converters were not at higher risk than nonconverters (P = 0.229). Eighty-seven percent of patients with active tuberculosis had IFN-γ values greater than 0.7 IU/ml, suggesting that these values are consistent with established Mtb infection. CONCLUSIONS: Implementation of optimized procedures and a more rigorous QuantiFERON-TB conversion definition (an increase from IFN-γ <0.2 to >0.7 IU/ml) would allow more definitive detection of recent Mtb infection and potentially improve identification of those more likely to develop disease.

Introducing the ESAT-6 free IGRA, a companion diagnostic for TB vaccines based on ESAT-6.

There is a need for an improved vaccine for tuberculosis. ESAT-6 is a cardinal vaccine antigen with unique properties and is included in several vaccine candidates in development. ESAT-6 is also the core antigen in the IFN-γ release assays (IGRA) used to diagnose latent infection, rendering IGRA tests unspecific after vaccination. This challenge has prompted the development of a companion diagnostic for ESAT-6 based vaccines, an ESAT-6 free IGRA. We screened a panel of seven potential new diagnostic antigens not recognized in BCG vaccinated individuals. Three highly recognized antigens EspC, EspF and Rv2348c were identified and combined with CFP10 in an ESAT-6 free antigen cocktail. The cocktail was prepared in a field-friendly format, lyophilized with heparin in ready-to-use vacutainer tubes. The diagnostic performance of the ESAT-6 free IGRA was determined in a cross-validation study. Compared IGRA, the ESAT-6 free IGRA induced a comparable magnitude of IFN-γ release, and the diagnostic performance was on par with Quantiferon (sensitivity 84% vs 79%; specificity 99% vs 97%). The comparable performance of the ESAT-6 free IGRA to IGRA suggests potential as companion diagnostic for ESAT-6 containing vaccines and as adjunct test for latent infection.

Thermostability of IFN-γ and IP-10 release assays for latent infection with Mycobacterium tuberculosis: A TBnet study.

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.

Adaptive Immunity against Streptococcus pyogenes in Adults Involves Increased IFN-γ and IgG3 Responses Compared with Children.

Each year, millions of people are infected with Streptococcus pyogenes, leading to an estimated 500,000 annual deaths worldwide. For unknown reasons, school-aged children have substantially higher infection rates than adults. The goal for this study was to provide, to our knowledge, the first detailed characterization of the human adaptive immune response against S. pyogenes in both children and adults. We report that all adults in our study, as well as most children, showed immunity against the two conserved group A streptococci (GAS) Ags, streptococcal C5a peptidase and immunogenic secreted protein. The response primarily consisted of three subsets of Th1 T cells, in which the TNF-α(+) and IL-2(+)TNF-α(+) subsets were most frequent. Humoral immunity was dominated by IgG1 and IgG3, whereas the Th2-associated IgG4 isotype was only detected at very low amounts. IgG3 levels correlated significantly with IFN-γ, but not with IL-5, IL-13, IL-17, or TNF-α. Interestingly, children showed a similar pattern of Ag-specific cytokine release, but displayed significantly lower levels of IgG3 and IFN-γ compared with adults. Thus, human immune responses against S. pyogenes consist of a robust Th1 cellular memory response in combination with IgG1/IgG3-dominated humoral immunity that increase with age. The significance of these data regarding both the increased GAS infection rate in children and the development of protective GAS vaccines is discussed.

Development of a one-step probe based molecular assay for rapid immunodiagnosis of infection with M. tuberculosis using dried blood spots.

BACKGROUND: Antigen specific release of IP-10 is the most promising alternative marker to IFN-γ for infection with M. tuberculosis. 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. AIM: To develop a robust IP-10 based molecular assay for the diagnosis of infection with M. tubercuolsis from whole blood and DBS. METHOD: 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 M. tuberculosis 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. RESULTS: 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.). CONCLUSION: We developed a rapid, robust and accurate molecular immunodiagnostic test for M. tuberculosis 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.

ELISA-based assay for IP-10 detection from filter paper samples.

IP-10 is a small pro-inflammatory chemokine secreted primarily from monocytes and fibroblasts. Alterations in IP-10 levels have been associated with inflammatory conditions including viral and bacterial infections, immune dysfunction, and tumor development. IP-10 is increasingly recognized as a biomarker that predicts severity of various diseases and can be used in the immunodiagnostics of Mycobacterium tuberculosis and cytomegalovirus infection. Here, we describe an ELISA-based method to detect IP-10 from dried blood and plasma spot samples.