Three seropositive pediatric recipients of allogeneic stem cell transplantation out of a group of 42 patients receiving T-cell–depleted, unrelated transplants and 37 patients receiving T-cell–depleted, haploidentical transplants were monitored longitudinally for human cytomegalovirus (HCMV) infection and the emergence of antiviral drug resistance. Early in the posttransplant course, all 3 patients developed HCMV mutations conferring drug resistance to ganciclovir. One child additionally developed multidrug resistance to foscarnet and cidofovir, with mutations in the viral phosphotransferase gene (UL97) and the DNA-polymerase gene (UL54) being found. These data show that resistant HCMV infection does not necessarily correlate with a severe clinical outcome. The early detection of genotypic resistance up to 129 days before the emergence of phenotypic resistance and the dissociation of resistance patterns among different body sites emphasize the importance of genotypic analyses of different DNA specimens for an efficient antiviral therapy. T-cell–depleted children having transplantation might be at an increased risk for the development of drug resistance.

Introduction

Despite the availability of effective antiviral drugs, human cytomegalovirus (HCMV) is still a serious infectious complication after allogeneic bone marrow transplantation (BMT).1 However, the limited number of reports concerning drug-resistant HCMV infection might suggest that this problem occurs very infrequently in this clinical setting.2 

In contrast, ganciclovir (GCV) resistance during antiviral treatment of HCMV retinitis in adults with acquired immunodeficiency syndrome (AIDS) has been shown to appear frequently after more than 3 months of therapy,3,4 and resistance to foscarnet (PFA)5 and cidofovir (CDV)6 has been described after even longer durations of therapy.

In the BMT setting, no reports exist about CDV resistance and only very few about GCV and PFA resistance.2 Nevertheless, there are indications that children with combined immune deficiencies after T-cell–depleted BMT are at a high risk of developing early GCV resistance.7 

Of 79 children who underwent allogeneic peripheral blood stem cell transplantation (PBSCT) at our institution, 3 subjects are presented who had a striking course of HCMV infection and development of resistance to various antiviral drugs. Because culture-based resistance screening is not a practical method for routine use,8,9therapy was monitored by molecular analysis of sequential specimens from various body sites for typical mutations conferring GCV resistance.

Study design

Transplantation procedure

The 3 patients belong to a sequence of pediatric patients of whom 42 (having transplantation between October 1995 and March 2000) received positively selected, highly purified CD34+peripheral blood stem cells from unrelated donors and 37 (having transplantation between August 1995 and March 2000) received these cells from familiarly mismatched donors. The CliniMACS (Miltenyi Biotec, Bergisch Gladbach, Germany) CD34+selection device was used for positive selection.10 The mode of transplantation is detailed in Table1. Because of the low T-cell number, no prophylaxis for graft-versus-host disease was necessary (Table 1). After transplantation, antiviral therapy was administered with acyclovir until day +100, Pneumocystis cariniiprophylaxis with trimethoprim-sulfamethoxazole until day +200, and antimycotic and antibiotic prophylaxis with fluconazole or itraconazole and colistin until day +100.

Table 1.

Clinical characteristics of recipients of highly purified peripheral blood stem cells

Patient no. Age Date at first diagnosis Underlying disease before PBSCT and primary treatment Conditioning regimen Donor HLA-A, -B, -DRβ1 match CD34+ cell
infusion ×
106cells/kg 
CD3+ cells × 103 cells/kg Donor leukocyte infusion after PBSCT* Disease after PBSCT 
Innate Innate T-cell defect, autoimmune hemolytic anemia
IV IG substitution 
Busulfan 4 × 4 mg/kg Mother; 3/6 antigens matched 50 18.7 No infusion Chronic
restrictive
lung disease  
Thiotepa 2 × 5 mg/kg  
Cyclophosphamide 4 × 50 mg/kg 
 ALG 3 × 20 mg/kg  
11 6 months before PBSCT AML M4 CR2; del(11)(q23); initial hyperleukocytosis/CNS infiltration
*AML—BFM93” regimen  
Busulfan 4 × 4 mg/kg
Cyclophosphamide 2 × 60 mg/kg
Melphalan 1 × 140 mg/m2
ATG 3 × 10 mg/kg  
Unrelated; 6/6 antigens matched 20.5
14.1 
Day +112
after PBSCT 
Gut and skin GvHD1-153;  autoimmune  hemolytic  anemia;  CMV  encephalitis  
5 months before PBSCT AML M2 CR1; t(8;21); initial infiltration of CNS, orbitae, pleura, lymph nodes
“AML—BFM93” regimen 
Busulfan 4 × 4 mg/kg Mother; 5/6 antigens matched 13.4 13.6 Days +72 and +100 after PBSCT Autoimmune hemolytic anemia  
Cyclophosphamide 2 × 60 mg/kg  
Thiotepa 2 × 5 mg/kg  
ATG 3 × 10 mg/kg  
 OKT3 + methylprednisolone for graft-rejection prophylaxis 
Patient no. Age Date at first diagnosis Underlying disease before PBSCT and primary treatment Conditioning regimen Donor HLA-A, -B, -DRβ1 match CD34+ cell
infusion ×
106cells/kg 
CD3+ cells × 103 cells/kg Donor leukocyte infusion after PBSCT* Disease after PBSCT 
Innate Innate T-cell defect, autoimmune hemolytic anemia
IV IG substitution 
Busulfan 4 × 4 mg/kg Mother; 3/6 antigens matched 50 18.7 No infusion Chronic
restrictive
lung disease  
Thiotepa 2 × 5 mg/kg  
Cyclophosphamide 4 × 50 mg/kg 
 ALG 3 × 20 mg/kg  
11 6 months before PBSCT AML M4 CR2; del(11)(q23); initial hyperleukocytosis/CNS infiltration
*AML—BFM93” regimen  
Busulfan 4 × 4 mg/kg
Cyclophosphamide 2 × 60 mg/kg
Melphalan 1 × 140 mg/m2
ATG 3 × 10 mg/kg  
Unrelated; 6/6 antigens matched 20.5
14.1 
Day +112
after PBSCT 
Gut and skin GvHD1-153;  autoimmune  hemolytic  anemia;  CMV  encephalitis  
5 months before PBSCT AML M2 CR1; t(8;21); initial infiltration of CNS, orbitae, pleura, lymph nodes
“AML—BFM93” regimen 
Busulfan 4 × 4 mg/kg Mother; 5/6 antigens matched 13.4 13.6 Days +72 and +100 after PBSCT Autoimmune hemolytic anemia  
Cyclophosphamide 2 × 60 mg/kg  
Thiotepa 2 × 5 mg/kg  
ATG 3 × 10 mg/kg  
 OKT3 + methylprednisolone for graft-rejection prophylaxis 

PBSCT indicates peripheral blood stem cell transplantation; IV, intravenous; IG, immunoglobulin; ALG, antilymphocyte globulin; AML, acute myeloblastic leukemia; CR2, second complete remission; CNS, central nervous system; ATG, antithymocyte globulin; GvHD, graft-versus-host disease; CMV, cytomegalovirus; CRI, first complete remission.

*

25 000 CD3+ cells/kg.

Patient underwent splenectomy.

On day +244, additional CD34+ cell infusion.

F1-153

Oral prednisone therapy since day +120 after PBSCT.

Monitoring of HCMV infection and development of resistance

All patients, except seronegative recipients of seronegative transplants, were screened weekly for HCMV infection by HCMV polymerase chain reaction (PCR) from plasma and leukocytes until day +100.11 Antiviral therapy with GCV was initiated (initiation, 5 mg/kg twice a day; maintenance, 5 mg/kg per day) when 2 consecutive PCR results from leukocytes were positive. GCV-resistant infection was alternatively treated with PFA (3 × 40 / 3 × 50 mg/kg per day) or CDV (5 mg/kg per week).

Especially in the 3 patients presented here, in the case of a high viral load during antiviral therapy and because of clinical suspicion, genotypic8 and phenotypic12 resistance screening was performed from all specimens available. Semiquantitative limiting-dilution nested PCR from native plasma was performed, as described,13 whenever HCMV plasma DNAemia was detected.

Sequence analyses of codons 439 to 696 of the UL97 gene and codons 365 to 1084 of the UL54 gene (viral polymerase) were performed by using BigDye-Terminator chemistry on an ABI 310 machine (PE Applied Biosystems, Weiterstadt, Germany).

Results and discussion

In the 3 pediatric patients, mutations conferring GCV resistance emerged early, after 25, 53, and 30 days of GCV therapy after PBSCT. Because patient 1 received GCV before PBSCT, he had had GCV therapy for 93 days when genotypic resistance was first detected.

The sudden emergence of mutations in blood specimens was observed within 12, 29, and 9 days after the last negative genotypic test result. After genotypic resistance in the 3 children had been detected for the first time, viral isolates were obtained 10, 124, and 129 days later and could be assayed retrospectively for phenotypic drug resistance (Table 2).

Table 2.

Longitudinal HCMV monitoring and the relation between antiviral therapy and development of mutations in UL97 and UL54

Patient no. Specimens (days after PBSCT) PCR Viral load* Therapy (cumulative days) IC50(μmol/L) UL97
mutations 
UL54
mutations 
GCV PFA CDV 
Leukocyte −183  GCV 0, PFA 0    Negative  
 Serum −67  GCV 29, PFA 0    Negative  
 Plasma −18 −  GCV 68, PFA 0      
 Plasma −12  GCV 68, PFA 0    Negative  
 Leukocyte +3  GCV 68, PFA 13    Negative  
 Plasma +17 ++ GCV 68, PFA 27    Negative  
 Serum +26 ++ GCV 75, PFA 30    Negative  
 Leukocyte +32 ++ GCV 81, PFA 30 1.4   Negative  
 Serum +44 +++ GCV 93, PFA 30    C603W  
 Urine +54 +++ GCV 103, PFA 30 13 100  C603W  
 Urine +149  GCV 103, PFA 125 9.8 77  C603W; A594V  
Serum −14 −        
 Throat −6      Negative  
 Leukocyte −1 GCV 0, PFA 0    Negative  
 Leukocyte +6 GCV 0, PFA 6    Negative  
 Leukocyte +42  GCV 24, PFA 18    Negative  
 Leukocyte +71  GCV 53, PFA 18    A591V  
 Leukocyte +100 ++ GCV 81, PFA 19    A591V Wild type  
 Leukocyte +167 GCV 141, PFA 56    A591V; C603W; M460I D515E; L516M; I521T 
 Leukocyte +195 ++++ GCV 156, PFA 56, CDV 14    A591V  
 Urine +195 ++++ GCV 156, PFA 56, CDV 14 29 388 A591V D515E; L516M; I521T; L802M 
 Leukocyte +251 ++ GCV 174, PFA 56, CDV 35    A591V  
 Urine +251 ++ GCV 174, PFA 56, CDV 35 29 551 A591V; D515E; L516M; I521T; L802M  
 Leukocyte +258  GCV 174, PFA 62, CDV 35    A591V  
 CSF +263  GCV 179, PFA 67, CDV 35    P521L D515E; L516M; I521T 
         A885T; P887S; N898D 
Plasma −3 −  GCV 0      
 Serum +30 −  GCV 0      
 Leukocyte +65  GCV 0    Negative  
 Leukocyte +71  GCV 6    Negative  
 Plasma +86 ++ GCV 21    Negative  
 Leukocyte +95  GCV 30    L595S  
 Plasma +95 −  GCV 30      
 Serum +106  GCV 41    L595S  
 Leukocyte +129  GCV 48, CDV 14    L595S  
 Leukocyte +169  GCV 48, CDV 35    Negative  
 Plasma +224 −  GCV 48, CDV 35      
 Throat +224  GCV 48, CDV 35 240 0.3 L595S  
Patient no. Specimens (days after PBSCT) PCR Viral load* Therapy (cumulative days) IC50(μmol/L) UL97
mutations 
UL54
mutations 
GCV PFA CDV 
Leukocyte −183  GCV 0, PFA 0    Negative  
 Serum −67  GCV 29, PFA 0    Negative  
 Plasma −18 −  GCV 68, PFA 0      
 Plasma −12  GCV 68, PFA 0    Negative  
 Leukocyte +3  GCV 68, PFA 13    Negative  
 Plasma +17 ++ GCV 68, PFA 27    Negative  
 Serum +26 ++ GCV 75, PFA 30    Negative  
 Leukocyte +32 ++ GCV 81, PFA 30 1.4   Negative  
 Serum +44 +++ GCV 93, PFA 30    C603W  
 Urine +54 +++ GCV 103, PFA 30 13 100  C603W  
 Urine +149  GCV 103, PFA 125 9.8 77  C603W; A594V  
Serum −14 −        
 Throat −6      Negative  
 Leukocyte −1 GCV 0, PFA 0    Negative  
 Leukocyte +6 GCV 0, PFA 6    Negative  
 Leukocyte +42  GCV 24, PFA 18    Negative  
 Leukocyte +71  GCV 53, PFA 18    A591V  
 Leukocyte +100 ++ GCV 81, PFA 19    A591V Wild type  
 Leukocyte +167 GCV 141, PFA 56    A591V; C603W; M460I D515E; L516M; I521T 
 Leukocyte +195 ++++ GCV 156, PFA 56, CDV 14    A591V  
 Urine +195 ++++ GCV 156, PFA 56, CDV 14 29 388 A591V D515E; L516M; I521T; L802M 
 Leukocyte +251 ++ GCV 174, PFA 56, CDV 35    A591V  
 Urine +251 ++ GCV 174, PFA 56, CDV 35 29 551 A591V; D515E; L516M; I521T; L802M  
 Leukocyte +258  GCV 174, PFA 62, CDV 35    A591V  
 CSF +263  GCV 179, PFA 67, CDV 35    P521L D515E; L516M; I521T 
         A885T; P887S; N898D 
Plasma −3 −  GCV 0      
 Serum +30 −  GCV 0      
 Leukocyte +65  GCV 0    Negative  
 Leukocyte +71  GCV 6    Negative  
 Plasma +86 ++ GCV 21    Negative  
 Leukocyte +95  GCV 30    L595S  
 Plasma +95 −  GCV 30      
 Serum +106  GCV 41    L595S  
 Leukocyte +129  GCV 48, CDV 14    L595S  
 Leukocyte +169  GCV 48, CDV 35    Negative  
 Plasma +224 −  GCV 48, CDV 35      
 Throat +224  GCV 48, CDV 35 240 0.3 L595S  

HCMV indicates human cytomegalovirus; PBSCT, peripheral blood stem cell transplantation; PCR, polymerase chain reaction; IC50, 50% inhibitory concentration; GCV, ganciclovir; PFA, foscarnet; CDV, cidofovir; CSF, cerebrospinal fluid.

*

Limiting-dilution nested PCR from native plasma/serum; + indicates undiluted (corresponding to 103 genome equivalents/mL), ++ indicates dilution of 10; +++ indicates dilution of 100.

Phenotypic resistance testing of viral isolates: cutoff values for resistance: GCV, IC50 > 6 μmol/L; CDV, IC50 > 4 μmol/L; PFA, IC50 > 400 μmol/L.

Mutated codon is located adjacent to known codons involved in drug resistance.

Patients

Clinical characteristics before and after transplantation are shown in Table 1.

Case 1: fatal clinical outcome in a patient with chronic restrictive lung disease.

The patient, as described elsewhere,8 was treated with GCV before PBSCT because of pneumonitis following recurrent HCMV infections. After transplantation, the development of resistance coincided with a 10-fold increase in the viral DNA titer in plasma; an increase is often suggested as a marker for resistance development.14 An immediate switch in therapy from GCV to PFA after the detection of genotypic resistance resulted in a prompt decrease in the viral load. However, HCMV could not be eliminated from the blood. Remarkably, an additional resistance mutation for GCV was found uniquely in a urine isolate (Table 2).Thereafter, the patient's pulmonary situation deteriorated and he died at day +170.

Case 2: fatal clinical outcome in a patient with multidrug resistance and HCMV encephalitis.

This patient had transplantation with a disseminated HCMV infection during the conditioning procedure. Upon observation of GCV resistance, an emerging HCMV encephalitis on day +100 was treated with PFA. After combined therapy with GCV and PFA, a renewed increase in the viral load and the detection of further mutations conferring GCV resistance on day +167 required the administration of CDV. Nevertheless, the HCMV-associated encephalitis worsened, and a high DNA titer in the cerebrospinal fluid (CSF) was measured. In this serious situation, GCV was given experimentally by intrathecal infusion (200 μg and 400 μg) because no UL97 mutations were found in the CSF by the restriction assay. However, antiviral therapy failed and the patient died on day +287. Sequencing analyses of the CSF sample revealed a new UL97 mutation restricted to this body site (Table 2). This finding demonstrates the difficulty of establishing effective antiviral therapy in the presence of different resistance mutations in various body sites. Interestingly, the patient developed multidrug resistance after antiviral therapy of 7 months—13 months earlier than in a reported case of multidrug resistance in an AIDS patient.15 

Case 3: patient with an asymptomatic HCMV infection and GCV resistance.

Initial HCMV leukoDNAemia manifest on day +65 was treated with GCV. When genotypic resistance was detected, the patient was negative for HCMV in the plasma by PCR. Therapy was switched to CDV for 5 weeks until day +150. When HCMV plasmaDNAemia disappeared, therapy was stopped. Since day +169, the mutation conferring resistance was no longer detectable in sporadically HCMV PCR–positive leukocyte specimens. The first viral isolate from this patient on day +224 showed phenotypic and genotypic GCV resistance. However, the patient never developed HCMV-related disease.

The persistence of a resistant strain in a throat isolate in patient 3 at day +224, after therapy had been stopped 72 days previously, suggests different body sites from which a mutant virus may arise if antiviral therapy is reinitiated. As a consequence, genotypic resistance screening restricted to blood specimens bears the risk of missing existing mutant strains.

Review of the literature

Until now, only one case of both genotypic and phenotypic antiviral drug resistance in the adult BMT setting has been documented.16 In contrast, Wolf et al7 showed the early development of resistance in 4 of 6 children after T-cell–depleted BMT. Our data strengthen the consideration that children having T-cell–depleted BMT or PBSCT may have an increased risk of resistance development. In contrast to the patients described by Wolf et al,7 our patients did not suffer from severe immunologic disorders. Additionally, our patients received highly purified stem cells, whereas their patients underwent T-cell–depleted BMT. Therefore, neither the mode of transplantation nor the general immune situation may solely explain the early emergence of drug resistance.

Rigorous T-cell depletion can increase the risk for opportunistic infections.17 As a consequence, the immune recovery, especially the recovery of specific immune cells, may play an important role in defending against these infections. In the case of an HCMV infection, the lack of a specific immune response could allow high viral replication rates during antiviral therapy. Thus, studies in pediatric recipients of T-cell–depleted transplants are needed to determine the relation between antiviral drug resistance in the context of HCMV-specific cellular immune reconstitution and immune cell function.

The publication costs of this article were defrayed in part by page charge payment. Therefore, and solely to indicate this fact, this article is hereby marked “advertisement” in accordance with 18 U.S.C. section 1734.

References

References
1
Hebart
H
Kanz
L
Jahn
G
Einsele
H
Management of cytomegalovirus infection after solid-organ or stem-cell transplantation. Current guidelines and future prospects.
Drugs.
55
1998
59
72
2
Erice
A
Resistance of human cytomegalovirus to antiviral drugs.
Clin Microbiol Rev.
12
1999
286
297
3
Jabs
DA
Enger
C
Dunn
JP
Forman
M
Cytomegalovirus retinitis and viral resistance: ganciclovir resistance. CMV Retinitis and Viral Resistance Study Group.
J Infect Dis.
177
1998
770
773
4
Gilbert
C
Handfield
J
Toma
E
Lalonde
R
Bergeron
MG
Boivin
G
Emergence and prevalence of cytomegalovirus UL97 mutations associated with ganciclovir resistance in AIDS patients.
AIDS.
12
1998
125
129
5
AIDS Clinical Trials Group (ACTG)
Studies of ocular complications of AIDS foscarnet-ganciclovir cytomegalovirus retinitis trial: 1. Rationale, design, and methods.
Control Clin Trials.
13
1992
22
39
6
Cherrington
JM
Fuller
MD
Lamy
PD
et al. 
In vitro antiviral susceptibilities of isolates from cytomegalovirus retinitis patients receiving first- or second-line cidofovir therapy: relationship to clinical outcome.
J Infect Dis.
178
1998
1821
1825
7
Wolf
DG
Yaniv
I
Honigman
A
Kassis
I
Schonfeld
T
Ashkenazi
S
Early emergence of ganciclovir-resistant human cytomegalovirus strains in children with primary combined immunodeficiency.
J Infect Dis.
178
1998
535
538
8
Prix
L
Hamprecht
K
Holzhüter
B
Handgretinger
R
Klingebiel
T
Jahn
G
Comprehensive restriction analysis of the UL97 region allows early detection of ganciclovir-resistant human cytomegalovirus in an immunocompromised child.
J Infect Dis.
180
1999
491
495
9
Baldanti
F
Simoncini
L
Sarasini
A
et al. 
Ganciclovir resistance as a result of oral ganciclovir in a heart transplant recipient with multiple human cytomegalovirus strains in blood.
Transplantation.
66
1998
324
329
10
Handgretinger
R
Schumm
M
Lang
P
et al. 
Transplantation of megadoses of purified haploidentical stem cells.
Ann N Y Acad Sci.
872
1999
351
361
11
Hamprecht
K
Steinmassl
M
Einsele
H
Jahn
G
Discordant detection of human cytomegalovirus DNA from peripheral blood mononuclear cells, granulocytes and plasma: correlation to viremia and HCMV infection.
J Clin Virol.
11
1998
125
136
12
Prix
L
Maierl
J
Jahn
G
Hamprecht
K
A simplified assay for screening of drug resistance of cell-associated cytomegalovirus strains.
J Clin Virol.
11
1998
29
37
13
Hamprecht
K
Mikeler
E
Jahn
G
Semi-quantitative detection of cytomegalovirus DNA from native serum and plasma by nested PCR: influence of DNA extraction procedures.
J Virol Methods.
69
1997
125
135
14
Boivin
G
Gilbert
C
Morissette
M
Handfield
J
Goyette
N
Bergeron
MG
A case of ganciclovir-resistant cytomegalovirus (CMV) retinitis in a patient with AIDS: longitudinal molecular analysis of the CMV viral load and viral mutations in blood compartments.
AIDS.
11
1997
867
873
15
Chou
S
Marousek
G
Guentzel
S
et al. 
Evolution of mutations conferring multidrug resistance during prophylaxis and therapy for cytomegalovirus disease.
J Infect Dis.
176
1997
786
789
16
Erice
A
Borrell
N
Li
W
Miller
WJ
Balfour
HH
Jr
Ganciclovir susceptibilities and analysis of UL97 region in cytomegalovirus (CMV) isolates from bone marrow recipients with CMV disease after antiviral prophylaxis.
J Infect Dis.
178
1998
531
534
17
Small
TN
Papadopoulos
EB
Boulad
F
et al. 
Comparison of immune reconstitution after unrelated and related T-cell-depleted bone marrow transplantation: effect of patient age and donor leukocyte infusions.
Blood.
93
1999
467
480

Author notes

Klaus Hamprecht, Med. Virologie und Epidemiologie der Viruskrankheiten, Universitätsklinik Tübingen, Calwerstr. 7/6, D-72076 Tübingen, Germany; e-mail: kshampre@med.uni-tuebingen.de.