At the Cutting Edge of Transplantation 2025 conference, Dr. Steve Kleiboeker, DVM, PhD, spoke about the evolving role of dual-biomarker monitoring in supporting precision immune surveillance and post-transplant care. Dr. Kleiboeker presented data about the use of donor-derived cell-free DNA and TTV viral load data in immunosuppression decision-making for transplant patients.

Dr. Kleiboeker is the Vice President of Research and Development at Eurofins Transplant Genomics. He is board certified as a high-complexity laboratory director and clinical consultant by the American Board of Bioanalysis, and he received his degrees from the University of Missouri. His research focuses on topics such as immunology and transplant monitoring.

The Clinical Dilemma of Balancing Immunosuppression Post-Transplant

Immunosuppression in post-transplant patients, particularly in the first few years, is a careful balance. Clinical teams must constantly monitor and evolve individual immunosuppression treatments to ensure patients are immunosuppressed enough to support graft health without putting them at too much risk of infection and other complications.

Historically, the trend has been toward over immunosuppression — not purposefully, but via a “better safe than sorry” approach to prevent rejection — that is backed off only when infection is noted. Clinicians have long relied on indirect and often insufficient tools for such decision-making, increasing risks associated with both acute rejection and serious infection. Long-term over immunosuppression also comes with risks such as malignancies.

Patient risks and needs also change throughout the years following a transplant. In the first few weeks, donor derived viruses tend to be the biggest issues. Throughout the first year, latent infections, relapses and opportunistic infections become more of a concern, and after the first year, community acquired infections can still create challenges for immunosuppressive balance.

Molecular biomarkers like dd-cfDNA and TTV may help clinical teams bridge the gaps in post-transplant care by better understanding immunosuppressive states earlier.

Understanding Torque Teno Virus (TTV) as a Marker of Immune Suppression Intensity

Research teams working on dd-cfDNA biomarkers discovered that the Torque Teno Virus (TTV) levels may be of use in making proactive immune suppression decisions.

TTV is a non-pathogenic virus with high prevalence in transplant recipients. In fact, research data indicates that TTV is present in more than 90% of normal, healthy adults. Depending on when post-transplant samples are taken, it’s present in around 99% of organ transplant recipients.

No human pathogenicity of TTV has ever been established, but it’s notable that TTV doesn’t respond to conventional antiviral drug therapy. However, it does respond to immunosuppressive treatment, which means TTV viral load can be used to understand how immunosuppressed a patient is.

Given this relationship, researchers hypothesized that TTV loads might be used as a biomarker to understand individual patient risk of infection and rejection. One meta-analysis looked at 23 studies consisting of kidney, liver and lung recipients to understand the relationship between TTV loads and risks. The authors of the study found an association between TTV loads and patient outcomes, including both infection and rejection.

Another study looked at how risk changes with TTV loads. For every 10-fold increase in TTV viral load:

-The odds of rejection decreased by 22%

-The odds of infection increased by 11%

The takeaway here is that TTV loads increase as immunosuppression increases. High levels of immunosuppression reduce the patient’s chances of graft rejection while increasing chances of infection. The insight into this relationship may help clinical teams walk the fine — and very individual — line of immunosuppression management for each patient.

Donor-Derived Cell-Free DNA: A Validated Biomarker of Graft Injury

Donor-derived cell-free DNA has emerged as a sensitive and specific marker for understanding graft injury, offering a non-invasive alternative to traditional diagnostics like biopsies. When integrated into post-transplant monitoring, dd-cfDNA allows teams to understand allograft health without the expense and inconvenience of routine biopsies.

Teams can collect cell-free DNA from plasma and assess it with next generation sequencing methods, identifying donor and recipient DNA so a percentage of donor DNA can be calculated. Donor DNA figures less than 0.7% are considered normal; anything higher is correlated with a high probability of graft injury.

While dd-cfDNA has been demonstrated to be a strong marker for graft injury, it doesn’t provide information about immune suppression levels. However, combining TTV loads with dd-cfDNA data can help clinicians get a more comprehensive picture of post-transplant patient status.

Introducing TRAC ID: A Combined Assay for Comprehensive Transplant Surveillance

TRAC ID combines dd-cfDNA and viral pathogen plus TTV levels in a single assay for a more holistic picture of allograft health through whole genome sequencing:

-Plasma is collected.

-Nucleic acid is isolated and optimized for dd-cfDNA.

-Sequencing library preparation occurs.

-Whole genome sequencing occurs.

Dual analyses are completed: human sequences to calculate the donor derived fraction of cfDNA and microbial sequences to identify viral infections (and TTV load).

Whole genome sequencing allows simultaneous quantification of dd-cfDNA and early detection of viral infection in a single test. The concordance is high for various virus detection:

-BKV – 96.1%

-CMV – 97.2%

-EBV – 97.8%

-TTV – 98.2%

Overall, that’s a 97.7% concordance of diagnostic performance of mNGS compared to qPCR detection.

Clinical Interpretation Framework for TRAC ID Results

The dual results provide a framework for decision-making in post-transplant care.

If dd-cfDNA < 0.7%

The team can look at TTV loads:

-If TTV is greater than or equal to 6.00 Log₁₀ genomic copies/mL there is potential over-immunosuppression. The evidence is stronger if TTV loads are greater than or equal to 10⁷ c/mL.

-If TTV is greater than or equal to 6.00 Log₁₀ genomic copies/mL and other viral pathogens are positive, there is potential over-immunosuppression. Teams should confirm pathogens with qPCR and treat relevant viral infections.

-If TTV is less than 6.00 Log₁₀ genomic copies/mL and viral pathogens are negative, teams should consider the possibility of under-immunosuppression. The risk is greater if TTV is less than 4.00 Log1₀ genomic copies/mL; in this case, teams should examine any trends in the dd-cfDNA results to assess the risks of allograft injury.

If dd-cfDNA > or = 0.7%

The team can also look at TTV loads:

-If TTV is greater than or equal to 6.00 Log₁₀ genomic copies/mL, immunosuppression is uncertain. Further diagnostics may be warranted, including biopsy.

-If TTV is greater than or equal to 6.00 Log₁₀ genomic copies/mL and other viral pathogens are positive, immunosuppression is uncertain. Teams should confirm pathogens with qPCR and treat relevant viral infections. They may also want to consider a biopsy to assess allograft health.

-If TTV is less than 6.00 Log₁₀ genomic copies/mL and viral pathogens are negative, under-immunosuppression may be at play, and there is a presumed alloimmune-mediated injury.

Future Applications and Ongoing Research in TTV-Guided Immunosuppression

Both the Maggi and R-GENE qPCR assays have looked at standardizing TTV measurements to better support clinical decision-making based on these findings. Recent studies and literature reviews using either assay have found high correlations between TTV loads and immunosuppression risks, creating solid foundations for future applications of these approaches.

As of early 2025, an ongoing clinical study is using TTV to guide tacrolimus (TAC) dosing. The study involves an active/intervention group and a control group. For the active group, TAC dosing is managed in part based on TTV loads:

-If TTV is above 6.2 log₁₀ genomic c/mL, TAC is stepped down by 2 ng/mL.

-If TTV is in the optimal range, TAC is unchanged.

-If TTV is below 4.6 log₁₀ genomic c/mL, TAC is stepped up by 2 ng/mL if adherent.

The control group is managed without TTV guidance using existing standards of care.

The results of this trial and how well TTV guidance works in managing tacrolimus dosing should be published in 2025 and will add to the evolving and growing landscape of research related to dual biomarkers.

Precision Monitoring Through Dual Biomarker Integration

The history of transplant medicine has been one of chasing immunosuppression to ensure an optimal environment for allograft and overall patient health. The integration of dd-cfDNA and TTV load monitoring marks a turning point in transplant care and may allow clinical teams to get more ahead of immunosuppression than they’ve ever been.

TRAC ID combines dd-cfDNA and TTV data to create one convenient, non-invasive test. This access to data about rejection and infection risk can reduce unnecessary biopsies, help teams optimize immunosuppression and, ultimately, improve patient outcomes.

The future of molecular diagnostics in transplantation is data-centric, bridging gaps that have existed in post-transplant care for decades. Transplant Genomics aims to improve transplant outcomes for recipients through non-invasive treatment options.