Quantification of Torque Teno Virus and Epstein-Barr Virus Is of Limited Value for Predicting the Net State of Immunosuppression After Lung Transplantation

Nordén, Rickard; Magnusson, Jesper; Lundin, Anna; Tang, Ka-Wei; Nilsson, Staffan; Lindh, Magnus; Andersson, Lars-Magnus; Riise, Gerdt C; Westin, Johan

To compare results regarding TTV levels between different transplantation centers, it is vital to reliably quantify the concentrations of TTV-DNA in a standardized manner. Here it was shown that the real-time PCR method was adequate as compared with the ddPCR method, which is considered a more reliable method for accurate quantification [32] . Nonetheless, real-time PCR is prone to interlaboratory differences, and utilization of ddPCR for quantification has been suggested to circumvent this problem, facilitating direct comparison of results between centers [33, 34] . Digital droplet PCR depends on partitioning of each master mix followed by end point PCR. Quantitation is determined using Poisson statistics to generate a result that is not dependent on a relation to a standard curve and should therefore exhibit lower variability between laboratories. However, the ddPCR assays still depend on amplification of a specific target sequence, and depending on the design of the primer and probe, the targeted sequence can vary. Recent work has shown that viral DNA in plasma samples is to a large extent free and not associated with viral particles and is also subjected to various degrees of degradation, influencing the results, depending on the amplicon length determined by the PCR-assay design [35] . Thus, standardization of the entire assay including primer and probe design, use of international standards, and assessment of the commutability of reference material is needed before a direct comparison of interlaboratory results can be made with absolute confidence, even with the use of ddPCR [36, 37] .