Does therapeutic drug monitoring (TDM) of trough concentrations suffice for optimizing preemptive therapy with ganciclovir of cytomegalovirus infections in non‐renal solid organ transplant recipients?

The aim of this study is to explore the relationship between ganciclovir exposure and clinical efficacy and/or safety in non‐renal solid organ transplant (SOT) recipients receiving preemptive therapy with ganciclovir/valganciclovir and undergoing therapeutic drug monitoring (TDM)‐guided dosing optimization.


INTRODUCTION
Cytomegalovirus (CMV) infection is responsible for remarkable morbidity and mortality among solid organ transplant (SOT) recipients, 1,2 making necessary the treatment of CMV reactivation in high-risk SOT for avoiding severe complications. 3,4nciclovir and its oral pro-drug valganciclovir are the main-stay agents for managing CMV reactivation or infection. 5In the last years, great efforts have been implemented for seeking which strategy could be the best for preventing severe CMV infections in SOT recipients.
Universal prophylaxis has been the standard approach for several years, but recently preemptive therapy, namely, implementation of antiviral treatment only after positive viremia detection, emerged as an effective strategy in high-risk SOT recipients. 1 Advantages of preemptive therapy versus universal prophylaxis may be reduced occurrence of late-onset CMV infection and minor both drug-related toxicity risk and drug acquisition costs. 6erapeutic use of ganciclovir/valganciclovir is quite challenging as it may be burdened on the one hand by the risk of dose-dependent myelotoxicity and on the other hand by that of CMV breakthrough resistance.Consequently, considering that in preclinical models ganciclovir concentrations ranging from 0.13 to 1.6 mg/L were shown to allow halving CMV replication, [7][8][9] therapeutic drug monitoring (TDM) has been proposed as a useful tool for optimizing preemptive therapy with ganciclovir. 102][13] Indeed, it could be argued that the conclusions of these studies could have been biased by some confounding factors.2][13] Besides, a recent position paper about the role of antimicrobial TDM in critically ill adult patients stated that currently no clear evidence exists for defining specific TDM target thresholds of ganciclovir. 14e aim of this study was to explore the relationship between ganciclovir exposure and clinical efficacy and/or safety in a homogeneous cohort of SOT recipients receiving preemptive therapy with ganciclovir/valganciclovir and undergoing TDM-guided dosing optimization.

METHODS
All adult non-renal SOT recipients (viz., liver, lung, or heart recipients) hospitalized and/or followed closely as outpatients at the  retrieved for each patient.ARC was defined as a measured CL CR ≥ 130 mL/min/1.73m 2 in males and ≥ 120 mL/min/1.73m 2 in females coupled with a normal serum creatinine value. 16Ganciclovir or valganciclovir dosage, time from transplant to initiation of antiviral therapy, number of TDM assessments per patient, time to first TDM assessment, and number of ganciclovir/valganciclovir dosing adjustments were also collected.Data on blood CMV-DNA load at baseline and throughout antiviral treatment were retrieved.CMV-DNA assay was performed on whole blood samples by using a commercial quantitative real-time polymerase chain reaction as previously described. 17e analytical sensitivity of the assay was 10 copies of target DNA per amplification reaction.The lower limit of quantification (LLQ) of the assay was 300 copies/mL whole blood.
Ganciclovir or valganciclovir was prescribed at the discretion of the attending physician or infectious disease consultant in terms of dosage and duration according to current guidelines 4  for dosing optimization in each single patient within the same day as previously described. 19,20Desired range of ganciclovir C min was set at 1.0-3.0mg/L according to recent findings, 11  tor; thrombocytopenia as a platelet count < 100 × 10 9 /L, a decrease ≥ 50%, compared to the baseline value, or the need for platelet administration; and anemia as a hemoglobin concentration of < 8.0 g/dL, a decrease ≥ 20%, compared to the baseline value, or the need for red blood cell transfusion. 21Hepatotoxicity was defined as an increase up to twice the upper limit of normal for serum alanine aminotrans-ferase or aspartate aminotransferase.Neurotoxicity was defined and assessed by subjective descriptions reported in electronic medical records.
Descriptive statistics were used to describe the patient sample, with continuous data presented as the median and interquartile range (IQR), whereas categorical variables were expressed as count and percentage.Univariate analyses were performed by using the Fisher exact test, χ 2 test, or Mann-Whitney U test as appropriate.The receiver operating characteristic (ROC) curve analysis was performed by selecting ganciclovir average C min as the test variable and the different efficacy/toxicity outcomes as the state variable, and area under the curve (AUC) along with 95% confidence interval (CI) were calculated.The optimal cut-off point was computed using the Youden Index method.
Youden Index was calculated according to the following equation: sensitivity (%) + specificity (%) − 100.Linear correlation between ganciclovir average C min and time to CMV negativity was also calculated.
A p-value < .05 was considered significant.Statistical analysis was performed using MedCalc for Windows (MedCalc statistical software, version 19.6.1,MedCalc Software Ltd.).

RESULTS
Among a total of 89 patients who underwent TDM-guided ganciclovir/valganciclovir therapy in the period March 2021-August 2022, 29 fulfilled the inclusion criteria and were selected (Figure 1).Demographics and clinical characteristics of the included patients are reported in Table 1.
The median age was 56 years (IQR 50-63 years), with male preponderance (86.2%).The median CL CR at baseline was 50.5 mL/min/1.73mOutcomes in terms of clinical efficacy and safety were reported in ROC analysis did not allow to identify an average C min cut-off predictive either of clinical efficacy or of toxicity (Supplementary Table S1).
Overall, three out of 29 patients (10.3%) had an average C min below recipients, compared to heart-or lung-transplant recipients (p = .006;Supplementary Table S4).No other significant differences emerged between patients with leukopenia occurrence, compared to those with no decrease in white blood cell count.

DISCUSSION
Our study explored the role of a TDM-guided strategy in optimizing ganciclovir/valganciclovir preemptive therapy in a cohort of non-renal SOT recipients.Our findings suggested that standard dosing regimens of ganciclovir/valganciclovir adjusted for renal function allowed the attainment of the desired trough level at first TDM assessment in approximatively half of the patients and that the need for further dosing adjustments concerned more than 30% of ECPAs during the overall treatment.These findings are consistent with those reported in previous recent real-world studies conducted in similar scenarios. 11,12tchie et al. 11  Platelet count 50% decrease from baseline 2 ( Hemoglobin < 8.0 g/dL 6 ( Hemoglobin 20% decrease from baseline Neurotoxicity 0 (0.0) Abbreviations: CMV, cytomegalovirus; IQR, interquartile range.
CMV-infected patients having hematological or autoimmune disorders, HIV infection, or being SOT recipients.Similarly, Martson et al. 12 reported that dosing adjustments were needed in 29% cases of SOT and HSCT recipients receiving ganciclovir or valganciclovir as prophylaxis or treatment.
Unfortunately, ROC analysis did not show any clear relationship between ganciclovir C min and clinical response in terms of undetectable CMV viral load at 30 days.2][13] Several reasons could explain why measuring C min could not be enough for this purpose.First, preclinical studies showed that the IC 50 needed for reducing CMV replication by 50% was quite variable, ranging from 0.1 to 1.7 mg/L. 7,8,22,23The IC 90 needed for reducing CMV replication by 90% could be a more clinically relevant concentration as it reflects what is aimed during treatment.However, the IC 90 was found to be as high as 3.5 mg/L, 24 namely, a value higher than the C min upper safety threshold that is currently applied in clinical practice for avoiding dose-dependent toxicity risk.Indeed, recent studies showed that the 24-h area under the concentration time curve (AUC 24h ) could be a better predictor of clinical outcome.A target AUC 24h of 40-50 mg•h/L was associated with decreased risk of CMV infection for adults undergoing CMV prophylaxis, 25 whereas that of 80-120 mg•h/L was suggested for granting efficacy in the treatment of active CMV disease. 26Unfortunately, previous studies showed that C min was quite poorly correlated with the AUC 24h , 12,25 and this furtherly strengthens the hypothesis that C min per se could not be a valuable predictor of CMV viral load decrease.
Dose-dependent ganciclovir/valganciclovir-related leukopenia occurred in more than two-thirds of SOT recipients included in our study.2][13] Unfortunately, ROC analysis did not allow to identify a threshold of ganciclovir C min helpful in predicting the likelihood of this adverse event.We found a trend toward lower leukopenia occurrence among patients having average values and the decrease in white blood cells count. 12A retrospective analysis found a significant correlation between ganciclovir C min and lymphopenia but not leukopenia among 46 SOT recipients. 13deed, it should also be mentioned that the remarkable proportion of leukopenia occurring among SOT patients may recognize several multifactorial causes other than ganciclovir/valganciclovir treatment, namely, therapy with immunosuppressants or with cotrimoxazole or the co-presence of other underlying diseases. 27Indeed, more than two-thirds of our patients received myelotoxic agents during ganciclovir/valganciclovir treatment, thus their role in contributing to the relevant proportion of leukopenia occurrence cannot be ruled out.
Although according to both our and previous findings the role of ganciclovir TDM could be questionable, as also stated in a recent position paper, 14 further studies investigating the relationship between ganciclovir/valganciclovir exposure (in terms of both C min and AUC 24h ) and efficacy and/or safety will be required for definitively assessing the clinical usefulness of a TDM-guided approach.
It is noteworthy that novel antiviral agents (i.e., letermovir, maribavir) have been recently issued in order to overcome resistance and toxicity occurrence reported with ganciclovir and/or foscarnet. 28though real-world evidence is still limited, preliminary evidence found a significantly lower rate of myelotoxicity and nephrotoxicity with the use of letermovir and/or maribavir, compared to traditional antiviral agents. 29,30 are aware of some limitations of our study.The retrospective study design and the limited sample size should be acknowledged.
Furthermore, CMV-DNA viral load was not reported in IU/mL as recommended by World Health Organization international guidelines. 6nversely, a strength element is represented by the fact that the analysis was carried out in a homogeneous cohort of patients composed entirely of non-renal SOT recipients.
and dosing adjustments were recommended whenever values were outside of this range.The primary outcome of clinical efficacy was reduction of CMV viral load below the LLQ at 30 days.Secondary outcomes included time to negativity, rate of persistent infection (defined as a drop of CMV viral load < 1-log after 2 weeks of treatment), the occurrence of resistance development with treatment escalation to foscarnet or other antivirals, and mortality rate during ganciclovir/valganciclovir treatment course.The primary outcome of toxicity was the occurrence of leukopenia, neutropenia, thrombocytopenia, or anemia at any time during treatment.Furthermore, the occurrence of hepatotoxicity and neurotoxicity during ganciclovir/valganciclovir was also assessed.Hematologic parameters were defined according to the Common Terminology Criteria for Adverse Events: leukopenia as a white blood cell count < 3.5 × 10 9 /L or a decrease ≥ 20%, compared to the baseline value; neutropenia as an absolute neutrophil count < 1.0 × 10 9 /L or administration of granulocyte colony-stimulating fac-

F I G U R E 1
Flowchart of patient inclusion and exclusion criteria.SOT, solid organ transplant; TDM, therapeutic drug monitoring.ganciclovir/valganciclovir dosing regimens were adjusted at the first TDM assessment in 15 out of 29 patients (51.7%, with 34.5% decreases and 17.2% increases).
found that ganciclovir C min was within the desired range of 1-3 mg/L in 55.2% of cases among a heterogeneous cohort of TA B L E 2 Clinical efficacy and safety outcomes of SOT recipients receiving ganciclovir/valganciclovir non-prophylactic dosing regimens.
and clinical practice implemented at our University hospital for each type of SOT.

Table 2 .
Reduction of CMV viral load below the LLQ at 30 days was in which leukopenia occurred, whereas in no case granulocyte colonystimulating factor was used.Thrombocytopenia and anemia were found in three (10.3%)and six (20.7%) patients, respectively.Concomitant agents causing myelotoxicity were used in 20 out of 29 patients (69.0%), being cotrimoxazole plus mycophenolate the most frequent (44.8%).Five patients (17.2%) developed hepatotoxicity during gan-ciclovir/valganciclovir treatment, whereas no case of neurotoxicity occurred.
Supplementary TableS2).No other difference in terms of clinical efficacy and/or safety outcomes emerged (Supplementary TableS2).A total of six out of 29 patients (20.7%) had an average C min > 3 mg/L, and no difference emerged in terms of clinical efficacy and/or safety outcomes, compared with those having an average C min < 3 mg/L (Supplementary TableS3).
1 mg/L and showed a trend toward a lower rate of leukopenia, compared with those having an average C min > 1 mg/L (0.0% vs. 65.4%;p= .06;ents(77.8%) showed a reduction of CMV viral load below the LLQ at 30 days, compared to lung-(53.3%)orheart-transplant recipients (40.0%), although not statistically significant.The occurrence of leukopenia was 100.0%, 86.7%, and 33.3% in heart-, lung-, and liver-transplant recipients, respectively.A lower leukopenia occurrence was reported among liver transplant TA B L E 1 Demographics and clinical variables of non-renal solid organ transplant (SOT) recipients treated with ganciclovir/valganciclovir non-prophylactic dosing regimen.
compared with those having C min ≥ 1 mg/L, but this disappeared when comparing average C min > 3 mg/L versus ≤ 3 mg/L.Available data in the literature are inconsistent with respect to such safety issues.Ritchie et al. did not find any significant association between leukopenia occurrence and serum ganciclovir peak and trough concentrations. 11Martson et al. found at multivariate analysis a significant relationship between the highest C min and AUC 24h Clinical features of patients developing ganciclovir resistance during CMV treatment.Univariate analysis comparing SOT recipients showing 30-day CMV negativization versus those with no 30-day CMV negativization.