Comparative evaluation of molecular methods for the quantitative measure of torquetenovirus (TTV) viremia, the new surrogate marker of immune competence

BACKGROUND: Torquetenovirus (TTV) viremia is emerging as a promising tool to assess functional immune competence, to predict post-transplant immune-related complications, and eventually to customize immunosuppression. METHODS: In this study, 327 blood samples were tested using two real-time PCR (rtPCR) assays both targeted to the untranslated region of TTV genome. The first assay was an in-house rtPCR developed by our group, the second one was the recently marketed TTV R-GENE® assay. RESULTS: In the validation study, the TTV R-GENE® showed good performances in precision and reproducibility, and a sensitivity as low as 12 TTV DNA copies/ml, like previously reported for the in-house rtPCR. Bland-Altman analysis showed that the mean difference between the two methods was -0.3 Log copies/ml. In the comparison study, 69% and 72% of samples were detected positive by rtPCR and TTV R-GENE®, respectively (94% concordance, κ = 0.88). Performances did not differ between the two rtPCRs by type of TTV group examined. When a newly-developed in-house digital droplet PCR was applied for TTV quantification and used as alternative method of comparison on 94 samples, the results strongly correlated with those obtained by the two rtPCR methods (99% concordance). CONCLUSION: In summary, all the molecular methods assayed are highly sensitive and accurate in quantitation of TTV DNA in blood samples.