A Molecular Assay for Sensitive Detection of Pathogen-Specific T-Cells

Ragon Institute of MGH, MIT and Harvard, Harvard Medical School, Boston, Massachusetts, United States of America.
PLoS ONE (Impact Factor: 3.23). 08/2011; 6(8):e20606. DOI: 10.1371/journal.pone.0020606
Source: PubMed


Here we describe the development and validation of a highly sensitive assay of antigen-specific IFN-γ production using real time quantitative PCR (qPCR) for two reporters--monokine-induced by IFN-γ (MIG) and the IFN-γ inducible protein-10 (IP10). We developed and validated the assay and applied it to the detection of CMV, HIV and Mycobacterium tuberculosis (MTB) specific responses, in a cohort of HIV co-infected patients. We compared the sensitivity of this assay to that of the ex vivo RD1 (ESAT-6 and CFP-10)-specific IFN-γ Elispot assay. We observed a clear quantitative correlation between the two assays (P<0.001). Our assay proved to be a sensitive assay for the detection of MTB-specific T cells, could be performed on whole blood samples of fingerprick (50 uL) volumes, and was not affected by HIV-mediated immunosuppression. This assay platform is potentially of utility in diagnosis of infection in this and other clinical settings.

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    • "More recently, BTB assays measuring changes in the abundance of IFNG messenger ribonucleic acid (mRNA) as a signal of immune activation have been developed for various species (Harrington et al., 2000; Sawyer et al., 2007; Bibova et al., 2012; Parsons et al., 2012). Moreover, alternative markers of immune activation have been investigated and include interferon-gamma-induced protein 10 (IP-10/CXCL10) (Farber, 1997; Kasprowicz et al., 2011) and monokine induced by gamma interferon (MIG/ CXCL9) (Kasprowicz et al., 2011). However, the use of such mRNA targets requires normalization to reference gene(s) that are expressed constitutively (Radoni c et al., 2005). "
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    ABSTRACT: Mycobacterium bovis infection, the cause of bovine tuberculosis (BTB), is endemic in wildlife in the Kruger National Park (KNP), South Africa. In lions, a high infection prevalence and BTB mortalities have been documented in the KNP; however, the ecological consequences of this disease are currently unknown. Sensitive assays for the detection of this infection in this species are therefore required. Blood from M. bovis-exposed, M. bovis-unexposed, M. tuberculosis-exposed and M. bovis-infected lions was incubated in QuantiFERON®-TB Gold (QFT) tubes containing either saline or ESAT-6/CFP-10 peptides. Using qPCR, selected reference genes were evaluated for expression stability in these samples and selected target genes were evaluated as markers of antigen-dependent immune activation. The abundance of monokine induced by gamma interferon (MIG/CXCL9) mRNA, measured in relation to that of YWHAZ, was used as a marker of ESAT-6/CFP-10 sensitization. The gene expression assay results were compared between lion groups, and lenient and stringent diagnostic cut-off values were calculated. This CXCL9 gene expression assay combines a highly specific stimulation platform with a sensitive diagnostic marker that allows for discrimination between M. bovis-infected and M. bovis-uninfected lions.
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    • "Amplification of nucleic acids is a powerful tool for sensitive detection of transcriptional changes in low sample volumes [18]. Also, it is a well proven diagnostic approach and has shown promise for the diagnosis of M. tuberculosis specific immune responses using mRNA encoding IFN-γ, IL-2 and other cytokines, but in particular IP-10 [15], [19]. Kinetic studies of IFN-γ gene expression suggests that the shorter incubation is vastly superior for diagnostic assays [20], but no detailed investigations have been attempted with IP-10 possibly having led to an underestimation of the potential of the technology. "
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    ABSTRACT: Background: Antigen specific release of IP-10 is the most promising alternative marker to IFN-gamma for infection with M. tuberculosis. Compared to Interferon-c 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-gamma 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-gamma 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-gamma 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.
    Full-text · Article · Sep 2014 · PLoS ONE
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    • "We used a highly sensitive assay of ESAT-6 and CFP-10-specific IFN-γ production using real time quantitative PCR (qPCR) for two reporters - monokine-induced by IFN-γ (MIG) and the IFN-γ inducible protein-10 (IP10) (RD1-qPCR). This assay was performed as described in Kasprowicz et al [31]. In brief, PBMCs (106 cells/condition) were stimulated with 10 ng/ml recombinant IFN-γ (positive control), 25 µl of R10 media (null), or ESAT-6 and CFP-10 peptide pools (final peptide concentration of 8 µg/ml) at 37° and 5% CO2 for 16 hours. "
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    ABSTRACT: Monitoring of latent Mycobacterium tuberculosis infection may prevent disease. We tested an ESAT-6 and CFP-10-specific IFN-γ Elispot assay (RD1-Elispot) on 163 HIV-infected individuals living in a TB-endemic setting. An RD1-Elispot was performed every 3 months for a period of 3-21 months. 62% of RD1-Elispot negative individuals were positive by cultured Elispot. Fluctuations in T cell response were observed with rates of change ranging from -150 to +153 spot-forming cells (SFC)/200,000 PBMC in a 3-month period. To validate these responses we used an RD1-specific real time quantitative PCR assay for monokine-induced by IFN-γ (MIG) and IFN-γ inducible protein-10 (IP10) (MIG: r = 0.6527, p = 0.0114; IP-10: r = 0.6967, p = 0.0056; IP-10+MIG: r = 0.7055, p = 0.0048). During follow-up 30 individuals were placed on ARVs and 4 progressed to active TB. Fluctuations in SFC did not correlate with CD4 count, viral load, treatment initiation, or progression to active TB. The RD1-Elispot appears to have limited value in this setting.
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