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.