Richard C. Miner

High-Dose Acyclovir Compared with Short-Course Preemptive Ganciclovir Therapy To Prevent Cytomegalovirus Disease in Liver Transplant Recipients: A Randomized Trial

Cytomegalovirus (CMV) is a major pathogen in liver transplant recipients. Although recent progress in the treatment of CMV disease has led to decreased mortality from this virus, a substantial number of patients still die of CMV-related complications, such as superinfection with bacterial and fungal agents in association with CMV infection [1-3], CMV-associated atherosclerosis in heart transplant recipients [4], bronchiolitis obliterans in lung transplant recipients [5], and chronic rejection (the vanishing bile duct syndrome) in liver transplant recipients [6]. Cytomegalovirus disease also substantially increases the cost of transplantation because it prolongs hospitalization [7]. In 1989, Balfour and colleagues [8] reported that high-dose oral acyclovir decreased the rate of CMV disease in kidney transplant recipients. Despite a small number of patients and an unusually high attack rate (100%) in the control patients, acyclovir-treated seronegative recipients of grafts from seropositive donors had the greatest protection from CMV disease. Based on this study, high-dose oral acyclovir is now also routinely used as prophylaxis for CMV in other organ transplant recipients, including liver recipients. Acyclovir, however, is inactive against CMV in vitro, its long-term administration is expensive, and CMV disease continued to occur at our institution despite such prophylaxis. Another potential problem with continuous, long-term administration of acyclovir is the possible emergence of CMV strains resistant to its closely related nucleoside analog, ganciclovir. Ganciclovir is several times more active against CMV in vitro than is acyclovir. Ganciclovir prophylaxis has been administered in various ways in organ transplant recipients [9], either as prophylaxis in all post-transplant patients, thereby unnecessarily exposing a large number of patients to the drug (most of whom do not develop CMV infection [10-12]) or as prolonged therapy (100 to 120 days), which causes hematologic toxicity, expense, and possibly a prolonged hospital stay [13, 14]. In this study, our approach was to identify or target the patients at highest risk for CMV disease. Viral excretion or shedding precedes CMV disease in transplant recipients [15]. Assuming that viral excretion is a predictor of CMV disease, we hypothesized that a short course (7 days) of ganciclovir instituted as preemptive antiviral therapy in patients shedding the virus would prevent progression of early asymptomatic CMV infection to more severe, invasive CMV disease. Thus, we did a randomized, controlled trial of standard high-dose acyclovir compared with short-course, pulse ganciclovir to be given only if CMV shedding was documented in asymptomatic patients using a CMV surveillance protocol. Methods Study Design All patients having liver transplantation at our institution were randomly assigned to one of the two prophylactic groups. Randomization was stratified by the CMV antibody status of the recipient and the donor. Patients in the control group received 800 mg of acyclovir orally, four times daily, beginning immediately after transplantation as described by Balfour and colleagues [8]; patients continued receiving acyclovir (Zovirax; Burroughs Wellcome, Research Triangle Park, North Carolina) for 24 weeks postoperatively. The dosage of acyclovir was adjusted for impaired renal function as follows: If the creatinine clearance was greater than 50 mL/min, then patients received 800 mg of acyclovir four times a day; if the creatinine clearance was 25 to 50 mL/min, they received 800 mg of acyclovir three times a day; if the clearance was 10 to 25 mL/min, they received 800 mg of acyclovir twice a day; and if the clearance was less than 10 mL/min, they received 800 mg of acyclovir once daily. Surveillance cultures for CMV (buffy coat and urine) were obtained at 2, 4, 6, 8, 12, 16, and 24 weeks postoperatively for all study patients using the shell vial culture method. The experimental group did not receive acyclovir, but intravenous ganciclovir (Cytovene; Syntex, Palo Alto, California), 5 mg/kg twice daily, was administered for 7 days only if surveillance cultures yielded CMV (Figure 1). Ganciclovir dosage was modified for abnormal creatinine clearance as follows: If the creatinine clearance was 80 mL/min or more, then patients received 5 mg/kg of ganciclovir twice daily; if the creatinine clearance was 50 to 79 mL/min, they received 2.5 mg/kg of ganciclovir twice daily; if the clearance was 25 to 49 mL/min, they received 2.5 mg of ganciclovir daily; if the clearance was less than 25 mL/min, they received 1.25 mg/kg of ganciclovir daily. The study was continued for 24 weeks postoperatively. Figure 1. Flow chart representing the study design. Immunosuppression All patients received 0.10 mg/kg of tacrolimus (Fujisawa, Deerfield, Illinois) as a continuous drip for 24 hours, until they were able to take oral medications. The oral dosage of tacrolimus was 0.10 mg/kg every 12 hours. Subsequent dosage adjustments were made as indicated by clinical course and plasma levels of tacrolimus. Methylprednisolone, 1 g, was given immediately after revascularization of the graft. Methylprednisolone, 20 mg, was given intravenously immediately after transplantation and daily thereafter until the oral route was established, at which time 20 mg of prednisone was administered daily. During the subsequent months, prednisone was slowly tapered. Rejection episodes were treated with boluses of 1 g of methylprednisolone with or without steroid recycles (prednisone decreasing daily by 40 mg from an initial starting dose of 200 mg). Muromonab-CD3 (Orthoclone OKT3, Ortho Pharmaceuticals, Raritan, New Jersey) was used for steroid-resistant rejection. Definition of Viral Infections Cytomegalovirus Infection Serologic test results for cytomegalovirus were determined using an enzyme immunosorbent assay, and titers of 0.79 or more were considered positive. All patients received blood products that were neither tested nor screened for CMV antibody. Primary infection was defined as isolation of virus or seroconversion in a patient who was seronegative before transplantation. Reactivation infection was diagnosed by isolation of virus in a seropositive recipient. Cytomegalovirus Disease Clinical diseases caused by CMV included the viral syndrome, localized CMV disease, and disseminated CMV disease. Identification of the viral syndrome caused by CMV required the following: 1) positive culture for CMV; 2) temperature of 38 C or more with no other source to account for it; and 3) one of the following findings: leukocyte count 4000/mm3 or less, atypical lymphocytes 3% or more, and platelets 100 000/mm3 or less. Localized CMV disease was defined as tissue invasion of a single organ determined histopathologically with or without culture of the virus from tissue. Disseminated disease was defined as tissue involvement of two or more noncontiguous organ sites. Identification of Other Viruses Antibodies against viral capsid antigen, early antigen, and Epstein-Barr virus (EBV) nuclear antigen were determined preoperatively in all patients. Patients were defined as having a symptomatic EBV infection if they had EBV-associated lymphoproliferative disease identified by the presence of EBV DNA in tissue using nucleic acid hybridization. Antibody titers to detect asymptomatic increases in EBV were not routinely determined. Herpes simplex virus infection was defined as the presence of typical symptomatic oral or genital ulcers. Varicella zoster virus infection was determined clinically by the presence of typical dermatomal lesions with or without viral isolation. Statistical Analysis Analysis was done using the Prophet System (BBN Systems and Technologies, Division of Research Resources, National Institutes of Health, Bethesda, Maryland). Baseline characteristics (age, Child-Pugh score) were compared using the Fisher exact or t-test. We estimated that at least 20 patients in each group would be needed to detect a decrease in CMV disease from 35% with standard acyclovir prophylaxis to 5% with ganciclovir ( = 0.05, power = 0.8). The incidence of infection or disease was compared using a two-tailed Fisher exact test. A Kaplan-Meier estimate was used to examine the number of days from transplant until first CMV infection for each group. The two curves were compared using the Mantel-Cox log-rank test. Similar curves were constructed for CMV disease. Results The study sample consisted of 47 consecutive adult male patients who received liver transplants at the Pittsburgh Veterans Affairs Medical Center during a 2-year period and who survived at least 72 hours postoperatively. These included 44 patients who had primary transplants and 3 who had re-transplants (2 transplanted once previously and another twice previously). Of 47 patients enrolled in the study, 24 were randomly assigned to the acyclovir group and 23, to the experimental group. The two patient groups were similar at entry in terms of all baseline characteristics measured (Table 1). The Child-Pugh scoring system was used to assess the severity of liver disease in the two groups before transplantation [16]. Table 1. Characteristics of the Study Group at the Time of Enrollment Cytomegalovirus Infection and Disease Shedding of CMV before the onset of CMV disease occurred in 25% (6 of 24) of patients receiving acyclovir prophylaxis and in 22% (5 of 23) of patients receiving no prophylaxis (experimental group) (Figure 2). Seventeen percent (4 of 24) of the patients in the acyclovir group and 4% (1 of 23) in the experimental group did not have previous shedding and developed CMV disease as the first manifestation of CMV infection. Thus, 42% (10 of 24) in the acyclovir group and 26% (6 of 23) in the experimental group had CMV infection (16% difference; 95% CI, 10% to 42%; P > 0.2). All CMV infections were diagnosed by viral isolation. One of 6 infections in the experimental group wa

Viral DNA Polymerase Mutations Associated with Drug Resistance in Human Cytomegalovirus

Certain mutations in the viral DNA polymerase (pol) gene are known to confer drug resistance when transferred to susceptible human cytomegalovirus (CMV) strains, whereas other putative resistance mutations remain unproven. A new marker-transfer technique was used to produce recombinant CMV strains, to determine the drug susceptibility phenotypes conferred by 10 pol mutations (9 observed in clinical isolates). Various degrees of resistance to ganciclovir and cidofovir were conferred by mutations D301N, N410K, D413E, T503I, and L516R, which are located within exonuclease functional domains where D301N and D413E affect highly conserved residues. Mutations A692S, E756K, and E756D, which are not located within recognized functional domains, each conferred foscarnet resistance. This study significantly increases the number of confirmed CMV pol resistance mutations, at both conserved and nonconserved loci, with implications for molecular mechanisms and the genotypic diagnosis of antiviral resistance.

Cytomegalovirus UL97 Phosphotransferase Mutations That Affect Susceptibility to Ganciclovir

Most ganciclovir (GCV)-resistant cytomegalovirus (CMV) isolates contain UL97 gene mutations at codon 460 or 520 or between codons 590 and 607, where an increasing variety of mutations have been detected, including deletions. To determine their phenotypic effect, 9 UL97 mutations not previously studied were transferred to drug-sensitive laboratory CMV strains that contained unique restriction sites developed for this purpose. Deletion of the entire codon range 591-607 conferred a 6-fold increase in GCV resistance, with little effect on viral replication. Some mutations found in clinical isolates, including C592G and A594T, conferred only 2-3-fold decreases in GCV susceptibility. For C592G, this phenotype was confirmed by transfer to different CMV strains and by restoration of full drug susceptibility after removal of the mutation. Low drug levels resulting from oral GCV therapy may predispose the virus to the initial selection of these low-grade UL97 resistance mutations and to later accumulation of other mutations and greater resistance.

Evolution of Mutations Conferring Multidrug Resistance during Prophylaxis and Therapy for Cytomegalovirus Disease

In a human immunodeficiency virus-infected subject, cytomegalovirus (CMV) isolated 9 months after the patient began oral ganciclovir prophylaxis was resistant to ganciclovir and cidofovir and contained mutations in both UL97 and Pol coding regions. At 1 year, retinitis developed, which progressed despite intravenous ganciclovir followed by foscarnet and then cidofovir. A subsequent buffy coat virus isolate was resistant to all three drugs and contained new mutations in UL97 and Pol. By individually transferring the observed mutations to laboratory strain AD169, it was shown that a mutation at codon 603 of UL97 conferred resistance to ganciclovir, a mutation at codon 412 of Pol conferred resistance to both ganciclovir and cidofovir, and a mutation at codon 802 of Pol conferred resistance to ganciclovir and foscarnet. This case illustrates the development of multidrug resistance during prolonged exposure to antiviral therapy for CMV and cross-resistance arising from point mutations in the CMV Pol gene.

Cytomegalovirus Infections in Homosexual Men: An Epidemiological Study

Levels of cytomegalovirus antibody (IgG and IgM) were measured and urine viral cultures were done in 237 homosexual men over a mean period of 14.1 months. The initial prevalence of cytomegalovirus IgG antibody was 86.9%. By the 9th month of follow-up, 71% of serosusceptible men had become infected with cytomegalovirus. During the study period cytomegaloviruria was noted in 32% of seropositive men. Cytomegalovirus IgM antibody was intermittently present in the serum of 95% of IgG-seropositive men, suggesting that frequent reactivation of latent infection or reexposure to exogenous virus had occurred. Of seven sexual practices investigated, only passive anal-genital intercourse correlated with the acquisition of cytomegalovirus infection (p = 0.008).

Phase I Dose Escalation Trial Evaluating the Pharmacokinetics, Anti-Human Cytomegalovirus (HCMV) Activity, and Safety of 1263W94 in Human Immunodeficiency Virus-Infected Men with Asymptomatic HCMV Shedding

ABSTRACT 1263W94 [maribavir; 5,6-dichloro-2-(isopropylamino)-1,β-l-ribofuranosyl-1-H-benzimidazole] is a novel benzimidazole compound for treatment of human cytomegalovirus (HCMV) infection and disease, with potent in vitro activity against HCMV and good oral bioavailability. A phase I study was conducted to determine the pharmacokinetics (PK), anti-HCMV activity, and safety of 1263W94 administered as multiple oral doses to human immunodeficiency virus type 1-infected adult male subjects with asymptomatic HCMV shedding. Subjects received one of six dosage regimens (100, 200, or 400 mg three times a day, or 600, 900, or 1,200 mg twice a day) or a placebo for 28 days. 1263W94 demonstrated linear PK, with steady-state plasma 1263W94 profiles predictable based on single-dose data. 1263W94 was rapidly absorbed following oral dosing, and values for the maximum concentration of the drug in plasma and the area under the concentration-time curve increased in proportion to the dose. 1263W94 demonstrated in vivo anti-HCMV activity in semen at all of the dosage regimens tested, with mean reductions in semen HCMV titers of 2.9 to 3.7 log10 PFU/ml among the four regimens evaluated for anti-HCMV activity. 1263W94 was generally well tolerated; taste disturbance was the most frequently reported adverse event over the 28-day dosing period.

A Standardized Plaque Reduction Assay for Determination of Drug Susceptibilities of Cytomegalovirus Clinical Isolates

ABSTRACT Twelve laboratories collaborated in formulating and testing a standardized plaque reduction assay for cytomegalovirus (CMV) cell-associated clinical isolates. Four characterized and plaque-purified CMV strains, as well as six coded clinical isolates obtained after antiviral therapy, were distributed and tested. Good agreement was obtained for four of the clinical isolates, but a broad distribution of results was obtained for two isolates. Analysis of these results indicates the problems associated with clinical isolates, including the large genetic variability and the highly cell-associated phenotype. This collaborative effort, by addressing these problems, represents a significant step toward the development of a standardized assay.

Mutation in Region III of the DNA Polymerase Gene Conferring Foscarnet Resistance in Cytomegalovirus Isolates from 3 Subjects Receiving Prolonged Antiviral Therapy

Three human immunodeficiency virus-infected subjects with progressive cytomegalovirus (CMV) retinitis despite prolonged antiviral therapy had buffy coat CMV isolates that were resistant to both ganciclovir and foscarnet. Genetic analysis of the resistant isolates showed that each contained a well-known ganciclovir resistance mutation in the viral UL97 phosphotransferase sequence, as well as a mutation (Ala to Val at codon 809, V809) in conserved region III of the DNA polymerase (Pol) sequence. A segment of the Pol sequence from one of the clinical isolates was transferred to CMV laboratory strain AD169 by homologous recombination. The recombinant virus containing V809 showed 6.3-fold increased foscarnet resistance and 2.6-fold increased ganciclovir resistance. Occurrence of the V809 mutation in 3 unrelated cases suggests that it is a clinically significant viral genetic marker for foscarnet resistance and decreased susceptibility to ganciclovir.

A Deletion Mutation in Region V of the Cytomegalovirus DNA Polymerase Sequence Confers Multidrug Resistance

A patient with AIDS and cytomegalovirus (CMV) retinitis received ganciclovir and foscarnet for 20 and 5 months, respectively, with evidence of periodic disease progression. After this therapy, a CMV isolate from the patient was resistant to ganciclovir, foscarnet, and cidofovir. Sequence analysis showed a known ganciclovir resistance mutation in the viral UL97 phosphotransferase (L595F) and a new mutation in conserved region V of the DNA polymerase gene (pol) sequence (codons 981-982 deleted). The pol mutation was transferred to a laboratory CMV strain (Towne) by homologous recombination and selection with either ganciclovir or foscarnet. Recombinant viruses containing this deletion showed a 6-8-fold increased ganciclovir resistance and a 3-5-fold increased resistance to both foscarnet and cidofovir, compared with the wild-type CMV. A single mutation in region V of CMV pol can, therefore, confer multiple drug resistance in a clinical isolate.

Frequency and duration of plasma CMV viremia in seroconverting blood donors and recipients

BACKGROUND : Both CMV‐seronegative blood and unscreened, filtered blood carry a low but definite risk of transmitting CMV infection. To explain this residual risk, evidence of cell‐free viremia was sought in seroconverting and seroprevalent blood donors and seroconverting transfusion recipients by means of a plasma‐based assay for CMV DNA.