INTRODUCTION
Cytomegalovirus (CMV) affects a large proportion of the general population without causing significant clinical symptoms. However, in patients having undergone solid organ transplantation, owing to immune suppression, the previously latent virus can reactivate and cause great morbidity and mortality.1,2
Ganciclovir and its oral prodrug, valganciclovir, are currently used as first-line therapies for the prevention and treatment of CMV infection.3,4 5
The pharmacokinetic properties of (val)ganciclovir are characterized by great interindividual variability in plasma levels. Therefore, therapeutic drug monitoring (TDM) is performed in numerous care facilities. However, clinically established target trough levels for efficacy or toxicity are lacking. Several studies have suggested an association between plasma levels and efficacy6–10 11–15 11,16–21 2
METHODS
Design and Patient Population
This retrospective, single-center cohort study was conducted at the University Medical Center in Utrecht using data retrieved from electronic medical records from January 2012 to January 2022.
Adult patients who had undergone a solid organ transplantation and were treated for CMV infection with (val)ganciclovir, and from whom at least 1 ganciclovir trough level (as part of routine care) and 2 CMV viral loads were examined were included in the study. Primo infection and reactivation, with or without systemic symptoms and organ damage were considered relevant for the study, with classification according to the criteria by Ljungman et al.22
Serum ganciclovir concentrations were determined using liquid chromatography coupled with mass spectrometry (LC-MS/MS). This assay uses ganciclovir-d5 as an internal standard and has proven linearity in the range of 0.2–15 mg/L. The lower limit of quantification was set at 0.2 mg/L, and the within-run and between-run precision at a concentration of 3.0 mg/L was 5.0% and 6.0%, respectively. Plasma levels were considered trough levels if they were obtained at most 60 minutes before the next administration. The trough levels were interpreted by hospital pharmacists, who made the dose adjustment recommendations. CMV viral loads in the plasma were determined using quantitative polymerase chain reaction based on a real-time TaqMan CMV-DNA polymerase chain reaction assay, as described previously.23
The following data were collected: patient characteristics (sex, age, underlying disease, type of transplant, and need for and type of renal replacement therapy), disease and treatment specifics (indication according to disease criteria by Ljungman et al,22 24 25
Study Endpoints
The primary endpoint was the difference in efficacy between target trough levels. At our center, a target trough level of 1–2 mg/L was originally used; however, from March 1, 2021 onward, trough levels of 2–4 mg/L were deemed adequate according to the study by Martson et al.2
Efficacy was defined as the absence of treatment failure for which a composite endpoint was formulated: > 1 log10 rise in viral load,26
Secondary endpoints were taken to be the association between trough levels and efficacy, toxicity, occurrence of resistance (21), and difference in time to reach viral decay (viral load <100 IU/mL) in different target trough level groups.
Statistical Analysis
For data collection and analysis, SPSS (IBM SPSS Statistics 28, Armonk, NY) and R (4.1.2.) were used. Population and TDM characteristics were summarized as descriptive statistics (mean and SD (SD), median and interquartile ranges (IQR), frequencies, and percentages).
To compare the efficacy between the target trough levels, the χ2 and Fisher exact tests were used for categorical data. Numerical data were compared using the t test for normally distributed data and Wilcoxon rank test for non-normally distributed data.
To determine the association between the composite endpoint and toxicity, repeated measures of ganciclovir levels were used to construct a general estimating equation (GEE) model. Independent correlations were also used. Because more frequent repeat measures are expected in patients who have low trough levels, interactions between the composite endpoint and time between trough level measurements were allowed to minimize confounding because of this phenomenon. The baseline viral load was also included in the model because previous studies have shown slower viral decay at higher baseline viral loads.26
Kaplan–Meier survival curves and a Cox regression model (with age, type of transplant, baseline viral load, and baseline kidney function as covariates) were constructed to assess differences in time taken for viral decay in patients with different target trough levels. Proportional hazard assumptions were tested using scaled Schoenfeld residuals and their independence over time. P values below 0.05 were deemed statistically significant.
RESULTS
Patient and TDM Characteristics
A total of 46 patients were enrolled, of whom 28 had undergone kidney transplantation, 11 had undergone lung transplantation, and 7 had undergone heart transplantation. The patients' characteristics are listed in Table 1 . Most patients had (val)ganciclovir prescribed for CMV infection (65.2%), 26.1% for CMV syndrome, and the remaining 8.7% for CMV disease. The patient characteristics did not differ significantly between the 2 different target trough level groups.
TABLE 1. -
Patient Characteristics
Variable
Total n = 46
1-2 mg/L Target Trough Level Group (n = 36)
2–4 mg/L Target Trough Level Group (n = 10)
P
Age (yr) mean ± SD
56 ± 14.4
54.6 ± 15.4
60.6 ± 9.5
0.42*
Female n (%)
21 (45.7)
18 (50.0)
3 (30.0)
0.30†
Type of transplant n (%)
0.55†
Kidney
28 (60.9)
21.0 (58.3)
7.0 (70.0)
Lung
11 (23.9)
9.0 (25.0)
2.0 (20.0)
Heart
7 (15.2)
6.0 (16.7)
1.0 (10.0)
CMV status∼ n (%)
0.88†
Infection
30 (65.2)
23.0 (63.8)
7.0 (70.0)
Syndrome
12 (26.1)
10.0 (27.8)
2.0 (20.0)
Disease
4 (8.7)
3.0 (8.3)
1.0 (10.0)
CMV load at baseline (IU/mL); median (IQR)
9,82E‡ ‡ ‡
1,07E‡ ‡ ‡
7,65E‡ ‡ ‡
0.33‡
CMV status of donor and recipient
0.28†
D-/R-
3
3
0
D-/R‡
8
7
1
D‡ ‡
10
9
1
D‡
25
17
8
Route of (val)ganciclovir administration n (%)
0.72†
Oral
9 (19.6)
7.0 (25.0)
2.0 (20.0)
Intravenously
9 (19.6)
9.0 (19.4)
0.0 (0.0)
Consecutively IV-PO
28 (60.9)
20.0 (55.6)
8.0 (80.0)
Duration of therapy (d); median (IQR)
107 (22–225)
89.0 (21.8–282.8)
143.0 (104.0–153.8)
0.91*
Leucocytes at baseline (10^9/mL)
5.6 (3.7–9.0)
4.8 (2.9–9.2)
7.4 (6.3–8.2)
0.25*
Lymphocytes at baseline (10^9/mL) median (IQR)
0.7 (0.4–1.4)
0.7 (0.4–1.3)
1.2 (0.5–2.6)
0.47‡
Neutrophils at baseline (10^9/mL) median (IQR)
3.3 (2.3–5.3)
3.1 (2.0–4.3)
4.5 (3.3–6.2)
0.12*
* Wilcoxon rank sum test.
† Fisher exact test.
‡ t test ∼ CMV infection: virus isolation or detection of viral proteins or nucleic acids in any body fluid or tissue specimen. CMV syndrome: CMV infection combined with fever ≥38°C for at least 2 d, new or increased malaise or new or increased fatigue, leukopenia or neutropenia, ≥5% atypical lymphocytes, thrombocytopenia, and elevation of aminotransferase level 2 times the upper limit of normal. CMV disease: CMV syndrome combined with end-organ disease (pneumonia, gastro-intestinal disease, hepatitis, retinitis, encephalitis, nephritis, cystitis, myocarditis, and pancreatitis).
Overall, 200 trough levels were obtained, with a median of 3 (IQR 2.0–5.8) per patient. The median (IQR) of the first obtained through level was 1.4 (0.7–2.0) mg/L for all patients. When divided as per patients who were treated per used target trough levels (1–2 mg/L versus 2–4 mg/L), they were 1.6 (0.8–2.0) mg/L and 1.1 (0.7–1.6) mg/L, respectively (P = 0.76).
The median (IQR) for all trough levels was 1.7 (0.9–1.9) for patients with 1–2 mg/L target trough levels and 1.5 (1.2–1.9) for those with 2–4 mg/L target trough levels (P = 0.74).
The TDM characteristics are summarized in Table 2 .
TABLE 2. -
TDM Characteristics; Divided by Used Target Trough Level
Target Trough Level 1-2 mg/L (n = 36)
Target Trough Level 2–4 mg/L (n = 10)
P *
Median (IQR)
Median (IQR)
All trough levels (mg/L)
1.7 (0.9–1.9)
1.5 (1.2–1.9)
0.74†
First trough level (mg/L)
1.6 (0.8–2.0)
1.1 (0.7–1.6)
0.76†
Number of trough levels obtained per patient
2.0 (2.0–4.0)
7.0 (3.3–8.8)
0.05‡
Time between start (v)GCV and reaching target trough of 1 mg/L (d)
10.0 (3.0–20.0)
6.0 (3.00–7.0)
075†
Time between start (v)GCV and reaching target trough of 2 mg/L (d)
12.0 (6.0–20.0)
9.0 (6.5–27.0)
0.95†
* Statistically significant, P < 0.05.
† t test
‡ Wilcoxon rank sum test.
A total of 28 patients (77.8%) reached their prespecified target trough level of 1–2 mg/L within 10.0 (3–20) days and 7 (70.0%) reached their target trough level of 2–4 mg/L within 9.0 (6.5–27.0) days (see Fig. 1 ). No statistically significant differences were observed (see Table 2 ).
FIGURE 1.: Trough levels over time (days), divided by target trough level used; blue line indicates median.
Primary Endpoint
The composite endpoint was reached by 23 of 36 patients, in whom a target trough level of 1–2 mg/L was used, and 6 of 10, in whom a level of 2–4 mg/L was used. No statistically significant differences were observed (P = 1.0).
Even when specified for patients who actually reached the target trough level of 2–4 mg/L (24 of 46), no statistically significant difference was found in reaching the composite endpoint (P = 1.0, see Table 3 ).
TABLE 3. -
Composite Outcome and Its Components
Occurrence n (%) Target Trough Level 1-2 mg/L N = 36
Occurrence n (%) Target Trough Level 2–4 mg/L N = 10
P *
Composite endpoint
23 (63.9)
6 (60.0)
1.0
>1 log10 rise in viral load
22 (61.1)
6 (60.0)
1.0
Initiation of foscarnet
8 (22.2)
0 (0)
0.17
Resistance analysis
10 (27.8)
4 (40)
0.46
Secondary Endpoints
Using the GEE model, no statistically significant correlation between trough levels and the composite endpoint was found (P = 0.75). Using a GEE model, a correlation between trough levels and toxicity could be established for lymphopenia (P = 0.01), but not for nephrotoxicity (P = 0.75), anemia (P = 0.66), thrombocytopenia (P = 0.69), leukopenia (P = 0.93), and neutropenia (P = 0.25).
The occurrence of toxicity and its distribution in the groups with different target trough levels are summarized in Table 4 . No statistically significant difference was found between the 2 groups.
TABLE 4. -
Occurrence of Toxicity Specified by Used Target Trough Levels
Occurrence n (%) Target Trough Level 1-2 mg/L
Occurrence n (%) Target Trough Level 2–4 mg/L
P *
Nephrotoxicity
9 (25.0)
5 (50.0)
0.71
Anemia
16 (61.5)
5 (50.0)
1
Thrombocytopenia
10 (27.8)
1 (10.0)
0.14
Leukopenia
13 (36.1)
0 (0.0)
0.06
Neutropenia
8 (22.2)
0 (0.0)
0.08
Lymphopenia
12 (33.3)
3 (30.0)
0.49
There was no statistically significant difference in the viral decay time between the target trough level groups (P = 0.93). In addition, Cox regression showed no statistically significant correlation between the target trough level and viral decay time, as shown in Figure 2 (P = 0.36, proportional hazards could be assumed according to Schoenfeld residuals (P = 0.44) and their independence of time).
FIGURE 2.: Viral decay time divided by used target trough level. Difference not statistically significant (P = 0.93, Kaplan–Meier curve).
Resistance analysis was performed in 14 patients, and resistant CMV strains were detected in 3 patients, all belonging to the 1–2 mg/L target trough level group (P = 0.5).
DISCUSSION
Our study could not establish a greater efficacy of (val)ganciclovir treatment of CMV infection in solid organ transplant recipients when a target trough level of 2–4 mg/L was used compared with the target trough levels of 1–2 mg/L. The initiation of foscarnet was noted more often in the 1–2 mg/L target trough level group, but this difference was not statistically significant. In addition, 3 versus 0 resistant strains of CMV were detected in the lower target trough level group, without any statistical significance. No correlation was found between repeated measures of ganciclovir trough levels and efficacy. A correlation between the occurrence of lymphopenia and trough level was demonstrated, but not with other forms of toxicity often attributed to (val)ganciclovir. Because of the low number of resistant strains detected, no statement can be made regarding the correlation between trough levels and the development of resistance.
In many care facilities, TDM of ganciclovir is routinely performed, although clinically established target trough levels are lacking. The in vitro established half-maximal inhibitory concentration (IC50) of ganciclovir is 0.1–2 mg/L.27,28 10 29
For ganciclovir to be biologically active, it must undergo several intracellular phosphorylation steps. In CMV-infected cells, ganciclovir is transformed to ganciclovir monophosphate by the viral protein kinase pUL97 and then to its active form, ganciclovir triphosphate, which is incorporated into viral DNA. Bilat et al showed great interindividual variability in intracellular concentrations of ganciclovir triphosphate and poor correlation with plasma concentrations of ganciclovir.13
In accordance with other studies,11,16–20 2,29 12,18 18 2
The difficulty in interpreting earlier studies lies in the fact that patients with various degrees of immunosuppression were included in the study. Viral clearance depends not only on adequate exposure to antivirals, but also on the function of the cellular immune system. Patients who underwent hematological stem cell transplantation had a higher degree of immunosuppression than patients who had undergone solid organ transplantation or chemotherapy. This is the strength of our study, which only included solid organ transplant recipients.
Our study had several limitations. The relatively small sample size makes it impossible to draw conclusions on the correlation between ganciclovir levels and toxicity and development of resistance because of the low occurrence of these outcomes. In addition, in accordance with other studies,2,12,30
Patients in the 1–2 mg/L target group were treated for as long as 10 years ago. Although no differences in patient or treatment characteristics were noted between these groups, there might have been differences that were not accounted for in our analysis, which might have introduced bias in our study.
The exact types of immunosuppressive regimens and concomitant drugs (possibly causing blood dyscrasias) were not recorded in our database. Although the different types of organ transplants were evenly distributed between the 2 target level groups, bias could have been introduced because the levels of immunosuppression and use of other drugs can differ between these types of patients.
To make a definite decision on whether TDM of ganciclovir contributes to higher cure rates in the treatment of CMV infections in solid organ transplant patients and what target trough levels should be adopted, more research is needed. Randomized clinical trials are not suitable for this; however, a study design comparable to ours could be used. Collaboration between clinics is advised to be able to achieve a representative sample size and, subsequently, statistical power can be achieved. Further research should focus on the intracellular measurement of ganciclovir triphosphate, although this technique is not yet readily available and no established target trough levels exist for this application.
Because of its great interindividual variability, especially in patients with decreased kidney function, TDM could be beneficial for this patient category.2
CONCLUSION
Our study could not establish a difference in clinical efficacy or toxicity in the use of a ganciclovir target trough level of 1-2 mg/L or 2–4 mg/L because of the lack of clinical differences between the compared groups. However, a correlation was found between repeated measurements of ganciclovir trough levels and lymphopenia, which warrants further research.
REFERENCES
1. Scott JC, Partovi N, Ensom MHH. Ganciclovir in solid organ transplant recipients. Ther Drug Monit. 2004;26:68–77.
2. Märtson AG, Edwina AE, Burgerhof JGM, et al. Ganciclovir therapeutic drug monitoring in transplant recipients. J Antimicrob Chemother. 2021;76:2356–2363.
3. Razonable RR, Humar A. Cytomegalovirus in solid organ transplant recipients—guidelines of the American society of transplantation infectious diseases community of practice. Clin Transpl. 2019;33:e13512.
4. Kotton CN, Kumar D, Caliendo AM, et al. The third international consensus guidelines on the management of cytomegalovirus in solid-organ transplantation. Transplantation. 2018;102:900–931.
5. Crumpacker CS. Ganciclovir. N Engl J Med. 1996;335:721–729.
6. Märtson AG, Touw D, Damman K, et al. Ganciclovir therapeutic drug monitoring: a case series. Ther Drug Monit. 2019;41:107–110.
7. Stockmann C, Roberts JK, Knackstedt ED, et al. Clinical pharmacokinetics and pharmacodynamics of ganciclovir and valganciclovir in children with cytomegalovirus infection. Expert Opin Drug Metab Toxicol. 2015;11:205–219.
8. Galar A, Valerio M, Catalán P, et al. Valganciclovir—ganciclovir use and systematic therapeutic drug monitoring. An invitation to antiviral stewardship. Antibiot. 2021;10:77.
9. Piketty C, Bardin C, Gilquin J, et al. Low plasma concentrations achieved with conventional schedules of administration of ganciclovir in patients with AIDS. J Infect Dis. 1996;174:188–190.
10. Piketty C, Bardin C, Gilquin J, et al. Monitoring plasma levels of ganciclovir in AIDS patients receiving oral ganciclovir as maintenance therapy for CMV retinitis. Clin Microbiol Infect. 2000;6:117–120.
11. Ritchie BM, Barreto JN, Barreto EF, et al. Relationship of ganciclovir therapeutic drug monitoring with clinical efficacy and patient safety. Antimicrob Agents Chemother. 2019;63:e01855.
12. Padullés A, Colom H, Bestard O, et al. Contribution of population pharmacokinetics to dose optimization of ganciclovir-valganciclovir in solid-organ transplant patients. Antimicrob Agents Chemother. 2016;60:1992–2002.
13. Billat PA, Woillard JB, Essig M, et al. Plasma and intracellular exposure to ganciclovir in adult renal transplant recipients: is there an association with haematological toxicity? J Antimicrob Chemother. 2016;71:484–489.
14. Perrottet N, Csajka C, Pascual M, et al. Population pharmacokinetics of ganciclovir in solid-organ transplant recipients receiving oral valganciclovir. Antimicrob Agents Chemother. 2009;53:3017–3023.
15. Balfour HH. Management of cytomegalovirus disease with antiviral drugs. Clin Infect Dis. 1990;12:S849–S860.
16. Giménez E, Solano C, Azanza JR, et al. Monitoring of trough plasma ganciclovir levels and peripheral blood cytomegalovirus (CMV)-Specific CD8
+ T cells to predict CMV DNAemia clearance in preemptively treated allogeneic stem cell transplant recipients. Antimicrob Agents Chemother. 2014;58:5602–5605.
17. Trevillyan J, Angus P, Shelton E, et al. Electronic estimations of renal function are inaccurate in solid-organ transplant recipients and can result in significant underdosing of prophylactic valganciclovir. Antimicrob Agents Chemother. 2013;57:4058–4060.
18. Perrottet N, Manuel O, Lamoth F, et al. Variable viral clearance despite adequate ganciclovir plasma levels during valganciclovir treatment for cytomegalovirus disease in D+/R- transplant recipients. BMC Infect Dis. 2010;10:2.
19. Welker H, Farhan M, Humar A, et al. Ganciclovir pharmacokinetic parameters do not change when extending valganciclovir cytomegalovirus prophylaxis from 100 to 200 days. Transplantation. 2010;90:1414–1419.
20. Fishman JA, Doran MT, Volpicelli SA, et al. Dosing of intravenous ganciclovir for the prophylaxis and treatment of cytomegalovirus infection in solid organ transplant recipients. Transplantation. 2000;69:389–394.
21. Erice A, Jordan MC, Chace BA, et al. Ganciclovir treatment of cytomegalovirus disease in transplant recipients and other immunocompromised hosts. JAMA. 1987;257:3082–3087.
22. Ljungman P, Boeckh M, Hirsch HH, et al. Definitions of cytomegalovirus infection and disease in transplant patients for use in clinical trials: table 1. Clin Infect Dis. 2017;64:87–91.
23. van Doornum GJJ, Guldemeester J, Osterhaus ADME, et al. Diagnosing herpesvirus infections by real-time amplification and rapid culture. J Clin Microbiol. 2003;41:576–580.
24. Khwaja A. KDIGO clinical practice guidelines for acute kidney injury. Nephron Clin Pract. 2012;120:c179–c184.
25. U.S. Department of Health and Human Services. National Institutes of Health, National Institute of Allergy and Infectious Diseases. Available at:
https://rsc.niaid.nih.gov/sites/default/files/daidsgradingcorrectedv21.pdf 26. Chemaly RF, Chou S, Einsele H, et al. Definitions of resistant and refractory cytomegalovirus infection and disease in transplant recipients for use in clinical trials. Clin Infect Dis. 2019;68:1420–1426.
27. Smee DF, Martin JC, Verheyden JP, et al. Anti-herpesvirus activity of the acyclic nucleoside 9-(1, 3-dihydroxy-2-propoxymethyl) guanine. Antimicrob Agents Chemother. 1983;23:676–682.
28. Mar EC, Cheng YC, Huang ES. Effect of 9-(1, 3-dihydroxy-2-propoxymethyl) guanine on human cytomegalovirus replication in vitro. Antimicrob Agents Chemother. 1983;24:518–521.
29. Wiltshire H, Paya CV, Pescovitz MD, et al. Pharmacodynamics of oral ganciclovir and valganciclovir in solid organ transplant recipients. Transplantation. 2005;79:1477–1483.
30. Jarrell AS, Crow JR, Strout SE, et al. Valganciclovir dosing for cytomegalovirus prophylaxis in solid-organ transplant recipients on continuous veno-venous hemodialysis. Clin Infect Dis. 2021;73:101–106.
