key: cord-1006906-0gao6r3u
authors: Jr, Jose Otto Reusing; Yoo, Jongwon; Desai, Amishi; Brossart, Katya; McCormick, Sarah; Malashevich, Allyson Koyen; Bloom, Michelle S.; Fehringer, Gordon; White, Roseann; Billings, Paul R.; Tabriziani, Hossein; Demko, Zachary P.; Gauthier, Philippe; Akkina, Sanjeev K.; David-Neto, Elias
title: Association between total cell free DNA and SARS-CoV-2 in Kidney Transplant Patients: A Preliminary Study
date: 2022-03-15
journal: Transplant Proc
DOI: 10.1016/j.transproceed.2022.02.027
sha: c1d9c8249b21fd5320ad15546e658262817cd26a
doc_id: 1006906
cord_uid: 0gao6r3u

Background : Kidney transplant (KT) recipients are at high-risk for developing severe COVID-19. Lowering immunosuppression levels in KT recipients with COVID-19 encourages native immune responses but can raise the risk of rejection. Donor-derived cell-free DNA (dd-cfDNA), reported as a fraction of total cfDNA, is a proven biomarker for KT rejection. Total cfDNA levels are elevated in COVID-19 patients, which may depress dd-cfDNA fractions, potentially leading to missed rejections. Methods : A retrospective analysis of 29 KT recipients hospitalized with COVID-19 between April and November 2020 examined total and dd-cfDNA levels. Blood samples were collected following onset of COVID-19, with follow-up samples collected from a subset of patients, when infection had likely subsided. Results : Following COVID-19 diagnosis, the median total cfDNA level was elevated [7.9 multiples of median (MoM)]. A significant decrease in total cfDNA levels was observed between the first and second time-points (6.2 MoM, 1.0 MoM; p=0.0009). A significant positive association was identified between total cfDNA levels and COVID-19 severity (p=0.02; R2=0.19). Two patients with biopsy-proven acute cellular rejection had dd-cfDNA fractions below the 1% cut-off for rejection (0.20% and 0.78%), with elevated total cfDNA levels of 7.9 MoM and 41.8 MoM, respectively. Conclusion : In this preliminary study,total cfDNA levels were elevated in KT patients with COVID-19, subsiding after resolution of infection. High total cfDNA levels may confound dd-cfDNA results, leading to failure to identify rejection. Considering total cfDNA levels is important in interpretation of dd-cfDNA tests for assessment of rejection in KT patients with COVID-19 or other infection.

immune responses but can raise the risk of rejection. Donor-derived cell-free DNA (dd-cfDNA), reported as a fraction of total cfDNA, is a proven biomarker for KT rejection. Total cfDNA levels are elevated in COVID-19 patients, which may depress dd-cfDNA fractions, potentially leading to missed rejections.

April and November 2020 examined total and dd-cfDNA levels. Blood samples were collected following onset of COVID-19, with follow-up samples collected from a subset of patients, when infection had likely subsided.

Results: Following COVID-19 diagnosis, the median total cfDNA level was elevated [7.9 multiples of median (MoM)]. A significant decrease in total cfDNA levels was observed between the first and second time-points (6.2 MoM, 1.0 MoM; p=0.0009). A significant positive association was identified between total cfDNA levels and COVID-19 severity (p=0.02;

Kidney transplantation (KT) is considered the ideal treatment for patients with end-stage kidney disease, and can lead to substantial improvements in patient survival and quality of life. 1 Unfortunately, recipient-mediated allograft damage and failure are common, with most patients experiencing acute kidney injury (AKI) within two years of transplant, 2, 3 an annual allograft failure of ~3-5% beyond the first year, and a 10-year transplant attrition rate of ~55%. 4 Acute rejection is a predominant cause of kidney allograft failure, most commonly, due alloimmunemediated injury. 5, 6 Chronic immunosuppression is the main treatment strategy to help prevent allograft rejection, functionally counteracting the inflammatory and immunological responses mounted by the recipients. 7, 8 The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) that causes COVID-19, has brought significant challenges to the treatment and management of KT recipients. 9 Chronic immunosuppression may place transplant recipients at a heightened risk of developing more severe courses of COVID-19, 10 and virus-positive transplant recipients have poorer survival outcomes compared to healthy individuals. 11 Consequently, physicians typically lower immunosuppression in COVID-19 patients, which increases the risk of allograft rejection.

Additionally, comorbidities common in KT patients, such as diabetes, obesity, hypertension and cardiac disease, are also major risk factors for severe COVID-19 symptoms and poor outcomes. 11, 12 Compounding this, SARS-CoV-2, itself, reportedly causes kidney damage, including acute kidney injury/failure (AKI/AKF) due to virally induced multiorgan failure, reduced renal perfusion, and cytokine storm. 13, 14 . Kidney damage is found to increase with COVID-19 severity, and AKI/AKF is associated with poor prognosis. 15 In severe SARS-CoV-2 infection, immunosuppressive treatments may help mitigate the cytokine storm and consequential kidney damage during the inflammatory stage of the disease. 16, 17 Stratification of virally infected KT patients into high-and low-risk groups for AKI/AKF could aid in physician decision making regarding patient management and treatment, including the use, dose, and timing of immunosuppressants.

Circulating, donor-derived cell-free DNA (dd-cfDNA) is now a proven biomarker that can detect AKI/AKF reliably, and with high sensitivity. 18-20 Due to its circulation in the blood, dd-cfDNA can be measured non-invasively, and serially through a simple blood test, and is reportedly more accurate than measurement of serum creatinine. 21 Current commercial tests generally report dd-cfDNA as a fraction of total cfDNA, with >1% considered high risk for rejection. Elevated total cfDNA levels associated with COVID-19 and other infections have been hypothesized to depress dd-cfDNA fractions, potentially confounding interpretation of dd-cfDNA test results.

Here, we present results of dd-cfDNA testing in a series of hospitalized KT patients with COVID-19, examining the effect of COVID-19 infection on the total cfDNA levels, and the effect on dd-cfDNA fractions. Patients had an initial dd-cfDNA test performed shortly after infection, with a subset of patients having a follow-up test after COVID-19 clearance. Demographic, clinical and outcome data was collected for each patient and de-identified prior to analysis.

Individuals who were under 18 years of age, had more than one organ transplanted, were pregnant, or had a blood transfusion within two weeks of enrollment were excluded. The inclusion of samples in the primary analysis were based on availability of adequate plasma to run the dd-cfDNA assay, and availability of clinical follow-up.

Blood samples were processed and analyzed at Natera, Inc.'s CLIA-Certified and College of American Pathologists (CAP) accredited laboratory (San Carlos, California, USA). Laboratory testing was performed as previously described, using massively multiplexed-PCR (mmPCR), targeting over 13,000 single nucleotide polymorphisms. 22 Next generation sequencing, with an average of 10-11 million reads per sample, was performed on the Illumina HiSeq 2500 on rapid run. For all patients, both the total cfDNA level (reported in multiples of the median; MoM, and copies/mL) and the donor-derived cfDNA (dd-cfDNA) fraction (analyzed as the percentage of total cfDNA) were measured.

Biopsy samples were analyzed and graded according to the standard practice at each site by their respective pathologists using Banff 2017 classification. 23 AKI was defined as increases in serum creatinine levels (either >2.0x baseline or ≥10% baseline), urine output <0.5 ml/kg/h for >12 hours, or "de novo" appearance of proteinuria (≥0.3g/day). 24 

Differences in either total cfDNA levels or dd-cfDNA fractions were assessed between tests performed closest to the onset of COVID-19 symptoms and the follow-up time-point (a proxy for baseline levels) using paired t-tests. To determine if elevated cfDNA levels are attributed to either AKI or renal replacement therapy (RRT), paired t-tests were performed across time periods and Wilcoxon rank sum tests were performed for intra-time period comparisons. All ttest and Wilcoxon tests were two-tailed and cut off for statistical significance was < 0.05.

Stepwise regressions were used to investigate associations of cfDNA measures (both total and dd-cfDNA) with COVID-19 severity scores (linear) and mortality (logistic regression). In addition to total cfDNA level and dd-cfDNA fraction, potential predictor variables included in these models were age, donor type and AKI. Donor type and AKI were entered as binary variables.

Total cfDNA, dd-cfDNA and age were entered into models as continuous variables. Variables were entered in models at P≤0.10 and retained at P<0.15. Body Mass Index (BMI) and baseline creatinine were considered for inclusion in analyses but were inestimable in all models. Clinical significance was based on the assessment of the observed effect and the magnitude of the appropriate p-value.

The study included 29 KT patients who were diagnosed with COVID-19 between April and November 2020. Six of these patients were admitted to the hospital for other reasons (two for KT surgery) and contracted COVID-19 nosocomially ( Table 1 ). The median age among the cohort was 58 years (range: 21 -73 years), with a median time from transplant to onset of COVID-19 of 781 days (range 6 -6694). The cohort was predominantly male (62.1%), white (41.4%), with allografts received from deceased donors (79.3%) ( Table 2 ). The median time from onset of COVID-19 symptoms to hospital admission was 6 days, with a range of 17 days before to 13 days after hospital admission.

AKI was diagnosed in 69.0% (20/29) of patients. RRT was required for 34.4% (10/29) of patients; three of these individuals were initiated on RRT prior to COVID-19 diagnosis due to delayed graft function (DGF) following KT. Biopsies were performed on five individuals with AKI, which confirmed acute cellular rejection in two of these patients and had inconclusive borderline findings in one individual who was nonetheless treated for possible acute rejection. Mechanical ventilation was initiated for 41% (12/29) of the cohort, of which 58.3% (7/12) patients died. The median time from onset of symptoms to death among these seven patients was 29 days (range: 20 -53 days).

At the time of COVID-19 diagnosis, the most common maintenance immunosuppressants among the cohort included mycophenolate mofetil (MMF) or mycophenolate sodium (MPS) for 90% (26/29) of patients; tacrolimus or tacrolimus extended release for 79% (23/29) of patients;

and prednisone for 72% (21/29) of patients. Lesser common treatments among the cohort included: maintenance with belatacept (1/29), sirolimus (1/29), azathioprine (2/29), and cyclosporine A (CsA; 4/29) ( Table 1 ). In the majority of patients, the change in immunosuppression due to COVID-19 was the decrease or discontinuation of MMF/MPS and the initiation/increase of steroid treatment (prednisone or hydrocortisone). For treatment of COVID-19, four patients received remdesivir; eight received dexamethasone or methylprednisolone; five were administered convalescent plasma; and one patient was treated with hydroxychloroquine (Table 1) .

Following admission to the hospital, all patients were monitored for allograft rejection using a dd-cfDNA test. For these patients, the median time from the onset of COVID-19 symptoms to the first dd-cfDNA test reading was 14 days (range: 5 -72) with 25 (86%) of these tests being n=9; p=0.01) and those who did not experience AKI (n=6; p=0.06). We had an inadequate sample size to assess this change in patients who did receive RRT (n=2; Table 3 ).

The median dd-cfDNA fraction among the initial test results from the 29 patients was 0.11% (range: 0.01% -1.54%) vs. 0.32% (range: 0.03% -0.98%) for the 15 follow-up tests; this difference was not significant for the 15 individuals with paired test results (p=0.67; Fig 1C) . 

Clinical COVID-19 severity scores in this cohort ranged from 3 (hospitalization with no oxygen therapy) to 8 (mortality), with a median score of 5. 25 Stepwise regression identified a significant positive association between total cfDNA levels and the COVID-19 severity score (R 2 =0.19; p=0.02; Fig 2) . No other covariates achieved the p≤0.10 level of significance required for inclusion in the model.

Stepwise regression analysis identified total cfDNA and dd-cfDNA fractions as the only predictors of mortality. A clinically relevant trend toward an association between each of these variables with mortality was observed, (dd-cfDNA: p=0.07; total cfDNA: p=0.11) but did not reach statistical significance. The probability of death increased with increasing total cfDNA levels ( Fig 3A) . In contrast, the probability of death increased with decreasing dd-cfDNA fractions, but only for dd-cfDNA values less than 0.25%; above this value, probability of death was estimated to be 0 (Fig 3B) . In contrast to total cfDNA levels, dd-cfDNA fractions at the first time-point were not elevated, and no significant difference in levels was observed between the first and second time-points. This is not surprising, as elevations in total cfDNA levels would be expected to depress the proportion of dd-cfDNA. Specifically, in this cohort only 3.4% of patients (1/29) had dd-cfDNA fractions at or above the 1% threshold for indication of allograft injury/rejection, as compared to clinical cohorts in which we previously identified test positivity rates of 32% (37/114) and 47%

(48/103) in individuals with surveillance and for-cause biopsies respectively. 18 Total cfDNA levels were higher at the first time point compared to the second in all sub-cohorts queried, regardless of RRT or AKI status. Although studies have implicated RRT such as hemodialysis in elevations in cfDNA, 36 our findings suggest that neither AKI nor RRT can fully account for the changes observed. While many of these changes in total cfDNA were statistically significant, they are likely not clinically useful.

Two individuals in our cohort with biopsy-confirmed active rejection had substantially elevated total cfDNA. The dd-cfDNA fractions for both individuals were below the standard 1% dd-cfDNA cut-off for high-risk for rejection, indicating that interpretation of the dd-cfDNA test results was confounded by the elevated total cfDNA. Studies have suggested that subtle changes in dd-cfDNA levels could be masked when reporting dd-cfDNA fractions due to fluctuations in total cfDNA. Additionally, measuring dd-cfDNA quantity (cp/mL) independent of total cfDNA, is valuable in assessing allograft rejection. 37, 38 We recently reported on a methodology that incorporates both the fraction and quantity dd-cfDNA for assessment of allograft rejection; 39 this algorithm would have identified high-risk for rejection for both of these cases during active infection with SARS-CoV-2 when total cfDNA levels were elevated. Further studies are needed to assess broad use of dd-cfDNA quantity to test for allograft rejection during viral infection.

Our analysis demonstrated a clinically significant correlation between total cfDNA levels and COVID-19 severity. These findings corroborate another study that similarly identified an association between cfDNA concentrations and WHO clinical progression scores in hospitalized patients. 10 While these findings are scientifically interesting, they are likely not clinically useful.

Limitations to our study include an analysis restricted to only hospitalized KT patients, thus we were unable to evaluate whether these trends are also present in allograft recipients with symptomatically mild COVID-19 cases. Additionally, serial dd-cfDNA testing and tracking of COVID-19 severity over the course of hospitalization in these individuals would enable a more precise understanding of the relationship between progression of COVID-19 and total cfDNA levels. As this is a preliminary study, future studies will be needed to validate the relationship between total cfDNA and dd-cfDNA levels and COVID-19 infection in KT patients.

In summary, the data presented herein indicate an association between elevated total cfDNA and COVID-19 infection and its severity in hospitalized KT patients. Additionally, when using dd-cfDNA testing for monitoring allograft rejection in individuals with COVID-19, consideration of total cfDNA levels along with the dd-cfDNA fraction is important, to ensure that cases of rejection are not missed.

assistance.

The results presented in this paper have not been submitted/being considered/published previously by any other journal.

The data that support the findings of this study are available on request from the corresponding author. The data are not publicly available due to privacy or ethical restrictions. 

Natera, Inc. provided support for study development, analysis, writing and reviewing the manuscript, and for use of Prospera TM in this study. 

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2021:ASN.2021050645. Figure Legends and Tables Fig 1: donor-derived, and total cfDNA levels in kidney transplant recipients with COVID-19. (A) Total cfDNA levels, represented as cp/mL, were plotted against time in days from onset of COVID-19 symptoms to date of blood draw for dd-cfDNA tests at both the initial time-point (yellow) and the follow-up time-point (blue). (B)

and the follow-up time-point (Draw 2), stratified by patients who had a single draw either due to death (red), or a second draw was unavailable (green

Grey dotted lines indicate medians for 15 paired values at first draw (3.46x10 4 cp/mL) and second draw (5.66x10 3 cp/mL). (C) dd-cfDNA levels at the initial time-point and the follow-up time-point, stratified as indicated in (B). Black lines connect paired tests. Grey dotted lines indicate medians for 15

The authors would like to thank Karina Soboleva, MD, (Natera, Inc.) for her assistance in data analysis and critical feedback and Meenakshi Malhotra, PhD, (Natera, Inc.) for her editorial