DEVELOPMENT OF GANCICLOVIR RESISTANCE DURING TREATMENT OF PRIMARY CYTOMEGALOVIRUS INFECTION AFTER LIVER TRANSPLANTATION1

Abstract

Despite recent advances in diagnosis and treatment, cytomegalovirus (CMV*) continues to be a common cause of infection and disease in solid organ transplant recipients. Risk factor analyses have provided important clues to identify those patients at high risk for symptomatic CMV infection (1). Preemptive therapy of CMV infection before the onset of overt disease is now a common practice, driven by the considerable morbidity and costs resulting from CMV disease (2). A recent study suggested that CMV could be almost completely eliminated as a significant pathogen in liver transplant patients by the posttransplant administration of ganciclovir, with only 1 of 124 patients (0.8%) developing symptomatic CMV disease (3). One concern about the frequent and early use of anti-CMV therapy in the posttransplant period is the possibility of selecting for drug-resistant viral strains. Ganciclovir-resistant CMV has been isolated from immunocompromised patients, usually with acquired immune deficiency syndrome or hematologic malignancies and after prolonged therapy (4, 5), but so far in vitro ganciclovir resistance has not been reported after ganciclovir therapy in solid organ recipients, even those in whom this therapy was unsuccessful in suppressing viremia (6). It has therefore been suggested that CMV drug resistance is unlikely to be a problem in the setting of organ transplantation. This report describes the clinical course of a liver transplant recipient who developed ganciclovir-resistant CMV after exposure to the drug in the posttransplant period. The emergence of antiviral resistance was rapidly detected by screening for viral resistance mutations, and this guided the selection of alternative therapy for the infection. A 17-year-old, CMV-seronegative male underwent orthotopic liver transplantation for end-stage liver disease secondary to Alagille's syndrome. The initial immunosuppression consisted of cyclosporine, azathioprine, prednisone, and antithymocyte globulin (15 mg/kg). Because the donor was CMV seropositive (D+/R-), the patient was treated with intravenous CMV immunoglobulin (Cytogam) at a dose of 150 mg/kg during the first 72 hr after liver transplantation and subsequently with 100 mg/kg every 2 weeks for six doses. In addition, the patient received intravenous ganciclovir 5 mg/kg every 12 hr for 10 days, followed by oral acyclovir 800 mg four times daily. On day 27, the patient developed biopsy-confirmed acute cellular rejection which did not respond to high-dose methylprednisolone (1 g every other day for 3 days). Intravenous ganciclovir (5 mg/kg) was administered during a 14-day course of OKT3 monoclonal antibody therapy (5 mg daily; Orthoclone OKT3, Ortho Pharmaceutical Corp.). Despite attempted preemptive therapy with CMV immunoglobulin, ganciclovir, and acyclovir, the patient developed primary CMV infection and disease manifested by leukopenia, fatigue, fever, and positive buffy coat CMV culture (Fig. 1). The CMV isolate was screened for UL97 ganciclovir resistance mutations (7) and none were found. Intravenous ganciclovir treatment was reinstituted and CMV immunoglobulin was given for 2 weeks (150 mg/kg every other day for six doses), with clearing of CMV viremia. The patient then developed another episode of allograft rejection which was treated with high-dose methylprednisolone and subsequently with tacrolimus (FK506 [Prograf]). Two weeks later, he presented with headache, fever, fatigue, and epigastric abdominal pain. CMV buffy coat cultures were again positive. A liver biopsy was positive for CMV by culture. Treatment with intravenous ganciclovir and CMV immunoglobulin was resumed, but 2 weeks later endoscopy revealed ulcerated erythematous gastric lesions which on biopsy stained positive for CMV antigen. By this time, results of UL97 mutation screening of the most recent CMV isolates were positive for ganciclovir resistance mutations V594 and S595 (7). Both mutations were present in each isolate, although plaque purification of the virus enabled the recovery of CMV strains carrying only one or the other of the mutations. This showed that each resistance mutation occurred in a separate viral genome. Cell culture (plaque reduction) susceptibility testing of one of the isolates later confirmed the expected ganciclovir resistance, with a drug concentration of 22 μM required to inhibit plaque formation by 50%, as compared with 6 μM for the control laboratory strain AD169 when tested simultaneously. Based on the genetic data from the CMV isolates, antiviral therapy was switched to foscarnet. Although a urine culture 1 month later was still positive for CMV, the symptomatic CMV disease resolved, and the patient eventually became culture negative for CMV in buffy coat and urine specimens as well (Fig. 1). This case illustrates the relatively rapid development of ganciclovir resistance mutations in CMV, after less than 120 days of cumulative exposure to the drug in the posttransplant setting. It is possible that oral acyclovir therapy also contributed to the selection of CMV mutants, although this has not been documented by any clinical observations to date (8). Usually, ganciclovir resistance is encountered in patients with sustained immunodeficiency, such as acquired immune deficiency syndrome, who receive prolonged maintenance therapy for CMV disease (5). It has been suggested that a limited duration of acyclovir or ganciclovir usage in the posttransplant period does not select for ganciclovir-resistant isolates of CMV in solid organ recipients (6), but some individuals could be at a higher risk for this development. Possible contributing factors in this patient are primary CMV infection and high levels of immunosuppressive therapy for rejection, both of which probably favor the emergence of resistance by increasing the amount of replicating virus. In addition, the recurrence of viremia and symptomatic disease after an initial period of therapy prompted the genetic screening of the CMV isolates. The role of drug resistance as a cause of treatment failure in CMV disease and the use of viral genetic analysis as a rapid diagnostic screening test require additional study. Preliminary evidence suggests that even the continued shedding of infectious CMV in the face of prolonged therapy does not necessarily imply drug resistance (5), and that some form of susceptibility testing is needed to guide therapeutic decisions. Unfortunately, classical cell culture-based susceptibility testing in CMV is too slow to be useful for this purpose. Recently, it has been shown that over 90% of ganciclovir-resistant clinical CMV isolates contain one or more mutations in the UL97 phosphotransferase gene, which plays a role in converting ganciclovir into its active antiviral metabolite (ganciclovir triphosphate) (7). These mutations are strongly clustered at codons 460, 520, and 591-595 of the UL97 gene, with just three codons (460, 594, and 595) accounting for the vast majority of observed resistance mutations (7, 9). Screening for mutations at these three codons (by polymerase chain reaction amplification of viral DNA, followed by restriction enzyme analysis; Fig. 2) has been proposed for diagnostic use (7), and in this case revealed the presence of the two most common ganciclovir resistance mutations (V594 and S595) in CMV isolates from this patient. Also of interest is the rapid disappearance of the mutations after discontinuation of ganciclovir (Fig. 1). We conclude from our experience with this case that ganciclovir resistance can occasionally be a clinically significant problem in solid organ transplantation, and that screening for drug resistance mutations may be a useful way of monitoring for this development in a manner that guides the selection of alternative therapy.Figure 1: Clinical and virological data. UL97 genotype: wt, wild type (no UL97 mutation detected); V594/S595, UL97 mutation detected (see text).Figure 2: Effect of UL97 mutations on restriction sites. The UL97 point mutations detected in this patient's CMV isolates were at codons 594 and 595. Mutations resulted in an amino acid change from Ala to Val (V594) and Leu to Ser (S595), respectively. They also caused a gain or loss of a restriction enzyme recognition site that could be used diagnostically. The V594 mutation altered an existing Hha I site (boxed), and the S595 mutation resulted in the gain of a Taq I restriction site (boxed) that was created by the mutation in combination with a mutant polymerase chain reaction primer (CPT1830M).