Torque Teno Virus (TTV) Plasma Load and Immune Reconstitution Post-Transplantation in Patients with Lymphoproliferative Disorders: A Systematic Review

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

The authors conducted the systematic review which included 21 studies evaluating Torque teno virus (TTV) plasma load as a biomarker of immune recovery after stem cell transplantation in lymphoproliferative disorder patients. It is interesting topic, however, I would like to provide the comments as below.

 

Major comments

1. Although the authors identify 21 studies, they did not perform the pooled analysis or standardized effect size calculations. They only summarize the descriptive findings narratively without structured statistical comparison across included studies. This substantially limits interpretability and fails to quantify association strength or heterogeneity.
2. Although the authors mentioned the methodological variation across studies (e.g., detection kits, PCR regions, cut-off thresholds, conditioning regimens), they did not perform the subgroup analysis. The authors should consider performing sub-group analysis.
3. The review lacks detail on whether extraction was double-checked or independently verified, how disagreements were resolved, whether standardized extraction tools were used, and specific criteria for evaluating correlation with immune markers.
4. The authors did not define the key clinical endpoints (immune reconstitution metrics, opportunistic infections, GVHD grading) consistently across studies.
5. The conclusion suggests that TTV plasma load “holds promise as a biomarker” for immune competence after HSCT. However, given that most reported results in the review are contradictory or inconclusive, this statement is not fully justified. The authors should provide the evidence for this conclusion.
6. The PRISMA flow diagram is included, but the search strategy lacks the details of complete search terms and timeframe, etc.
7. Study quality scoring (ROBVIS) is described, but results are reported roughly without detailed explanation of high-risk study sources or sensitivity exclusion.

Author Response

Answers to reviewer 1

The authors conducted the systematic review which included 21 studies evaluating Torque teno virus (TTV) plasma load as a biomarker of immune recovery after stem cell transplantation in lymphoproliferative disorder patients. It is interesting topic, however, I would like to provide the comments as below.

 

Major comments

1. Although the authors identify 21 studies, they did not perform the pooled analysis or standardized effect size calculations. They only summarize the descriptive findings narratively without structured statistical comparison across included studies. This substantially limits interpretability and fails to quantify association strength or heterogeneity.

 

Thank you for the suggestion. We have performed a metanalysis by using Meta-Mar version 4.0.2 (A next-generation meta-analysis platform) (https://doi.org/10.1186/s12888-020-2442-7). Here are the results:

The meta-analysis was performed on studies, which explored the same outcome (correlation between TTV viral load and lymphocytes number. It included 12 studies in order to assess correlation effect sizes using a Fisher’s z-transformed correlation approach. We used a random-effects model for the analysis, applying Restricted Maximum Likelihood (REML) to estimate tau and an inverse-variance method to combine effect sizes. The pooled effect size was 0.15 (95% CI: –0.05 to 0.33; Z = 1.51, p = 0.13), indicating that the association was not statistically significant. The prediction interval ranged from –0.52 to 0.70, underlining a high level of ambiguity about what future studies might find. The results evidenced high heterogeneity (I² = 83%, χ² = 43.44, df = 11, p < 0.001), with various results across studies. In fact, some studies showed strong positive associations (for example, Focosi et al., 2010: 0.62 [95% CI: 0.41 to 0.77]), while others detected negative effects (such as Pradier et al., 2020: –0.27 [95% CI: –0.42 to –0.10]). Because of the presence of this high variability and according to the PRISMA 2020 guidelines, we decided to use a narrative synthesis of results. This approach allowed us to summarize the findings clearly and transparently without relying on potentially misleading pooled estimates.

 

 

1. Although the authors mentioned the methodological variation across studies (e.g., detection kits, PCR regions, cut-off thresholds, conditioning regimens), they did not perform the subgroup analysis. The authors should consider performing sub-group analysis.

 

We performed a subgroup analysis between the different detection limits of TTV, between adults and children, and between in-house and commercial assays to explore potential moderators. PCR regions subgroup analysis was not performed because they were the same in all the included studies; furthermore, we did not perform subgroup analysis for conditioning regimens because they differed within the same population under study. Here are the results:

Age Subgroup analysis:

Adults (11 studies) showed a small, non-significant pooled effect (Fisher’s z = 0.10; 95% CI: –0.12 to 0.31) with high heterogeneity (I² = 83%), whereas children (2 studies) demonstrated a significant moderate effect (z = 0.41; 95% CI: 0.20 to 0.59) with minimal heterogeneity (I² = 1%). The test for subgroup differences was significant (p = 0.04), suggesting age may moderate the effect. However, the imbalance in subgroup sizes (11 vs. 2 studies) limits the robustness of this conclusion.

Detection Limit Subgroup analysis:

Stratification by LoD indicated variability in effect sizes, with LoD = 100 copies showing a moderate positive correlation (z = 0.36) and LoD = 10 copies showing a smaller, non-significant effect (z = 0.10). The LoD = 25 copies subgroup contained only one study with a negative association. Although the test for subgroup differences was significant (p < 0.001), the small number of studies per subgroup and high heterogeneity (I² up to 85%) reduce confidence in these findings.

Assay Type Subgroup analysis:

In-house assays (11 studies) showed a small, non-significant effect (z = 0.18) with high heterogeneity (I² = 84%), while the commercial assay subgroup included only one study, preventing meaningful interpretation. The test for subgroup differences was not significant (p = 0.11), and the extreme imbalance in subgroup sizes further limits conclusions.

 

Overall, these subgroup analyses suggest potential moderators (age and detection limit), but their interpretation is limited by high heterogeneity within subgroups, small sample sizes in certain subgroups, imbalance in subgroup composition and therefore we decided not to include them in the study.

 

1. The review lacks detail on whether extraction was double-checked or independently verified, how disagreements were resolved, whether standardized extraction tools were used, and specific criteria for evaluating correlation with immune markers.

 

Thank you for the suggestions; all these points have been included.

 

1. The authors did not define the key clinical endpoints (immune reconstitution metrics, opportunistic infections, GVHD grading) consistently across studies.

 

Thank you, we have added clinical endpoints for each study in Table 1.

 

1. The conclusion suggests that TTV plasma load “holds promise as a biomarker” for immune competence after HSCT. However, given that most reported results in the review are contradictory or inconclusive, this statement is not fully justified. The authors should provide the evidence for this conclusion.

 

Thank you for your comment. We have changed this sentence in conclusion section.

 

1. The PRISMA flow diagram is included, but the search strategy lacks the details of complete search terms and timeframe, etc.

 

Complete search term used were already included in search strategy and the timeframe with the following sentence: “No time window was applied to the search”. However, if further data is needed, we are available to provide it.

 

1. Study quality scoring (ROBVIS) is described, but results are reported roughly without detailed explanation of high-risk study sources or sensitivity exclusion.

 

Thank you for your comment. A more thorough description of high-risk study sources has been added. A random-effects model (DerSimonian–Laird) was used to conduct a leave-one-out sensitivity analysis. This method is based on repeating the analysis each time after the progressive exclusion of each individual study to demonstrate their influence on the pooled effect size and heterogeneity (I², τ²). The results indicate that the overall effect size, ranging from 0.099 to 0.189 (r = 0.10–0.19), was the same even when individual studies were removed from the analysis. Throughout each exclusion step, it was found that heterogeneity remained high (I² > 80%), and the confidence intervals often included zero. Only the exclusion of one study (Pradier et al., 2020) resulted in a significant borderline effect (p = 0.045), but this did not decrease heterogeneity in a significant way. These findings suggest that the observed overall effect is not driven by any single study and that the results are not robust, underlining the need for cautious interpretation.

 

 

 

Author Response File: Author Response.pdf

Reviewer 2 Report

Comments and Suggestions for Authors

This is an extensive and well written review regarding TTV serum loads in patients undergoing hematopoietic stem cell transplantation. In contrast to solid organ transplantation, this field is muddled. The authors did a good job of pointing out the discrepancies and similarities in published research.

My comments are minor:

“However, its role in hematopoietic stem cell transplantation (HSCT) recipients is less understood”. –  the word “role” implies an active function whereas TTV load just passively reflects the  immune status and/or number of available permissive cells. Please rewrite.

“Therefore, while TTV holds promise as a non-invasive biomarker of immunocompetence” – what are the invasive markers?

“supervised by the Professor in Infectious and Tropical Diseases and the Professor in Microbiology”. – provide names

“of 41 allo-HSCT recipients after 6 months of transplant and found that TTV was detected in 68%” - missing text

“TTV DNAemia”  - viral load is preferred

“TTV viral load and lymphocyte numbers might be established a correlation, but the results were contradictory” (Table 2) – provide r and p values in the table

Author Response

Answers to reviewer 2:

This is an extensive and well written review regarding TTV serum loads in patients undergoing hematopoietic stem cell transplantation. In contrast to solid organ transplantation, this field is muddled. The authors did a good job of pointing out the discrepancies and similarities in published research.

We would like to thank the reviewer for his/her positive feedback on our work

My comments are minor:

“However, its role in hematopoietic stem cell transplantation (HSCT) recipients is less understood”. –  the word “role” implies an active function whereas TTV load just passively reflects the  immune status and/or number of available permissive cells. Please rewrite.

Thank you for your comment; we have revised the sentence

“Therefore, while TTV holds promise as a non-invasive biomarker of immunocompetence” – what are the invasive markers?

Thank you we have deleted the term “non-invasive”

“supervised by the Professor in Infectious and Tropical Diseases and the Professor in Microbiology”. – provide names

Thank you, we have revised it

“of 41 allo-HSCT recipients after 6 months of transplant and found that TTV was detected in 68%” - missing text

Thank you, we have inserted the first sentence that was located above the table

“TTV DNAemia”  - viral load is preferred

Thank you, we have corrected it

“TTV viral load and lymphocyte numbers might be established a correlation, but the results were contradictory” (Table 2) – provide r and p values in the table

We have inserted r and p values in table 2 as suggested by the reviewer

Reviewer 3 Report

Comments and Suggestions for Authors

The manuscript addresses an important and timely topic concerning the potential value of Torque teno virus (TTV) plasma load as a biomarker of immune competence in patients undergoing hematopoietic stem cell transplantation. The authors present a comprehensive systematic review, drawing attention to the clinical relevance of the question, given the need for reliable tools to evaluate immune reconstitution and guide post-transplant management. The work is generally well-written, based on an adequate literature search, and follows the PRISMA guidelines. Tables and figures appropriately summarize the included studies and help visualize the reported kinetics of TTV DNAemia.

 Although heterogeneity between the included studies is acknowledged, the discussion does not sufficiently analyze how this heterogeneity influences conflicting results. In particular, the interpretation of discrepancies in the associations between TTV viral load and lymphocyte counts, opportunistic infections, and GVHD remains limited. Greater attention should be given to mechanistic and methodological explanations, such as the impact of different PCR targets, sampling timepoints, conditioning regimens, T-cell depletion strategies, and prophylaxis protocols. Furthermore, the review does not address publication bias, a common issue in systematic reviews.

The clinical implications of the findings also require further development. While the authors state that TTV “holds promise” as a biomarker, the conclusion remains general. It does not help readers understand how TTV monitoring could be implemented in routine HSCT follow-up or decision-making. A more detailed synthesis of when TTV appears to reflect immune recovery versus immunosuppression would strengthen the translational value of the manuscript. Finally, minor issues should be corrected, including typographical errors, inconsistencies in abbreviations, and readability of figures.

With revisions that expand the critical interpretation of heterogeneous results, clarify clinical applicability, and address methodological limitations, the manuscript has the potential to make a valuable contribution to the field. At the current stage, a significant revision is recommended.

Author Response

Answers to reviewer 3

 

The manuscript addresses an important and timely topic concerning the potential value of Torque teno virus (TTV) plasma load as a biomarker of immune competence in patients undergoing hematopoietic stem cell transplantation. The authors present a comprehensive systematic review, drawing attention to the clinical relevance of the question, given the need for reliable tools to evaluate immune reconstitution and guide post-transplant management. The work is generally well-written, based on an adequate literature search, and follows the PRISMA guidelines. Tables and figures appropriately summarize the included studies and help visualize the reported kinetics of TTV DNAemia.

Thank you very much for your positive feedback on this paper

 Although heterogeneity between the included studies is acknowledged, the discussion does not sufficiently analyze how this heterogeneity influences conflicting results. In particular, the interpretation of discrepancies in the associations between TTV viral load and lymphocyte counts, opportunistic infections, and GVHD remains limited. Greater attention should be given to mechanistic and methodological explanations, such as the impact of different PCR targets, sampling timepoints, conditioning regimens, T-cell depletion strategies, and prophylaxis protocols. Furthermore, the review does not address publication bias, a common issue in systematic reviews.

Thank you for your suggestion. We have expanded Discussion section with a more detailed description of study limitation due to the high heterogeneity of the included studies and publication bias has been addressed

The clinical implications of the findings also require further development. While the authors state that TTV “holds promise” as a biomarker, the conclusion remains general. It does not help readers understand how TTV monitoring could be implemented in routine HSCT follow-up or decision-making. A more detailed synthesis of when TTV appears to reflect immune recovery versus immunosuppression would strengthen the translational value of the manuscript. Finally, minor issues should be corrected, including typographical errors, inconsistencies in abbreviations, and readability of figures.

The conclusion section has been rewritten according to the reviewer’s suggestion. Typo errors and inconsistencies in abbreviations have been corrected. Figures have been provided to Journal as zip file with high resolution.

With revisions that expand the critical interpretation of heterogeneous results, clarify clinical applicability, and address methodological limitations, the manuscript has the potential to make a valuable contribution to the field. At the current stage, a significant revision is recommended.

 

Round 2

Reviewer 1 Report

Comments and Suggestions for Authors

The authors responded to my previous concerns and revised the manuscript properly. No new comments are added. However, I would like to suggest to start the conclusion with the following sentence in exchange of current first sentence of conclusion if agreeable. 

"TTV plasma viral load may reflect the degree of immunosuppression and immune reconstitution after HSCT, and therefore could serve as a candidate biomarker in patients with immunoproliferative disorders, although current evidence remains heterogenous."

Author Response

We thank the reviewer  and according to his/her suggestion, we have changed the first sentence of our conclusions

Reviewer 3 Report

Comments and Suggestions for Authors

Thank you to the authors for thoroughly addressing all comments. The revisions have substantially improved the manuscript.

Author Response

We would like to thank the reviewer for helping us to improve our manuscript