Cytomegalovirus (CMV) and human immunodeficiency virus (HIV) burden, CMV end-organ disease, and survival in subjects with advanced HIV infection (AIDS Clinical Trials Group Protocol 360).

We undertook a prospective study to analyze cytomegalovirus (CMV) end-organ disease (EOD) in subjects with advanced human immunodeficiency virus (HIV) infection. Of 403 individuals without prior CMV EOD who were followed up for a median of 151 weeks, 56 died and 21 developed CMV EOD. Twenty of the subjects with CMV EOD had CD4 cell counts of < or =50 cells/mm3 and HIV RNA level of >10,000 copies/mL of plasma at baseline; in these 20 subjects, an increase of CMV DNA level to greater than the quantification limits was associated with CMV EOD. A CD4 cell count of < or =100 cells/mm3 and an HIV RNA level of >10,000 copies/mL of plasma at baseline, a CMV DNA level of >200 copies/mL of blood during follow-up, or development of CMV EOD were all associated with decreased survival. HIV-infected subjects with CD4 cell counts of < or =50 cells/mm3 and HIV RNA levels of >10,000 copies/mL of plasma should have blood fractions screened for CMV DNA; if CMV DNA is detected, CMV prophylaxis might be considered.

Analysis of HIV-1 reverse transcriptase and protease sequences in paired plasma and lymphoid tissue specimens from HIV-1 infected individuals.

OBJECTIVES: To determine how representative the genotype of HIV-1 circulating in plasma is of the genotype of the virus present in lymphoid tissue. METHODS: Paired plasma and tonsillar tissue samples were prospectively obtained from patients with various levels of plasma HIV-1 RNA who were receiving combination antiretroviral therapy. HIV-1 reverse transcriptase and protease sequences were amplified from plasma and lymphoid tissue specimens by nested polymerase chain reaction and analyzed using an automated sequencing system. Results were compared with consensus HIV-1 sequences to determine whether drug-resistance mutations were present in the regions analyzed. RESULTS: HIV-1 protease sequences were compared in 11 plasma/tissue pairs obtained from eight patients; HIV reverse transcriptase sequences were compared in 12 plasma/tissue pairs obtained from nine patients. Sequence homology between plasma and tissue RNA, tissue RNA and DNA, and plasma and tissue DNA ranged from 97% to 100%. Few discrepancies were found when the percentage of mutant sequences at resistance codons was compared among paired samples. In most instances, tissue RNA or plasma contained a higher percentage of mutant sequences than did tissue DNA. CONCLUSION: The genotype of plasma HIV-1 is similar to the genotype of the virus in lymphoid tissue. Resistance studies using plasma samples should provide accurate information regarding the genotype of HIV-1 in lymphoid tissues.

Comparison of quantitative and qualitative PCR assays for cytomegalovirus DNA in plasma.

We analyzed the performance characteristics of the qualitative AMPLICOR CMV Test (Roche Molecular Systems, Pleasanton, Calif.) and quantitative COBAS AMPLICOR CMV MONITOR Test (Roche Molecular Systems) assays and compared the performance of the AMPLICOR quantitative assay with an in-house-developed cytomegalovirus (CMV) DNA PCR assay. The quantitative AMPLICOR assay was found to be more sensitive than the qualitative AMPLICOR assay. The quantitative AMPLICOR assay has a lower limit of sensitivity of 400 CMV DNA copies/ml of plasma and is linear to 50,000 CMV DNA copies/ml of plasma. Compared to the in-house PCR assay, the AMPLICOR quantitative assay gave lower viral load values at all concentrations tested, but the difference between the two assays was not consistent across the entire dynamic range of the AMPLICOR quantitative assay. At the lower end of the assay, the viral load values obtained with the in-house PCR assay were three- to fivefold (0.5 to 0.7 log units) higher than those measured with the AMPLICOR assay. At higher input concentrations, the differences between the two assays approached 10-fold. This direct comparison of the in-house assay and the quantitative AMPLICOR assay provides the ability to compare previously published in-house data with an assay widely available for future research and clinical monitoring of patients with CMV infections.

Performance characteristics of the QUANTIPLEX HIV-1 RNA 3.0 assay for detection and quantitation of human immunodeficiency virus type 1 RNA in plasma.

The QUANTIPLEX HIV-1 RNA assay, version 3.0 (a branched DNA, version 3.0, assay [bDNA 3.0 assay]), was evaluated by analyzing spiked and clinical plasma samples and was compared with the AMPLICOR HIV-1 MONITOR Ultrasensitive (ultrasensitive reverse transcription-PCR [US-RT-PCR]) method. A panel of spiked plasma samples that contained 0 to 750,000 copies of human immunodeficiency virus type 1 (HIV-1) RNA per ml was tested four times in each of four laboratories (1,344 assays). Negative results (<50 copies/ml) were obtained in 30 of 32 (94%) assays with seronegative samples, 66 of 128 (52%) assays with HIV-1 RNA at 50 copies/ml, and 5 of 128 (4%) assays with HIV-1 RNA at 100 copies/ml. The assay was linear from 100 to 500,000 copies/ml. The within-run standard deviation (SD) of the log(10) estimated HIV-1 RNA concentration was 0.08 at 1,000 to 500,000 copies/ml, increased below 1,000 copies/ml, and was 0.17 at 100 copies/ml. Between-run reproducibility at 100 to 500 copies/ml was <0.10 log(10) in most comparisons. Interlaboratory differences across runs were /=50 copies/ml when they were retested by the US-RT-PCR assay (median, 86 copies/ml; range, 50 to 217 copies/ml). Estimation of bDNA 3.0 values of <50 copies/ml by extending the standard curve of the assay showed that these samples with discrepant results had higher HIV-1 RNA levels than the samples with concordant results (median, 34 versus 17 copies/ml; P = 0.0051 by the Wilcoxon two-sample test). The excellent reproducibility, broad linear range, and good sensitivity of the bDNA 3.0 assay make it a very attractive method for quantitation of HIV-1 RNA levels in plasma.

Viability and functional activity of cryopreserved mononuclear cells.

Factors that influence viability and function of cryopreserved peripheral blood mononuclear cells (PBMC) were identified on 54 samples from 27 AIDS Clinical Trial Units. PBMC viability ranged from 1 to 96% with a median of 70%, was higher in laboratories with experienced staff, and was not significantly associated with CD4 cell number. Function of cryopreserved PBMC, measured by lymphocyte proliferation, was associated with viability. Preparations with viability greater than or equal to 70% had consistent proliferative responses and were suitable for functional analyses.

CMV antigenemia following bone marrow transplantation: risk factors and outcomes.

Cytomegalovirus (CMV) infection remains a major problem in blood and bone marrow transplant (BMT) recipients. Recent efforts have been directed at prevention, early diagnosis, and treatment of CMV disease following BMT. Assay for CMV early antigen pp65 on circulating leukocytes has been shown to be sensitive, and specific in detecting early CMV infection. We examined the frequency, risk factors, and outcomes of a positive CMV antigen assay in 118 consecutive BMT patients. Forty-three (36%) of the 118 patients developed CMV antigenemia a median of 26 days post-BMT (range, -6 to 209 days). The incidence of antigenemia in autologous, related donor, and unrelated donor BMT recipients was 15%, 50%, and 48%, respectively (P < .01) and was lower in CMV-seronegative patients (19% versus 51% in seropositive patients; P < .01). Patients with grade II to IV acute graft-versus-host disease (GVHD) had 2.2 times the risk of antigenemia of patients with no or only limited GVHD (P = .03). Age at transplantation, underlying disease, CMV prophylaxis regimen, and GVHD prophylaxis regimen did not affect the risk of CMV antigenemia. Ten of the 43 antigenemic patients, all CMV-seropositive allogeneic BMT (alloBMT) recipients, developed CMV organ disease a median of 101 days (range, 28-283 daya) post-BMT. These data suggest that CMV-seropositive alloBMT patients are at highest risk for CMV antigenemia and for organ disease as well. CMV disease may occur before antigenemia is detectable in leukopenic patients and may also develop late post-BMT, even in patients still receiving antiviral prophylaxis. In high-risk groups, intensive surveillance continuing for more than 6 months after BMT may be indicated.

A standardized plaque reduction assay for determination of drug susceptibilities of cytomegalovirus clinical isolates.

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.

Recovery and analysis of human immunodeficiency virus type 1 (HIV) RNA sequences from plasma samples with low HIV RNA levels.

Amplification of human immunodeficiency virus type 1 (HIV) reverse transcriptase (RT) and protease (PT) sequences from plasma is difficult when HIV RNA levels are low, and it usually cannot be accomplished in samples with <1,000 HIV RNA copies/ml. Because the RNA extraction step is critical for the success of subsequent amplifications and sequence analyses, two RNA extraction methods were compared to study plasma samples with low HIV RNA levels. Forty-four plasma samples containing <500 HIV RNA copies/ml in a branched-DNA (bDNA) assay (Quantiplex HIV RNA assay version 2.0 [Chiron Corp., Emeryville, Calif.]) were studied. RNA was extracted by using two commercial kits (QIAamp Viral RNA kit [Qiagen, Hilden, Germany] and NucliSens kit [Organon Teknika, Boxtel, The Netherlands]). Fragments (1,144 bp) encompassing HIV PT and RT sequences were amplified by nested PCRs. Amplified products were sequenced by using a commercial kit (Applied Biosystems). HIV RNA was recovered from a total of 21 plasma samples, including 20 samples after extraction by the NucliSens method, and 8 samples after extraction by the QIAamp method (P < 0.05). Mean HIV RNA levels in these samples, measured by an ultrasensitive bDNA assay (Quantiplex HIV RNA assay version 3.0; Chiron Corp., Emeryville, Calif.), were 848 copies/ml (median, 666; range, 154 to 2,606 copies/ml). Analysis of RT and PT sequences in five samples demonstrated an average of 3.8 and 2.4 resistance mutations in these regions, respectively. The NucliSens RNA extraction kit is a valuable method for obtaining HIV RNA for genotypic studies from plasma fractions of individuals with low HIV RNA levels.

Neurological outcomes in late HIV infection: adverse impact of neurological impairment on survival and protective effect of antiviral therapy. AIDS Clinical Trial Group and Neurological AIDS Research Consortium study team.

OBJECTIVE: In a large multi-center clinical trial of combination reverse transcriptase inhibitors (RTIs), we assessed the impact of antiretroviral therapy on neurological function, the relationship between neurological and systemic benefit, and the prognostic value of neurological performance in late HIV-1 infection. DESIGN: Neurological evaluations incorporated in a randomized, multi-center trial of combination antiretroviral therapy. SETTING: Forty-two AIDS Clinical Trials Group sites and seven National Hemophilia Foundation sites. PATIENTS: Adult HIV-infected patients (n = 1313) with CD4 counts < 50 x 10(6) cells/l. INTERVENTIONS: Four combinations of reverse transcriptase inhibitors consisting of zidovudine (ZDV), alternating monthly with didanosine (ddl), or in combination with zalcitabine (ddC), ddl or ddl and nevirapine. MAIN OUTCOME MEASURES: Mean change from baseline of a four-item quantitative neurological performance battery score, the QNPZ-4, administered to 1031 subjects. RESULTS: Triple therapy and ZDV/ddl combination preserved or improved neurological performance over time compared with the alternating ZDV/ddl and ZDV/ddC regimens (P < 0.001), paralleling their impact on survival in the same trial as previously reported. QNPZ-4 scores were predictive of survival (P < 0.001), after adjusting for CD4 counts and HIV-1 plasma RNA concentrations. CONCLUSIONS: Combination antiretroviral therapy can have a salutary effect on preserving or improving neurological function. Superior systemic treatments may likewise better preserve neurological function. The significant association of poor neurological performance with mortality, independent of CD4 counts and HIV-1 RNA levels indicates that neurological dysfunction is an important cause or a strong marker of poor prognosis in late HIV-1 infection. This study demonstrates the value of adjunctive neurological measures in large therapeutic trials of late HIV-1 infection.

Virologic responses to a ritonavir--saquinavir-containing regimen in patients who had previously failed nelfinavir.

OBJECTIVES: The effectiveness of a second protease inhibitor in patients who failed an initial protease inhibitor is unclear but believed to be low. It has been postulated, however, that patients who fail nelfinavir may respond differently. We therefore assessed the virologic response to a ritonavir-saquinavir-containing regimen in patients who had previously failed nelfinavir. METHODS: A total of 26 patients enrolled in the nelfinavir clinical trials AG506 and AG511 at our two sites who failed (two consecutive HIV viral loads > 5000 copies/ml; branched DNA assay) were switched to a combination of stavudine 40 mg twice daily, lamivudine 150 mg twice daily, ritonavir 400 mg twice daily and saquinavir 400 mg twice daily. RESULTS: The mean viral load at enrollment in this study was 46 674 copies/ml (range, 1075-146400 copies/ml). The median CD4 cell count was 222 x 10(6)/l (range, 82-448 x 10(6)/l). The median duration of nelfinavir use with a detectable viral load before the switch occurred was 48 weeks. Two patients discontinued the study at 3 weeks. All of the remaining patients (n = 24) reached undetectable viral loads (< 500 copies/ml) that were sustained at week 24 in 17 (71%) out of 24 subjects. The most frequent baseline mutations in the protease gene prior to switching were D30N (13 out of 18), N88D (eight out of 18) and M36I (eight out of 18). The presence or absence of these mutations was not predictive of a short-term virologic response. CONCLUSIONS: Most patients who failed a nelfinavir-containing regimen responded to a switch to a combination regimen with saquinavir-ritonavir.

Resistance of human cytomegalovirus to antiviral drugs.

Resistance of cytomegalovirus (CMV) to antiviral agents is a well-recognized phenomenon that has been observed in the laboratory and in the clinical setting. Infections caused by antiviral-resistant CMV have been found exclusively among immunocompromised individuals, including patients with AIDS, bone marrow and solid-organ transplant recipients, and patients with hematologic malignancies, and in individuals with primary immunodeficiencies. The majority of these infections have been described to occur in patients with AIDS receiving prolonged antiviral therapy for CMV end-organ disease. Antiviral agents currently licensed for the treatment of CMV infections include ganciclovir, foscarnet, and cidofovir. Resistance of CMV to ganciclovir is related to mutations in the UL97 region of the viral genome and/or mutations in the viral DNA polymerase. Resistance to foscarnet and cidofovir is associated with mutations in the viral DNA polymerase. Antiviral susceptibility of CMV strains containing DNA polymerase mutations is dependent on the region of the DNA polymerase where the mutations are located. Some DNA polymerase mutant viruses are cross-resistant to ganciclovir, foscarnet, and cidofovir. The recognition that specific UL97 and UL54 mutations are associated with resistance to antiviral agents has led to the development of molecular methods for detection of mutant viruses. This article reviews the mechanisms of resistance of CMV to antiviral agents, the laboratory methods for detection of resistant CMV, and the clinical aspects of infections caused by antiviral-resistant CMV.

Are there cytopathic features associated with cytomegalovirus infection predictive of resistance to antiviral therapy?

Recent awareness of the broader spectrum of morphologic changes induced by cytomegalovirus (CMV) infection prompted us to investigate whether the concurrent introduction of effective antiviral therapy is in some way related to the appearance of atypical cytopathic features. These changes may be easily misinterpreted on histologic examination as reactive or degenerative. In addition, since resistant strain to antiviral therapy has emerged and its demonstration is a laborious process performed in highly specialized laboratories, it is important to determine if resistance to antiviral drugs can be predicted from the microscopic examination of infected tissues. The population consisted of seven immunosuppressed patients with documented CMV infection from which 18 tissue samples were obtained at autopsy or endoscopically. Antiviral susceptibility to ganciclovir was determined by plaque reduction assay and/or a DNA-DNA hybridization method. Eleven tissue specimens from patients harboring resistant strains were compared with seven specimens from patients infected with sensitive strains. Cytopathic changes were classified as typical or atypical according to previously published criteria. Of the 18 biopsy specimens, the cytopathic changes were distributed as follows: typical 1, typical and atypical 5, and atypical 12. Atypical inclusions were found in 10 of 11 and 7 of 7 sensitive and resistant cases, respectively. In conclusion, there are no specific morphologic features in CMV-infected tissues of patients with infections caused by ganciclovir resistant strains.

Cytomegalovirus UL97 and glycoprotein B (gB) sequences in tissues from immunocompromised patients with ganciclovir-resistant virus infection.

The pathogenesis of ganciclovir-resistant cytomegalovirus (CMV) was investigated by analysing UL97 and gB sequences in tissues obtained from 4 immunocompromised patients with infections caused by ganalciclovir-resistant virus. UL97 and gB sequences were obtained by automated sequencing of CMV DNA amplified from lysates prepared from deparaffinized tissue sections. Patient 1 contained wild-type UL97 and gB3 sequences. Patient 2 harboured genetically distinct viruses in his lung: one with a ganciclovir-resistance UL97 mutation and a gB3 genotype, and another without UL97 mutations and a gB1 genotype. In patient 3, a ganciclovir-resistant UL97 mutant virus with a gB1 genotype was cultured from the lung, whereas the CMV in the brain did not contain mutations and its genotype was gB2. In patient 4, ganciclovir-resistance UL97 sequences were found in oesophageal tissue prior to the isolation of a ganciclovir-resistant CMV from the blood. All viruses in this patient had a gB3 genotype. CMV containing ganciclovir-resistance UL97 mutations may cause end-organ disease in immunocompromised individuals. In these subjects, CMV circulating in the blood may have similar or different UL97 and gB genotypes than the virus causing end-organ disease.

A randomized, controlled, double-blind study comparing the survival benefit of four different reverse transcriptase inhibitor therapies (three-drug, two-drug, and alternating drug) for the treatment of advanced AIDS. AIDS Clinical Trial Group 193A Study Team.

OBJECTIVE: The primary objective was to compare the effects of dual or triple combinations of HIV-1 reverse transcriptase inhibitors with respect to survival. The time to new HIV disease progression or death, toxicities, the change in CD4 cells, and plasma HIV-1 RNA concentrations in a subset of study subjects were evaluated. DESIGN: This was a multicenter randomized, double-blind, placebo-controlled study. SETTING: The study was conducted among 42 adult AIDS Clinical Trials Group sites and 7 National Hemophilia Foundation centers. PATIENTS: 1313 HIV-infected patients with CD4 counts < or = 50 cells/mm3 participated in this study, which was conducted from June 1993 to June 1996. INTERVENTION: Patients were randomized to one of four daily regimens containing 600 mg of zidovudine: zidovudine alternating monthly with 400 mg didanosine; zidovudine plus 2.25 mg of zalcitabine; zidovudine plus 400 mg of didanosine; or zidovudine plus 400 mg of didanosine plus 400 mg of nevirapine (triple therapy). MAIN OUTCOME MEASURES: The main outcome was survival (i.e., time to death). RESULTS: A significant difference in survival time was found between the four treatment groups, favoring those assigned to triple therapy (p = .02). A significant difference was also found in the delay of disease progression or death among the four treatment arms favoring the group assigned to triple therapy (p = .002). Baseline CD4 cell counts and plasma HIV-1 RNA concentrations as well as changes of CD4 counts at week 8 predicted survival for subjects in the virology substudy. CONCLUSIONS: In the pre-protease inhibitor era, a combination of triple reverse transcriptase inhibitors prolonged life and delayed disease progression in AIDS patients with advanced immune suppression.

Interlaboratory concordance of DNA sequence analysis to detect reverse transcriptase mutations in HIV-1 proviral DNA. ACTG Sequencing Working Group. AIDS Clinical Trials Group.

Thirteen laboratories evaluated the reproducibility of sequencing methods to detect drug resistance mutations in HIV-1 reverse transcriptase (RT). Blinded, cultured peripheral blood mononuclear cell pellets were distributed to each laboratory. Each laboratory used its preferred method for sequencing proviral DNA. Differences in protocols included: DNA purification; number of PCR amplifications; PCR product purification; sequence/location of PCR/sequencing primers; sequencing template; sequencing reaction label; sequencing polymerase; and use of manual versus automated methods to resolve sequencing reaction products. Five unknowns were evaluated. Thirteen laboratories submitted 39043 nucleotide assignments spanning codons 10-256 of HIV-1 RT. A consensus nucleotide assignment (defined as agreement among > or = 75% of laboratories) could be made in over 99% of nucleotide positions, and was more frequent in the three laboratory isolates. The overall rate of discrepant nucleotide assignments was 0.29%. A consensus nucleotide assignment could not be made at RT codon 41 in the clinical isolate tested. Clonal analysis revealed that this was due to the presence of a mixture of wild-type and mutant genotypes. These observations suggest that sequencing methodologies currently in use in ACTG laboratories to sequence HIV-1 RT yield highly concordant results for laboratory strains; however, more discrepancies among laboratories may occur when clinical isolates are tested.

Concentration-controlled zidovudine therapy.

BACKGROUND: Heterogeneity in the response to antiretroviral therapy has been attributed to pharmacologic, immunologic, and virologic differences between patients. Currently available antiretroviral agents used for the treatment of human immunodeficiency virus (HIV) infection in adults are administered in standard fixed doses. The active moiety of nucleoside anti-HIV drugs is the intracellular anabolite. Therefore the heterogeneity in response to nucleoside agents may arise as a result of pharmacologic variability at both the systemic and cellular level. OBJECTIVES: To determine whether a novel concentration-controlled zidovudine regimen could improve anti-HIV response compared with the standard fixed-dose approach. DESIGN: At the Outpatient Clinic of the General Clinical Research Center at the University of Minnesota, 20 persons with HIV infection received an oral regimen of zidovudine designed to achieve a target concentration in plasma of 0.7 mumol/L and the 500 mg/day standard dose in a randomized, crossover 24-week study. RESULTS: The concentration-controlled regimen achieved overall higher systemic concentrations with reduced interpatient variability: steady-state average zidovudine plasma concentrations were 0.76 mumol/L (coefficient of variation, 12%) versus 0.62 mumol/L (coefficient of variation, 32%) for the standard regimen. There was no difference in safety and tolerance between regimens. Intracellular zidovudine triphosphate concentrations averaged 160 fmol/10(6) peripheral blood mononuclear cells (PBMCs) with concentration-controlled versus 92 fmol/10(6) PBMCs for standard therapy. The percentage change from baseline in CD4 cells was a 22% increase for the concentration-controlled regimen versus a 7% decrease with standard therapy. CONCLUSIONS: These data indicate that pharmacologic variability affects antiretroviral response. Furthermore, these findings provide a framework to characterize the pharmacologic determinants of effect and quantitate their contribution to the heterogeneity in clinical response to optimize therapeutic benefit.

Phenotypic and genotypic characterization of acyclovir-resistant herpes simplex viruses from immunocompromised patients.

Phenotypic and genotypic analyses were done on 30 acyclovir-resistant and 5 acyclovir-susceptible herpes simplex virus (HSV) isolates (22 HSV type 1 and 13 HSV type 2) recovered from 24 subjects. All isolates were susceptible to foscarnet. The phenotypes of the acyclovir-resistant HSV isolates were as follows: 17 were thymidine kinase (TK) deficient, 12 had decreased TK activity (produced low amounts of viral TK) or TK with altered substrate specificity, and 1 was undetermined. Sequencing analysis of the HSV TK gene revealed that 14 (46.7%) of 30 acyclovir-resistant isolates had an insertion or deletion of 1 or 2 nucleotides, especially in homopolymer runs of Gs, Cs, and rarely in As. On the other hand, 16 (53.3%) of 30 acyclovir-resistant isolates had point mutations in conserved or nonconserved regions of the TK gene. In conclusion, HSV can develop multiple strategies to exhibit acyclovir resistance, including, in about half of the cases, frameshift mutations in homopolymer nucleotide stretches of the TK gene.

Ganciclovir susceptibilities and analysis of UL97 region in cytomegalovirus (CMV) isolates from bone marrow recipients with CMV disease after antiviral prophylaxis.

Ganciclovir susceptibilities and UL97 sequences were analyzed in 20 cytomegalovirus (CMV) isolates recovered from 15 bone marrow transplant recipients with active CMV infection after prophylaxis with acyclovir (group I; 12 isolates) or after acyclovir prophylaxis followed by ganciclovir therapy (group II; 8 isolates). All group I isolates were susceptible to ganciclovir. Five group II isolates were susceptible to ganciclovir, and 3 isolates (all from the same person) were resistant to ganciclovir (IC50 > 12 microM). Ganciclovir resistance UL97 mutations were found in 4 group II isolates, including a ganciclovir-susceptible isolate obtained from 1 patient after 41 days of therapy with ganciclovir and 3 ganciclovir-resistant isolates obtained from another patient after 73, 116, and 132 days of treatment with ganciclovir. Ganciclovir-resistant CMV isolates may emerge rapidly in bone marrow transplant recipients who are treated with ganciclovir after receiving prophylaxis with acyclovir.