Drug-resistant human cytomegalovirus infection in children after allogeneic stem cell transplantation may have different clinical outcomes

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.

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.

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).

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).

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

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.

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 

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.

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.