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Related Collections Brief Reports Clinical Trials and Observations Social Bookmarking                   What's this?  Previous Article  |  Table of Contents  |  Next Article  Blood, 1 November 2000, Vol. 96, No. 9, pp. 3286-3289 BRIEF REPORT Drug-resistant human cytomegalovirus infection in children after allogeneic stem cell transplantation may have different clinical outcomes Tobias Eckle, Lothar Prix, Gerhard Jahn, Thomas Klingebiel, Rupert Handgretinger, Barbara Selle, and Klaus Hamprecht From the Department of Medical Virology and Epidemiology of Viral Diseases, University Hospital Tübingen, Tübingen, Germany; the Department of Pediatric Oncology and Hematology, University of Tübingen, Germany; and the Department of Pediatric Oncology and Hematology, University of Heidelberg, Germany.     Abstract Top Abstract Introduction Study design Results and discussion References 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. (Blood. 2000;96:3286-3289) © 2000 by The American Society of Hematology.     Introduction Top Abstract Introduction Study design Results and discussion References 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,8,9 therapy was monitored by molecular analysis of sequential specimens from various body sites for typical mutations conferring GCV resistance.     Study design Top Abstract Introduction Study design Results and discussion References 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 Table 1. 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 carinii prophylaxis with trimethoprim-sulfamethoxazole until day +200, and antimycotic and antibiotic prophylaxis with fluconazole or itraconazole and colistin until day +100.                                View this table: [in this window] [in a new window]   Table 1. Clinical characteristics of recipients of highly purified peripheral blood stem cells 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 Top Abstract Introduction Study design Results and discussion References 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).                                View this table: [in this window] [in a new window]   Table 2. Longitudinal HCMV monitoring and the relation between antiviral therapy and development of mutations in UL97 and UL54 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 months13 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.     Footnotes Submitted October 22, 1999; accepted July 3, 2000. 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. Reprints: Klaus Hamprecht, Med. Virologie und Epidemiologie der Viruskrankheiten, Universitätsklinik Tübingen, Calwerstr. 7/6, D-72076 Tübingen, Germany; e-mail: kshampre{at}med.uni-tuebingen.de.     References Top Abstract Introduction Study design Results and discussion 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. 1998;55:59-72[Medline] [Order article via Infotrieve]. 2. Erice A. Resistance of human cytomegalovirus to antiviral drugs. Clin Microbiol Rev. 1999;12:286-297[Abstract/Free Full Text]. 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. 1998;177:770-773[Medline] [Order article via Infotrieve]. 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. 1998;12:125-129[Medline] [Order article via Infotrieve]. 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. 1992;13:22-39[Medline] [Order article via Infotrieve]. 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. 1998;178:1821-1825[Medline] [Order article via Infotrieve]. 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. 1998;178:535-538[Medline] [Order article via Infotrieve]. 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. 1999;180:491-495[Medline] [Order article via Infotrieve]. 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. 1998;66:324-329[Medline] [Order article via Infotrieve]. 10. Handgretinger R, Schumm M, Lang P, et al. Transplantation of megadoses of purified haploidentical stem cells. Ann N Y Acad Sci. 1999;872:351-361[Medline] [Order article via Infotrieve]. 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. 1998;11:125-136[Medline] [Order article via Infotrieve]. 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. 1998;11:29-37[Medline] [Order article via Infotrieve]. 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. 1997;69:125-135[Medline] [Order article via Infotrieve]. 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. 1997;11:867-873[Medline] [Order article via Infotrieve]. 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. 1997;176:786-789[Medline] [Order article via Infotrieve]. 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. 1998;178:531-534[Medline] [Order article via Infotrieve]. 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. 1999;93:467-480[Abstract/Free Full Text]. © 2000 by The American Society of Hematology.   CiteULike    Connotea    Del.icio.us    Digg    Reddit    Technorati    What's this? 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