Risk Assessment of Neutropenia during Low-Dose Valganciclovir Prophylaxis for Heart Transplant Recipients

Mai Otokubo; Kyoichi Wada; Megumi Ikura; Kotoka Hayase; Takaya Uno; Kazuki Nakagita; Naoki Hayakawa; Takuya Watanabe; Osamu Seguchi; Norihide Fukushima; Tsutomu Nakamura

doi:10.1248/bpb.b21-00860

Abstract

The aim of this study was to investigate whether low-dose valganciclovir (VGCV) prophylaxis for cytomegalovirus (CMV) infection increased the risk of developing neutropenia in heart transplant recipients (HTRs). Forty-three HTRs receiving VGCV were divided into two groups: those who received VGCV prophylaxis (n = 22) and those who did not (n = 21). Neutropenia was defined as an absolute neutrophil count ˂1500/µL and was monitored for approximately one year post-transplantation. In the prophylaxis group, 77.3% (17/22) of HTRs experienced neutropenia, which was significantly higher than that in the no prophylaxis group (42.9% [9/21], p = 0.031). No significant differences in the duration of VGCV administration and cumulative dose up to the first neutropenia episode were observed between the groups. Meanwhile, the cumulative dose of mycophenolate mofetil was significantly higher in the prophylaxis group than in the no prophylaxis group (p = 0.018); the daily maintenance dose and regularly measured area under the concentration–time curve (AUC) of mycophenolic acid did not significantly differ between groups. In conclusion, the risk of developing neutropenia was higher in HTRs receiving low-dose VGCV prophylaxis than it was in those not receiving prophylaxis, probably not attributed to dosing period and cumulative dose of VGCV until the onset of neutropenia.

INTRODUCTION

Cytomegalovirus (CMV) infection after solid organ transplantation is a frequent and major complication that contributes to allograft dysfunction by accelerating endothelial dysfunction; it is one of the primary causes of morbidity and mortality in patients undergoing heart transplantation (HTx).^1–6) Although the routine monitoring of CMV DNA and/or antigen is performed after HTx, preventive strategies for managing CMV infection and disease in heart transplant recipients (HTRs) differ based on the pretransplant CMV serologic status, categorized into high-risk (recipient negative/donor positive), intermediate-risk (recipient positive), and low-risk (recipient negative/donor negative). In CMV high-risk HTRs, the initiation of antiviral prophylaxis within 10 d of HTx and continuation for three to six months are recommended; in CMV intermediate-risk HTRs, 3-month antiviral prophylaxis or preemptive therapy is recommended worldwide.^7,8)

Valganciclovir (VGCV), an orally administered prodrug of ganciclovir, has recently been widely employed for universal prophylaxis and preemptive therapy for CMV. However, VGCV may induce neutropenia, a common and serious complication of solid organ transplantation, including HTx.⁹⁾ The duration of VGCV prophylaxis tends to exceed preemptive treatment. Accordingly, CMV high-risk HTRs may be at an increased risk of developing severe neutropenia when compared with CMV intermediate/low-risk HTRs. Concerning the relationship between the duration of prophylaxis and adverse effects in HTRs, some reports have documented the use of standard-dose VGCV (900 mg daily) for CMV high-risk HTRs. Echenique et al. reported that irrespective of the recipient’s serostatus, extended-duration VGCV prophylaxis (i.e., 12 months versus six months) was not associated with the number of patients whose absolute neutrophil count dropped below 1800 per µL during the study period.¹⁰⁾ In addition, Imaly et al. reported that there was no statistically significant difference between the incidence of severe neutropenia (absolute neutrophil count of less than 500 or 1000 per µL, or requiring granulocyte-colony stimulating factor) after 3- and 6-month VGCV prophylaxis in CMV high-risk HTRs.¹¹⁾

Meanwhile, a meta-analysis was performed to compare standard and low-dose (450 mg daily) VGCV with ganciclovir use for CMV prophylaxis in solid organ transplant patients, including HTRs. A significantly increased risk of neutropenia was documented in the standard-dose VGCV group, whereas the risk for the low-dose VGCV group was relatively low.¹²⁾ In CMV high-risk kidney transplant recipients receiving 6-month VGCV prophylaxis, leukopenia was more likely to develop during standard-dose prophylaxis compared with the lower dose prophylaxis.¹³⁾ Although it remains unclear whether early intervention with a lower VGCV dose would be a safe strategy for CMV prophylaxis in HTRs and its safety is under consideration, VGCV has been administered prophylactically at our institute, with half the therapeutic dosage administered for approximately one year after HTx in CMV high-risk HTRs (Fig. 1).

Fig. 1. The Prophylaxis and Preemptive Therapy with VGCV for CMV Infection and Diseases

VGCV has been administered prophylactically at the National Cerebral and Cardiovascular Center (NCVC) in Japan with half the therapeutic dosage administered for approximately one year after heart transplantation in CMV high-risk HTRs. The prophylactic VGCV administration was initiated in the CMV high-risk HTRs depending on the patient’s condition, whereas in CMV intermediate/low-risk HTRs, a comprehensive judgment about the clinical necessity of the therapeutic indication for prophylactic therapy was made. VGCV, valganciclovir; CMV, cytomegalovirus; HTRs, heart transplant recipients.

In the present study, we retrospectively obtained data on patient background and characteristics during prophylactic and pre-emptive VGCV therapy from medical records and compared the incidence of neutropenia between the VGCV prophylaxis and no prophylaxis groups to assess the possible application of low-dose VGCV prophylaxis in this setting.

PATIENTS AND METHODS

Patient Selection and Study Protocol

Out of consecutive 80 patients who underwent HTx at the National Cerebral and Cardiovascular Center (NCVC) in Japan from January 2012 to March 2019, we retrospectively reviewed the medical records of the HTRs who received VGCV for prophylaxis and treatment against CMV within one year post-HTx. This study was conducted in accordance with the principles of the Declaration of Helsinki. The study protocol was approved by the local ethics committee of NCVC (NCVC IRB number, M27-044). This study was a retrospective observational study, and informed consent to participate was obtained via the opt-out method. We also excluded patients who were ˂18 years of age at the time of approval by the local ethics committee of the NCVC.

Forty-three HTRs receiving VGCV were divided into two groups: those who received VGCV prophylaxis (prophylaxis group; n = 22) and those who did not (no prophylaxis group; n = 21).

Clinical Parameters, Data Collection, and Assessment

All data were collected using an electronic medical record system. We assessed the following patient clinical characteristics before HTx (pre-HTx) and the initiation of VGCV administration: sex, age, body weight, body mass index, white blood cell (WBC) count, neutrophil count, lymphocyte count, and serum levels of albumin, alanine aminotransferase, aspartate aminotransferase, blood urea nitrogen, C-reactive protein, hematocrit, hemoglobin, serum creatinine (Scr), total bilirubin, total protein, dose and duration of VGCV, use of immunosuppressive medications (tacrolimus, mycophenolate mofetil (MMF), everolimus, prednisolone, and basiliximab), and trimethoprim-sulfamethoxazole combination. The estimated glomerular filtration rate (eGFR) was calculated using the Japanese Society of Nephrology equation: eGFR = 194 × Scr^−1.094 × Age^−0.287 (×0.739 if female).¹⁴⁾

Neutropenia was defined as an absolute neutrophil count ˂1500/µL, leukopenia was defined as a WBC count ˂3000/µL, and each episode was followed up for around one year after HTx.

Immunosuppressive Therapy

All patients received standard triple-drug immunosuppression therapy, including the regular-release formulation of tacrolimus, MMF, and prednisolone, immediately after HTx.¹⁵⁾ At our institute, tacrolimus is generally initiated at a dose of 1 mg/d divided into two doses (0.5 mg) on the first or second postoperative days (PODs). Twenty-four patients (55.8%) with impaired renal function pre-HTx or high risk of rejection underwent induction therapy with basiliximab; tacrolimus initiation was delayed, and its dosage and trough level slowly increased during weeks 1–3 post-HTx. The standard target trough levels of tacrolimus to be maintained during the first year post-HTx were 9–12 ng/mL. MMF was initiated at an initial dose of 1.5–2.0 g/d divided into two doses on the first or second POD. The MMF dose was adjusted based on WBC and neutrophil counts and the area under the concentration–time curve (AUC) of mycophenolic acid. In our institution, trough level and AUC monitoring of immunosuppressants have been performed for appropriate immunosuppressive therapy when HTRs are hospitalized for routine endomyocardial biopsies to assess graft rejection, coronary angiography, and coronary intravascular ultrasound with the development of cardiac allograft vasculopathy. Typically, conversion from MMF to everolimus is considered when worsening renal function or vascular compromise of the heart graft progresses during the maintenance period after HTx. Steroid administration was started immediately after HTx; thereafter, the steroid dosage was slowly tapered to 10 mg/d for six months post-HTx. Most patients were treated with steroids for approximately one year, while the duration of steroid use was adjusted depending on the patient’s condition. In cases where methylprednisolone was used, the data were calculated assuming that 4 mg of methylprednisolone was equivalent to 5 mg of prednisone.¹⁶⁾ If cardiac allograft rejection was detected on regular myocardial biopsy after HTx, the patient was treated with augmented immunosuppression and intravenous steroids.

Management of CMV Infection and Diseases

In HTRs, CMV infection and diseases were continuously monitored, while CMV high-risk recipients were routinely administered a 5-g dose of CMV immunoglobulin immediately after HTx, which was continued until five days post-HTx. All 43 HTRs in this study received CMV immunoglobulin. Additionally, the prophylactic VGCV administration was initiated in the CMV high-risk group depending on the patient’s condition (e.g., renal function and WBC count), whereas in CMV intermediate/low-risk HTRs, a comprehensive judgment about the clinical necessity of the therapeutic indication for prophylactic therapy was made. Two CMV high-risk HTRs did not receive prophylactic VGCV, whereas one CMV low-risk HTR received VGCV prophylaxis, because it took time to decide the CMV serological status. The prophylactic dose was set at half the therapeutic dose (450 mg/d) and was administered for a one-year period after HTx. In the prophylaxis group, if the results of CMV antigenemia and real-time PCR tests were positive, VGCV dose was increased to a therapeutic dose (900 mg/d) as treatment when accompanied by clinical symptoms or as preemptive therapy in asymptomatic cases when CMV DNA exceeded the threshold set for active CMV infection (Fig. 1). On the other hand, in no prophylaxis group, VGCV administration was initiated at a half or full therapeutic dose (450 or 900 mg/d) as CMV therapy shown above (Fig. 1). The VGCV dose was adjusted according to the patient’s condition such as renal function and counts of white blood cells and neutrophils.

Statistical Analysis

Fisher’s exact test was used to examine the differences in categorical variables between the groups. In addition, paired and unpaired continuous data were compared using the Wilcoxon signed-rank test and Mann–Whitney U test, respectively. The pairwise comparison tests were also applied with Bonferroni correction as a post-hoc test only when Friedman’s test revealed overall significant differences. Data were analyzed using JMP 15 (SAS Institute Inc., Cary, NC, U.S.A.), with p < 0.05, considered to indicate statistical significance.

RESULTS

Patient Clinical Characteristics and Use of Concomitant Medications

A total of 43 HTRs were included in the study. The preoperative patient characteristics are listed in Table 1, and clinical and laboratory characteristics of HTRs in pre-HTx, at the start of VGCV administration, and six months post-HTx are shown in Table 2. After the patient’s perioperative conditions were comprehensively judged, 21 and 1 HTRs at high and low risk for CMV infection, respectively, received VGCV prophylaxis, whereas two high-risk HTRs did not. Statistically significant differences in preoperative age and CMV serologic status were noted between the prophylaxis and no-prophylaxis groups. To prevent the deterioration of pre-HTx renal dysfunction of the HTRs, induction therapy using basiliximab was introduced to 24 HTRs, and the proportion of HTRs receiving basiliximab did not significantly differ between the two groups (40.9 and 71.4%, respectively; p = 0.067). After surgery, there was a significant increase in the eGFR values in both groups, most likely due to improved postoperative hemodynamics, although such improvement of renal function was not observed in some HTRs. In one CMV high-risk HTR, the administration of tacrolimus was initiated later than that of VGCV, and was started five days later. VGCV was started within three months after surgery in all HTRs in the present study.

Table 1. Preoperative Patient Characteristics

Variable	Prophylaxis (n = 22)	No prophylaxis (n = 21)	p
Male/Female	17/5	13/8	0.332
Age (years)	35.3 (27.9–43.7)	55.4 (42.0–60.2)	0.002
Primary indication for HTx
Dilated cardiomyopathy	13	13	0.604
Hypertrophic cardiomyopathy	4	3
Ischemic cardiomyopathy	0	2
Other	5	3
LVAD support pre-HTx	20	21
Type of LVAD support
Paracorporeal LVAD	4	4	0.610
Implantable LVAD	16	17
No-LVAD support pre-HTx	2	0
Donor/Recipient CMV serostatus
D+/R−	21	2	<0.001
D+/R+	0	15
D−/R+	0	3
D−/R−	1	1
Basiliximab induction	9	15	0.067
Start of VGCV administration after HTx (PODs)	11 (7–15)	37 (33–51)	<0.001

a) Data are expressed as median (interquartile range) for continuous values and number of subjects for categorical values. b) Categorical data were analyzed using the Fisher’s exact test. c) Continuous data were compared between the prophylaxis and no-prophylaxis groups using the Mann–Whitney U test. d) HTx, heart transplantation; LVAD, left ventricular assist device; CMV, cytomegalovirus; D + /R−, donor positive/recipient negative; D + /R+, donor positive/recipient positive; D − /R+, donor negative/recipient positive; D − /R−, donor negative/recipient negative; POD, postoperative day.

Table 2. Pre- and Post-operative Clinical and Laboratory Characteristics

Variable	Prophylaxis (n = 22)					No prophylaxis (n = 21)
	Pre-HTx	At start of VGCV administration	Six months Post-HTx	p		Pre-HTx	At start of VGCV administration	Six months Post-HTx	p
	Pre-HTx	At start of VGCV administration	Six months Post-HTx	Friedman test	At start of VGCV administration vs. six months post-HTx	Pre-HTx	At start of VGCV administration	Six months Post-HTx	Friedman test	At start of VGCV administration vs. six months post-HTx
Body weight (kg)	59.8 (52.6–65.6)	60.0 (50.1–67.0)	54.8 (49.6–63.9)	0.072	n.a.	64.0 (54.8–73.4)	58.4 (51.4–69.9)	59.0 (52.7–69.7)	<0.001	0.225
Body mass index (kg/m²)	22.1 (18.2–23.6)	21.9 (18.6–23.6)	20.1 (18.1–23.4)	0.072	n.a.	23.2 (21.3–25.1)	21.5 (19.7–23.3)	21.4 (20.1–24.0)	<0.001	0.225
WBC count (×10³/µL)	6.3 (5.8–7.4)	9.6 (8.3–10.8)	4.4 (3.3–5.1)	<0.001	<0.001	6.6 (5.0–7.8)	5.6* (4.1–6.9)	4.3 (3.3–6.5)	0.013	0.274
Neutrophil count (×10³/µL)	4.40 (3.83–5.34)	7.68 (6.86–8.95)	2.74 (2.02–3.44)	<0.001	<0.001	4.96 (3.81–5.71)	4.03* (2.96–5.84)	2.98 (1.96–4.63)	0.084	n.a.
Lymphocyte count (×10³/µL)	1.17 (0.82–1.49)	0.75 (0.51–0.98)	1.06 (0.66–1.46)	<0.001	<0.001	0.95 (0.59–1.23)	0.53 (0.37–0.86)	0.76 (0.55–1.27)	0.084	n.a.
Hemoglobin (g/dL)	12.5 (10.3–13.6)	11.2 (10.3–12.2)	12.2 (11.0–13.6)	0.040	0.051	11.9 (10.2–12.9)	10.8 (10.1–11.4)	11.8 (11.2–12.8)	0.007	0.002
Hematocrit (%)	37.8 (31.7–39.9)	33.5 (30.4–37.5)	37.9 (32.1–41.1)	0.003	0.035	35.9 (30.9–39.0)	32.1 (30.0–34.1)	35.7 (34.5–38.9)	0.018	0.003
C-reactive protein (mg/dL)	0.18 (0.10–0.59)	0.51 (0.35–0.85)	0.02 (0.01–0.11)	<0.001	<0.001	0.15 (0.05–1.04)	0.14 (0.07–0.57)	0.03 (0.01–0.06)	<0.001	<0.001
Albumin (g/dL)	4.4 (4.1–4.5)	3.5 (3.1–3.8)	4.4 (4.1–4.6)	<0.001	<0.001	4.3 (4.0–4.6)	3.7 (3.5–3.9)	4.4 (4.2–4.6)	<0.001	<0.001
Total protein (g/dL)	7.4 (6.6–7.8)	6.0 (5.7–6.3)	6.7 (6.4–6.9)	<0.001	0.001	6.9 (6.5–7.5)	6.1 (5.7–6.7)	6.6 (6.3–7.1)	<0.001	0.001
Total bilirubin (mg/dL)	0.6 (0.5–1.0)	0.9 (0.6–1.3)	0.5 (0.3–0.6)	<0.001	<0.001	0.7 (0.6–1.2)	0.6 (0.5–0.8)	0.5 (0.4–0.8)	0.011	0.984
Aspartate aminotransferase (IU/L)	32 (25–55)	26 (17–40)	22 (19–32)	0.009	0.170	32 (23–44)	20 (15–31)	21 (17–31)	0.037	0.601
Alanine aminotransferase (IU/L)	17 (13–24)	34 (20–70)	16 (13–37)	<0.001	<0.001	17 (14–27)	31 (17–40)	17 (12–36)	0.092	n.a.
eGFR (mL/min/1.73 m²)	80.6 (63.3–103.4)	93.0 (63.5–129.4)	84.1 (62.2–94.0)	0.016	0.003	59.8* (46.2–72.6)	72.2 (64.8–83.4)	55.2* (51.0–65.2)	0.001	<0.001
Scr (mg/dL)	0.78 (0.68–1.09)	0.68 (0.52–1.00)	0.80 (0.70–1.00)	0.022	0.020	1.02 (0.82–1.16)	0.78 (0.66–0.96)	0.98 (0.86–1.12)	0.002	0.003
Blood urea nitrogen (mg/dL)	12 (11–14)	13 (11–20)	14 (12–16)	0.030	0.852	15* (13–19)	15 (10–20)	18* (16–23)	0.116	n.a.

a) Data are expressed as median (interquartile ranges). b) Paired continuous data were compared using the Wilcoxon signed-rank test. The pairwise comparison was applied with Bonferroni correction as a post-hoc test only when Friedman’s test revealed overall significant differences (p < 0.05). c) Unpaired continuous data were compared between the prophylaxis and no prophylaxis groups using a Mann–Whitney U test with Bonferroni correction. * p < 0.01. d) HTx, heart transplantation; VGCV, valganciclovir; WBC, white blood cell; eGFR, estimated glomerular filtration rate; Scr, serum creatinine; n.a., not applied. e) The eGFR was calculated according to the equation 194 × Scr^−1.094 × Age^−0.287 (×0.739 if female).

At the start of VGCV administration, as shown in Table 2, the median WBC count and neutrophil count were significantly higher in the prophylaxis group than in the no prophylaxis group. There was no statistically significant difference in renal function based on the values of eGFR and Scr at the start of VGCV administration between the two groups (Table 2). Meanwhile, a comparison of the laboratory test values at the start of VGCV administration and six months post-HTx revealed a significant reduction in renal function in both groups (Table 2). None of six HTRs with eGFR values less than 60 mL/min/1.73 m² at the start of VGCV administration developed neutropenia. In contrast, 15.0% (3/20) and 23.5% (4/17) of HTRs in the prophylaxis and no prophylaxis groups, respectively, with eGFR values greater than 60 mL/min/1.73 m² at the start of VGCV administration presented eGFR values less than 60 mL/min/1.73 m² at the first onset of neutropenia after HTx.

Table 3 shows the daily maintenance doses of tacrolimus, MMF, and prednisolone at the start of VGCV administration and six months post-HTx; no significant difference in doses, trough level of tacrolimus, or AUC of mycophenolic acid at six months post-HTx were observed between the two groups, while the doses of tacrolimus, MMF, and prednisolone were adjusted appropriately according to the immunosuppression protocol and the patient’s conditions. The proportion of HTRs in whom MMF was switched to everolimus at six months post-HTx in the prophylaxis group was lower than that in the no prophylaxis group, but there was no statistically significant difference between the two groups (9.1 and 33.3%, respectively; p = 0.069). There was also no significant difference in age between HTRs in whom MMF was switched to everolimus and was not (median, 48.2 years [interquartile range (IQR) 38.1–59.6 years] and 40.8 years [29.3–54.0 years], respectively; p = 0.229). All HTRs received a trimethoprim-sulfamethoxazole combination.

Table 3. Drug Doses at the Start of VGCV Administration and Six Months Post-HTx

Variable	Prophylaxis (n = 22)			No prophylaxis (n = 21)
Variable	At start of VGCV administration	Six months Post-HTx	p	At start of VGCV administration	Six months Post-HTx	p
Tacrolimus dose (mg/d)	2.1 (1.2–3.0)	3.2 (1.9–5.0)	0.002	3.0 (1.9–3.8)	3.2 (2.1–5.3)	0.404
(mg/kg/d)	0.039 (0.022–0.048)	0.059 (0.026–0.086)	0.002	0.045 (0.034–0.068)	0.058 (0.036–0.089)	0.590
Tacrolimus trough level (ng/mL)	8.3 (5.1–9.5)	8.9 (7.3–11.2)	0.085	10.1* (8.7–12.3)	9.6 (6.6–10.7)	0.013
MMF dose (mg/d)	1625 (1250–2000)	750 (500–1000)	<0.001	1500 (500–2000)	500 (0–1250)	0.008
(mg/kg/d)	27.8 (22.1–31.5)	14.1 (8.7–19.5)	<0.001	21.6 (9.2–30.1)	11.3 (0.0–19.0)	0.007
Cumulative dose of MMF (g)	13.1 (10.0–20.3)	179.9 (144.4–236.9)	<0.001	56.0* (38.5–84.4)	224.5 (118.5–275.0)	<0.001
AUC of mycophenolic acid (µg·h/mL)	—	22.9 (11.3–32.9)	n.a.	—	20.1 (0.0–42.0)	n.a.
Prednisolone dose (mg/d)	32.5 (20.0–50.0)	7.5 (6.9–10.0)	<0.001	15.0* (12.5–17.5)	10.0 (7.5–10.0)	<0.001
VGCV dose (mg/d)
900	0	2	0.233	7	1	0.045
450	22	19		14	19
0	0	1		0	1
Everolimus use	0 (0.0%)	2 (9.1%)	0.488	0 (0.0%)	7 (33.3%)	0.009
Trimethoprim-sulfamethoxazole combination use	8 (36.4%)	21 (95.5%)	<0.001	20* (95.2%)	20 (95.2%)	1.000

a) Data are expressed as median (interquartile range) for continuous values and number of subjects and n (%) for categorical values. b) Categorical data were analyzed using the Fisher’s exact test. c) Paired continuous data at the start of VGCV administration and six months post-HTx were compared using the Wilcoxon signed-rank test. d) Unpaired continuous data were compared between the prophylaxis and no prophylaxis groups using a Mann–Whitney U test with Bonferroni correction. * p < 0.01. e) HTx, heart transplantation; VGCV, valganciclovir; MMF, mycophenolate mofetil; AUC, area under the curve; n.a., not applied. f) The values of trough level and AUC were taken from the latest available measurements prior to the onset of neutropenia. g) AUC monitoring of mycophenolic acid was not performed at the start of VGCV administration. h) In cases where methylprednisolone was used, the data were calculated assuming that 4 mg of methylprednisolone was equivalent to 5 mg of prednisone.¹⁶⁾

CMV Infection Status and VGCV Administration

In the prophylaxis group, seven out of 22 HTRs (31.8%) developed positive CMV antigenemia, and were switched from prophylaxis to preemptive treatment. In the no prophylaxis group, VGCV preemptive therapy started in 19 and 2 HTRs who developed positive CMV antigenemia and positive real-time PCR, respectively. The median time to detect positive antigenemia post-HTx in the prophylaxis group was significantly longer than that in the no prophylaxis group (83 [IQR 41–120] and 37 [IQR 32–57] PODs, respectively; p = 0.015).

In the prophylaxis group, VGCV administration for prophylaxis was initiated at a median of 11 (IQR: 7–15) PODs, which was significantly earlier than that for CMV therapy in the no prophylaxis group (37 [IQR 33–51] PODs) (Table 1). During the study period, the median duration of exposure to VGCV in the prophylaxis group was 357 (IQR: 339–362) days, and the value was significantly longer than that in the no prophylaxis group (321 [IQR 262–334] days; p < 0.001). For all 22 HTRs in the prophylaxis group, the initial VGCV dose was 450 mg/d (Table 3), and the dose was increased at least once in seven patients (31.8%) to a maximum of 900 mg/d for treatment of CMV infection during the study period. In the no prophylaxis group, the starting VGCV doses were 450 and 900 mg/d in 14 and 7 HTRs (67 and 33%), respectively, and 12 out of the 21 HTRs finally received a dose of 900 mg/d.

Incidence of Neutropenia/Leukopenia and Use of Concomitant Medications

As shown in Table 4, neutropenia occurred in 26 out of 43 HTRs during the study period. In the prophylaxis group, 77.3% (17/22) of HTRs experienced neutropenia, which was significantly higher than that in the no prophylaxis group (42.9% [9/21], p = 0.031). In the prophylaxis group, there was no statistically significant difference in the frequency of neutropenia between the switch from prophylaxis to preemptive treatment (6/7, 85.7%) and no switch (11/15, 73.3%). Six out of the 22 HTRs in the prophylaxis group presented a neutrophil count of less than 1000, whereas none in the no prophylaxis group did. There was no significant difference in time from the start of VGCV administration to the neutropenia onset between the two groups. Additionally, no significant difference in the duration of VGCV administration and the cumulative dose was observed between the two groups until the onset of neutropenia (Table 4). Leukopenia occurred in 15 and 11 HTRs (68.2 and 52.4%) in the prophylaxis and no prophylaxis groups, respectively; however, no statistically significant differences were detected between the two groups (p = 0.358).

Table 4. Clinical and Laboratory Characteristics and Daily Maintenance Dose of Immunosuppressive Drugs at the First Neutropenia after HTx

	Prophylaxis (n = 17)	No prophylaxis (n = 9)	p
Body weight (kg)	58.8 (49.4–63.0)	54.8 (53.8–61.9)	1.000
Body mass index (kg/m²)	20.4 (17.3–21.7)	21.3 (19.6–23.0)	0.178
WBC count (×10³/µL)	2.7 (2.4–3.2)	2.1 (2.0–2.5)	0.038
Neutrophil count (×10³/µL)	1.30 (1.15–1.43)	1.38 (1.19–1.43)	0.705
Lymphocyte count (×10³/µL)	0.95 (0.80–1.38)	0.65 (0.46–0.77)	0.006
Hemoglobin (g/dL)	12.3 (11.0–13.0)	11.3 (9.65–11.7)	0.138
Hematocrit (%)	38.9 (33.7–40.2)	33.6 (30.1–35.4)	0.063
C-reactive protein (mg/dL)	0.04 (0.02–0.28)	0.13 (0.02–0.69)	0.446
Albumin (g/dL)	4.3 (4.2–4.6)	3.9 (3.7–4.1)	0.007
Total protein (g/dL)	6.5 (6.0–6.8)	6.0 (5.9–6.6)	0.232
Total bilirubin (mg/dL)	0.5 (0.4–0.7)	0.6 (0.5–1.0)	0.048
Aspartate aminotransferase (IU/L)	20 (16–32)	21 (15–34)	0.850
Alanine aminotransferase (IU/L)	14 (11–35)	16 (13–25)	0.589
eGFR (mL/min/1.73 m²)	74.7 (63.9–99.2)	61.1 (53.2–89.0)	0.241
Scr (mg/dL)	0.86 (0.73–1.03)	0.80 (0.65–0.99)	0.647
Blood urea nitrogen (mg/dL)	14 (11–18)	11 (10–14)	0.048
Time up to the first neutropenia (PODs)	141 (104–250)	140 (70–199)	0.396
Tacrolimus dose (mg/d)	4.2 (2.2–6.1)	3.4 (2.0–6.2)	0.935
(mg/kg/d)	0.064 (0.050–0.117)	0.063 (0.037–0.101)	0.978
Tacrolimus trough level (ng/mL)	10.2 (9.0–11.4)	10.3 (9.2–11.3)	0.893
MMF dose (mg/d)	1000 (500–1250)	750 (500–1250)	0.321
(mg/kg/d)	16.0 (10.4–24.8)	11.6 (8.8–19.8)	0.269
Cumulative dose of MMF (g)	165.0 (124.4–282.3)	112.0 (62.8–157.5)	0.018
AUC of mycophenolic acid (µg·h/mL)	30.9 (21.5–39.6)	20.1 (7.1–44.3)	0.215
Prednisolone dose (mg/d)	10.0 (5.0–11.3)	10.0 (6.3–12.5)	0.579
Everolimus use	0 (0.0%)	1 (11.1%)	0.346
VGCV dose at onset (mg/d)
900	4	1	0.628
450	13	8
Duration of VGCV administration (days)	133 (94–224)	74 (11–164)	0.085
Cumulative dose of VGCV (g)	69.8 (45.9–113.9)	50.4 (5.0–73.8)	0.118

a) Data are expressed as median (interquartile range) for continuous values and number of subjects and n (%) for categorical values. b) Categorical data were analyzed using the Fisher’s exact test. c) Continuous data were compared between the prophylaxis and no-prophylaxis groups using the Mann–Whitney U test. d) HTx, heart transplantation; WBC, white blood cell; eGFR, estimated glomerular filtration rate; Scr, serum creatinine; POD, postoperative day; MMF, mycophenolate mofetil; AUC, area under the curve; VGCV, valganciclovir. e) The eGFR was calculated according to the equation 194 × Scr^−1.094 × Age^−0.287 (×0.739 if female).

No significant difference in doses was observed in the daily maintenance doses of tacrolimus, MMF or prednisolone at the onset of neutropenia between the two groups (Table 4). There was also no significant difference in the trough level of tacrolimus and the AUC of mycophenolic acid at six months post-HTx between the two groups, although the doses of tacrolimus, MMF, and prednisolone were adjusted appropriately according to the immunosuppression protocol and the patient’s conditions. On the onset of neutropenia post-HTx, the cumulative dose of MMF was significantly higher in the prophylaxis group than that in the no prophylaxis group (median, 165.0 g [IQR: 124.4–282.3 g] and 112.0 g [62.8–157.5 g], respectively) (p = 0.018).

DISCUSSION

VGCV prophylaxis in CMV high-risk HTRs has been performed worldwide. At our institute, VGCV has been administered prophylactically at half the therapeutic dosage for approximately one year post-HTx. The positive rate of CMV antigenemia was 31.8% during the study period in the VGCV prophylaxis group, and the median time to detect positive antigenemia was significantly longer than that in the no prophylaxis group (83 and 37 PODs, respectively; p = 0.015). Recently, Imaly et al. reported that CMV disease occurred in 22.4% of CMV high-risk HTRs, who received VGCV prophylaxis at the standard dose for three or six months, within one year post-HTx.¹¹⁾ Although it appeared that the results of the two studies were comparable, direct comparison was not performed because we did not collect information about clinical signs and symptoms to confirm CMV disease in the present study. The low-dose VGCV prophylaxis regimen could also contribute to the reduction of the incidence of CMV infection and disease in HTRs, although further studies are required to clarify this.

Meanwhile, the duration of exposure to VGCV in prophylaxis group tended to be longer than that in no prophylaxis group (median, 357 and 321 d, respectively; p < 0.001), and there is concern regarding CMV high-risk HTRs being exposed to an increased risk for neutropenia when compared with CMV intermediate/low-risk HTRs.⁹⁾ We retrospectively investigated whether the incidence of neutropenia differed between the prophylaxis and no prophylaxis groups. Based on our findings, the incidence rate of neutropenia in HTRs was significantly higher in the prophylaxis group than in the no prophylaxis group (77.3 and 42.9%, respectively) (Table 4). Accordingly, the low-dose VGCV prophylactic regimen could be associated with an increased risk of neutropenia, even if VGCV was administered at half the therapeutic dosage. Conversely, a trend of relatively longer VGCV administration and a higher cumulative dose was observed in the prophylaxis group, with no statistically significant differences observed when compared with those in the no prophylaxis group (Table 4). These findings suggest that the higher rate of neutropenia incidence in the prophylaxis group could not be comprehensively explained by the relatively longer VGCV administration.

Neutropenia after solid organ transplantation can be caused by factors other than VGCV.^17,18) Reportedly, CMV infection has been associated with neutropenia.^17,18) In the present study, as shown in Table 2, neutrophil and WBC counts at the start of VGCV administration were significantly lower in the no prophylaxis group than in the prophylaxis group. This is likely to be associated with CMV infection status at that time point. Meanwhile, as mentioned earlier, the rate of positive CMV antigenemia was lower in the prophylaxis group than in the no prophylaxis group during the study period; therefore, CMV infection was unlikely to largely contribute to the higher incidence of neutropenia in the prophylaxis group.

Drug-induced myelosuppression is another factor that influences the risk of neutropenia. Almost all concomitant medications used for immunosuppression and infection prophylaxis are likely to be associated with neutropenia, which has been well summarized in reviews concerning renal transplant recipients.^9,19) In the present study, the doses of tacrolimus, MMF, and prednisolone were carefully adjusted based on blood concentrations, laboratory tests, and/or clinical status, and their daily maintenance doses at the onset of neutropenia did not significantly differ between the prophylaxis and no-prophylaxis groups (Table 4). Furthermore, no significant difference was observed in the trough level of tacrolimus and the AUC of mycophenolic acid at that time between the two groups. Herein, a gradual dose reduction of MMF according to changes in the patient’s conditions, such as decreased neutrophil count, was performed after HTx, and its cumulative dose was approximately 1.5-fold higher in the prophylaxis group than in the no prophylaxis group. This finding may be associated with a higher likelihood of neutropenia.

In contrast, Pazdernik et al. reported that the addition of VGCV and trimethoprim-sulfamethoxazole combination to standard triple immunosuppressive therapy in HTRs was significantly associated with decreased leukocyte and lymphocyte counts, but not erythrocyte, thrombocyte, and neutrophil counts. In addition, the authors observed that the reduction or discontinuation of MMF led to the normalization of leukocyte and lymphocyte counts in some cases.²⁰⁾ MMF is strongly suspected to be associated with myelosuppression, although there have been reports that switching from tacrolimus to cyclosporine contributes to the recovery of the neutrophil count in solid organ transplant recipients, including HTRs who developed neutropenia.^21–24) Other immunosuppressive agents used post-HTx, such as everolimus,²⁵⁾ and basiliximab²⁶⁾ could also induce neutropenia and leucopenia. In the present study, no significant difference in the incidence of leukopenia was observed during the study period, suggesting that myelosuppression during VGCV treatment was more markedly reflected in the neutrophil count than in the WBC count. However, it remains unclear how MMF and tacrolimus alone or in combination with other concomitant drugs are associated with neutropenia in HTRs. Therefore, studies that compare the incidence of neutropenia in HTRs without VGCV administration are needed to appropriately evaluate these effects.

Following oral administration, VGCV is biotransformed into ganciclovir and is mainly excreted via glomerular filtration and tubular secretion. Previous studies have reported a prolonged elimination half-life and decreased clearance of ganciclovir in patients with renal failure.^27,28) The association between renal function and VGCV and ganciclovir-induced neutropenia and leukopenia has also been reported,^29–31) and lowered renal function can lead to higher systemic ganciclovir exposure, thereby increasing the incidence of neutropenia and leukopenia. In contrast, VGCV and tacrolimus may induce renal damage, and the prolonged administration of these drugs may trigger a deterioration of renal function.^32–34) In the present study, with or without VGCV prophylaxis, no significant association between eGFR values and the incidence of neutropenia was observed six months post-HTx, almost corresponding to the median time to the first neutropenia episode after HTx (data not shown). Therefore, it is considered that earlier exposure to low-dose prophylactic VGCV for CMV in HTRs did not increase the risk of renal damage during immunosuppressive therapy after HTx when compared with the risk in the no prophylaxis group.

Moreover, other factors increase the risk of developing neutropenia. For example, baseline neutrophil and WBC counts at treatment initiation^17,31,35,36) and body mass index^29,37) reportedly influence VGCV and ganciclovir-induced neutropenia and leukopenia. In the present study, the prophylactic administration of VGCV was not applicable for HTRs who had decreased renal function and lower counts of white blood cells and neutrophils, which are confounding factors that may have an influence on the occurrence of neutropenia. Decreased renal function may cause increased exposure to ganciclovir after VGCV administration, thereby increasing the risk of neutropenia, and lower counts of white blood cells and neutrophils are also risk factors for neutropenia during immunosuppressive therapy after HTx. This could explain the higher neutrophil and WBC counts at the start of VGCV administration in the prophylaxis group, comparing to that in the no prophylaxis group. It is considered that the HTRs in whom VGCV prophylaxis was avoided or who received preemptive therapy at half the standard therapeutic dose would have a relatively higher risk of neutropenia, but the present result was the opposite (Table 4). Meanwhile, in the prophylaxis group, VGCV administration for prophylaxis was initiated significantly earlier than that for treatment in the no prophylaxis group, and higher exposure of VGCV into the body was expected. In such patients, the risk of neutropenia increased, but the dosing period and cumulative dose of VGCV were not associated with the incidence of neutropenia in the present study (Table 4).

In conclusion, the results of the present study demonstrate that HTRs receiving VGCV prophylaxis presented a greater risk of developing neutropenia than those not receiving prophylaxis, and this risk was not associated with earlier exposure to low-dose VGCV. In addition, the prophylactic administration of low-dose VGCV did not increase the risk of renal damage during immunosuppressive therapy after HTx. The incidence of neutropenia in the prophylactic group may be largely affected by MMF administration. Although this study was limited by its retrospective design, single-center implementation, and small sample size, the low-dose VGCV regimen is effective for CMV prophylaxis in CMV high-risk HTRs. Simultaneously, careful dose adjustment of immunosuppressive drugs is recommended.

Acknowledgments

We gratefully acknowledge the patients, physicians, nurses, pharmacists, and all medical staff at the National Cardiovascular Center who contributed to this study.

Conflict of Interest

The authors declare no conflict of interest.

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