Reproducibility and Potential for Input Reduction for Torque Teno Virus DNA Quantification in Rheumatoid Arthritis

Background/Objectives: Torque teno virus (TTV) is small non-pathogenic virus, currently under investigation as a potential biomarker to monitor immunocompetence in patients with rheumatoid arthritis (RA) receiving various immunosuppressive therapies. To allow for large-scale assessment of existing pan-European RA cohorts, minimal specimen (serum) input for TTV DNA detection through quantitative PCR (qPCR) needs to be identified in relation to the type and quantity of immunosuppression. Methods: TTV qPCR analysis was performed by measuring 1:1, 1:2, 1:4 and 1:8 dilutions of 278 human serum samples derived from patients with RA under different therapies. The smallest detectable difference (SSD) as well as the intra-class correlation (ICC) were assessed. The reproducibility of results was determined by measuring an additional 19 sample duplicates. Results: RA patient sera showed a mean TTV viral load equivalent of 2.9 log10 genome copies/mL (SD ± 1.47). A total of 32 samples (11.6%) were negative for TTV; 54 (19.6%) were below 2.4 log10, the manufacturer's lower limit of quantification; 44 (16%) were between 2.4 and 3.0 log10; and 145 (52.7%) showed a high viral load above 3.0 log10. Dilution and replication experiments showed a high stability and reproducibility of TTV measurements (overall ICC: 0.95 (95%CI: 0.951 to 0.966)). TTV differed between undiluted and 4-fold- as well as 8-fold-diluted samples (1 vs. 2: p = 0.993; 1 vs. 4: p < 0.001; 1 vs. 8: p < 0.001), aligned with decreasing ICC and less sensitivity. RA disease activity did not influence reproducibility. Conclusions: Quantitative TTV DNA detection in serum samples from RA patients yields highly reproducible results, including 2-fold-diluted serum samples.