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The prevalent use of ganciclovir and its oral prodrug valganciclovir as prophylaxis and therapy for cytomegalovirus (CMV) infection has increased the awareness of drug resistance, the risk of which increases with prolonged drug exposure, impaired host defenses, or insufficient drug delivery [1]. In high-risk populations such as seronegative recipients of a CMV-seropositive donor organ, the incidence of ganciclovir resistance among treated individuals is estimated to be in the 5%–12% range. Drug resistance is suspected when rising plasma viral loads or progressive disease occur during prolonged antiviral treatment. Although ganciclovir resistance was originally documented by phenotypic testing of CMV culture isolates, this is a slow process, unsuitable in contemporary clinical practice where viral isolation is rarely performed. Instead, genotypic resistance testing is now standard, and is based on the detection of diagnostic mutations in the UL97 kinase gene involved in the initial phosphorylation of ganciclovir or in the UL54 DNA polymerase gene that encodes the antiviral drug target for ganciclovir. The accuracy of genotypic resistance testing depends on a comprehensive and validated database linking specific mutations with levels of drug resistance. Extensive clinical experience has defined a set of UL97 mutations that are the most frequent initial laboratory markers of ganciclovir resistance (M460V/I, H520Q, C592G, A594V, L595S, and C603W), accounting for about 80% of documented cases [1]. In the remaining cases, less common UL97 mutations (clustered at codons 590–607) or UL54 mutation may be the first genetic marker of ganciclovir resistance. UL54 mutations typically add to pre-existing UL97 mutation to increase the level of ganciclovir resistance and confer cross-resistance to other anti-CMV drugs. An ongoing difficulty with interpretation of UL54 mutations is the relatively frequent occurrence of natural sequence polymorphisms unrelated to drug resistance. This difficulty is compounded in prophylaxis studies, where no baseline comparator sequence is available. UL97 and UL54 sequence variants remain incompletely documented despite several surveys of clinical CMV sequences [1–6]. A randomized nonblinded treatment trial comparing 3 weeks of oral valganciclovir and intravenous ganciclovir, followed by 4 weeks of valganciclovir, in solid organ transplant recipients (VICTOR study, NCT00431353) involved 321 subjects [7], of which 275 were included in a published resistance substudy [8]. Study subjects included those with a prior antiviral treatment history. Based on genotypic testing of prospectively collected samples, 13 subjects (4.7% of those tested) were considered to have confirmed or probable ganciclovir resistance mutations during the treatment period (0–49 days). Among the 13 cases, 10 had UL97 mutations only, 1 had both UL97 and UL54 mutations, and 2 were considered to have a probable mixture of resistance-associated and wild-type UL54 sequences without UL97 change [8]. Left unresolved were 110 UL54 amino acid sequence variants that could not be classified as known resistance mutations or natural sequence polymorphism. This study aimed to resolve the status of these sequence variants in relation to ganciclovir resistance. The extensive recombinant phenotyping needed for this purpose relied on recent technical advances in the construction and testing of cloned CMV strains.

Background. In a phase III controlled trial IMproved Protection Against Cytomegalovirus in Transplantation (IMPACT) comparing 200 with 100 days of valganciclovir prophylaxis in 318 cytomegalovirus D+/R— kidney transplant recipients, an equal number of patients (n=3 per arm) had known ganciclovir resistance mutations detected during viral breakthrough. In addition, many other viral sequence variants were observed that were of unknown significance for ganciclovir resistance. Recombinant phenotyping was performed to determine whether the previously uncharacterized genotypic changes affected ganciclovir susceptibility, especially in those receiving the longer duration of prophylaxis. Methods. Sequences encoding individual amino acid substitutions in the UL97 kinase or UL54 DNA polymerase gene were transferred by recombination into a cloned cytomegalovirus laboratory strain, followed by reporter-based yield reduction phenotypic assay of the resulting virus for ganciclovir susceptibility. Results. Twenty-six uncharacterized amino acid substitutions were detected, 2 in UL97 and 24 in UL54. All 10 substitutions in the 200-day arm and 9 of 17 substitutions in the 100-day arm (prioritized based on location and conservation) were selected for phenotyping; one substitution was detected in both subsets. Results were generated for nine of ten 200-day and eight of nine 100-day substitutions, with no substitution demonstrating a significant reduction in ganciclovir susceptibility. The two remaining amino acid substitutions, both in UL54, were not evaluated because of poor viral viability. Conclusion. Phenotypic evaluation of previously uncharacterized viral genotypes in the 200-day valganciclovir prophylaxis group showed no evidence of an increased incidence of genotypic ganciclovir resistance when compared with those in the 100-day prophylaxis group.

Objectives: The identification and in vitro characterization of novel protease mutations strongly associated with known protease resistance mutations. Methods: The association between pairs of protease amino acid substitutions was identified using a database of protease sequences derived from protease inhibitor-experienced patients (n = 803). In vitro characterization included drug susceptibility and viral replication studies performed on recombinant viruses harbouring site-directed mutations. Results: The K55R mutation, which is not a natural polymorphism, was identified to be strongly associated with protease mutations M46I/L and to a lesser extent L24I, I54V and V82A/T/S/F. In vitro characterization of the K55R substitution indicated a primary role for this substitution in increasing replicative capacity in the presence of specific protease mutations. Conclusions: The K55R mutation is a secondary drug resistance mutation that can improve viral replication capacity in the presence of other primary protease mutations.

Objectives: To investigate the emergence of resistance to GW433908 (908), a protease inhibitor (PI) with demonstrated antiviral efficacy, safety and tolerability, when administered once daily (q.d.) with low dose ritonavir (908/r). Design: A 48-week Phase III open-label study (SOLO, APV30002) in which antiretroviral therapy-naive patients (n = 649) were treated with 908/r, (1400 mg/200 mg, q.d.) or nelfinavir [1250 mg, twice daily (b.i.d.)] with two nucleoside reverse transcriptase inhibitors (NRTI), abacavir (300 mg, b.i.d.) and lamivudine (150 mg, b.i.d.). Methods: Viral genotype and phenotype were analysed at baseline and on treatment up to 48 weeks and beyond. Results: Emergence of genotypic resistance was significantly different between the 908/r q.d. and the nelfinavir b.i.d. treatment arms for both PIs (0 versus 50%; P < 0.001) and the NRTI (13% versus 69%; P < 0.001) received. In the nelfinavir arm the key protease mutations D30N and/or L90M were frequently observed. The absence of protease resistance mutations and reduced incidence of NRTI resistance mutations in the 908/r q.d. arm was confirmed by phenotyping, which showed a lack of PI cross-resistance. Conclusions: The absence of resistance to 908 or cross-resistance to other PIs, and reduced NRTI resistance, following a 908/r q.d. regimen supports the use of this boosted PI early in therapy.

Previous data have indicated that the development of resistance to amprenavir, an inhibitor of the human immunodeficiency virus type 1 protease, is associated with the substitution of valine for isoleucine at residue 50 (I50V) in the viral protease. We present further findings from retrospective genotypic and phenotypic analyses of plasma samples from protease inhibitor-naïve and nucleoside reverse transcriptase inhibitor (NRTI)-experienced patients who experienced virological failure while participating in a clinical trial where they had been randomized to receive either amprenavir or indinavir in combination with NRTIs. Paired baseline and on-therapy isolates from 31 of 48 (65%) amprenavir-treated patients analyzed demonstrated the selection of protease mutations. These mutations fell into four distinct categories, characterized by the presence of either I50V, I54L/I54M, I84V, or V32I+I47V and often included accessory mutations, commonly M46I/L. The I50V and I84V genotypes displayed the greatest reductions in susceptibility to amprenavir, although each of the amprenavir-selected genotypes conferred little or no cross-resistance to other protease inhibitors. There was a significant association, for both amprenavir and indinavir, between preexisting baseline resistance to NRTIs subsequently received during the study and development of protease mutations ( P = 0.014 and P = 0.031, respectively). Our data provide a comprehensive analysis of the mechanisms by which amprenavir resistance develops during clinical use and present evidence that resistance to concomitant agents in the treatment regimen predisposes to the development of mutations associated with protease inhibitor resistance and treatment failure.

Background The IMPACT study was a randomized, double-blind study comparing 100 to 200 days of VGCV prophylaxis (900 mg once daily) in D+/R− kidney transplant recipients. Although extending the duration of prophylaxis resulted in a significant reduction in confirmed cytomegalovirus (CMV) disease (100-day: 36.8% vs 200-day: 16.1% 1 ), the consequence of extending the duration of prophylaxis on the development of viral resistance remains unknown. Objective To determine whether extending valganciclovir prophylaxis from 100 days to 200 days increased the incidence of ganciclovir resistance. Study design Genotypic analysis of CMV UL97 and UL54 was conducted on virus isolated from patients meeting the predefined resistance analysis criteria (RAC). Results A greater number of patients met the RAC in the 100 day prophylaxis arm (50/163; 31%) compared to the 200 day prophylaxis arm (22/155; 14%). Sequence data were successfully generated for all 200-day patients and 48/50 100-day patients. Three patients in each treatment arm (100 day: 3/163 (1.8%) vs 200 day: 3/155 (1.9%)) had a single known valganciclovir resistance mutation detected (100 day: UL97 gene: M460V, C592G twice; 200 day: UL97 gene: C603W, M460V and UL54 gene: P522S). Overall, a resistance mutation was more likely to be detected if the patient met the RAC during prophylaxis (5/12 (42%)) compared to post-prophylaxis (1/58 (2%)). All six patients with known ganciclovir resistance mutations cleared the virus; three cleared virus without treatment and three cleared virus following treatment. Conclusions Extending valganciclovir prophylaxis from 100 days to 200 days did not significantly affect the incidence of ganciclovir resistance.

The HIV-1 genome encodes an essential protease enzyme which is one of the major targets of antiviral therapy (1–3). Protease inhibitors (PIs) have been proven to be potent antiviral agents and their introduction in 1995 led to the era of highly active antiretroviral therapy, the most potent and prescribed treatment of HIV infections today (4, 5). Although resistance to HIV-1 reverse transcriptase inhibitors had been described in the late 1980s (6), it was originally thought that PIs would be much less prone to drug evasion because of intrinsic genetic and structural constraints. Contrary to these expectations however, a substantial number of patients in the initial studies with PIs experienced drug failure due to the accumulation of multiple mutations in the HIV-1 protease gene (7–15). To understand the mechanisms leading to PI resistance better, it is important to fi rst briefl y review the general structure of the enzyme as well as the interactions involved in inhibitor binding.

Development of in vitro resistance to GW640385, a new human immunodeficiency virus type 1 protease inhibitor, was studied. Variants characterized included one with 50-fold resistance and amino acid substitutions L10F/G16E/E21K/A28S/M46I/F53L/A71V (protease) and L449F/P453T (Gag). The A28S substitution substantially reduced replication capacity.