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

Medical Sciences, vol. 14, pp. 467

Abstract

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.

Authors 10

1. Paul Studenic

   Medical University of Vienna

   Affiliation as printed

   Division of Rheumatology, Department of Internal Medicine III, Medical University of Vienna, 1090 Vienna, Austria

2. Chaimae Akile Aachen LUCID Dept of Medical Microbiology & Infection Prevention

   Leiden University Medical Center

   Affiliation as printed

   LUCID Department of Medical Microbiology & Infection Prevention, Leiden University Medical Center, 2300 RC Leiden, The Netherlands

3. C.A. Hana

   Medical University of Vienna

   Affiliation as printed

   Division of Rheumatology, Department of Internal Medicine III, Medical University of Vienna, 1090 Vienna, Austria

4. Daniela Sieghart

   Medical University of Vienna

   Affiliation as printed

   Division of Rheumatology, Department of Internal Medicine III, Medical University of Vienna, 1090 Vienna, Austria

5. Thi Lan Vi Tran

   Medical University of Vienna

   Affiliation as printed

   Division of Rheumatology, Department of Internal Medicine III, Medical University of Vienna, 1090 Vienna, Austria

6. Josef Baliko

   Medical University of Vienna

   Affiliation as printed

   Division of Rheumatology, Department of Internal Medicine III, Medical University of Vienna, 1090 Vienna, Austria

7. François Bonnay

   Medical University of Vienna

   Affiliation as printed

   Division of Rheumatology, Department of Internal Medicine III, Medical University of Vienna, 1090 Vienna, Austria

8. Souzi Makri
   Affiliation as printed

   Cyprus League of People with Rheumatism, Nicosia 1056, Cyprus

9. Daniel Aletaha

   Medical University of Vienna

   Affiliation as printed

   Division of Rheumatology, Department of Internal Medicine III, Medical University of Vienna, 1090 Vienna, Austria

10. Mariet C.W. Feltkamp Aachen LUCID Dept of Medical Microbiology & Infection Prevention

    Leiden University Medical Center

    Affiliation as printed

    LUCID Department of Medical Microbiology & Infection Prevention, Leiden University Medical Center, 2300 RC Leiden, The Netherlands