Table 1.
Baseline.
Figure 1.
Overview of the RT-qPCR based method for immunodiagnosis of M. tuberculosis infection.
A. Blood collection and incubation at 37C. B. Extraction of RNA. C. One-step probe based RT-qPCR and calculation of IP-10 mRNA upregulation using ΔΔCt. D. Optional RNA storage on dried blood spots for easy sample transportation.
Figure 2.
Comparison of mRNA extraction from whole blood (WB) and dried blood spots (DBS).
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 p = 0.003.
Figure 3.
IP-10 and IFN-γ expression profiles.
A: Whole blood from two TB patients and two persons with known QFT-TB positivity was incubated in QFT-TB tubes for up to 48 hours at 37°C. Every second hour for 12 hours and at 18, 24 and 48 hours post stimulation, dried blood spots were prepared for later mRNA extraction and plasma was isolated for protein analysis except for 2, 4 and 6 hours post stimulation. IP-10 and IFN-γ gene expression specified as mRNA fold change was determined using our RT-qPCR assay and IP-10 protein levels were determined using an in-house IP-10 ELISA assay. The black bars represent median IP-10 mRNA upregulation in fold change, the white bars represent the IFN-γ mRNA upregulation and the grey line represents the median IP-10 protein expression. IFN-γ protein expression was not measured in this experiment. B: Whole blood from 12 TB patients and 8 LTBI persons was incubated in QFT-TB tubes for up to 20 hours at 37°C. Dried blood spots were made after 8 hours incubation and after 20 hours incubation. mRNA was subsequently extracted and IP-10 mRNA fold change was determined using our RT-qPCR assay. The difference was analysed using a Wilcoxon matched pairs test p = 0.0003.
Figure 4.
IP-10 mRNA expression and IP-10 and IFN-γ protein release.
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.
Figure 5.
Comparison of the diagnostic potential of IP-10 RT-qPCR, IP-10 protein and IFN-γ protein.
ROC curve comparison of antigen-specific IP-10 mRNA expression and IP-10 and IFN-γ protein release. Cases comprised 30 TB patients and 13 LTBI persons and controls were 96 persons with no known exposure to M. tuberculosis. IP-10 gene expression was analysed from DBS and IP-10 and IFN-γ protein levels were analysed in plasma samples. AUCs was comparable at 0.88, 0.91 and 0.97 for IP-10 mRNA, IP-10 protein and QFT-TB respectively (p<0.0001). Cut offs were selected at the point rendering high sensitivity without compromised specificity.