Rate of Emergence of Cytomegalovirus (CMV) Mutations in Leukocytes of Patients with Acquired Immunodeficiency Syndrome Who Are Receiving Valganciclovir as Induction and Maintenance Therapy for CMV Retinitis

Boivin, Guy; Gilbert, Clare; Gaudreau, Annie; I, Greenfield; Sudlow, Rebecca; Roberts, Noel A.

doi:10.1086/324672

Cited by 119 publications

(71 citation statements)

References 15 publications

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“…Of note, new drugs such as oral valganciclovir with improved, but still limited, bioavailability compared with that of their intravenous formulations will be increasingly prescribed. The detection of low CMV loads during prophylactic or preemptive therapy may lead to treatment adjustment and prevent the risk of emergence of resistant strains (1,6).…”

Section: Discussionmentioning

confidence: 99%

Monitoring of Cytomegalovirus Infection in Solid-Organ Transplant Recipients by an Ultrasensitive Plasma PCR Assay

Hadaya¹,

Wunderli²,

Deffernez³

et al. 2003

J Clin Microbiol

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Early and accurate monitoring of cytomegalovirus (CMV) infection in solid-organ transplant recipients is of major importance. We have assessed the potential benefit of an ultrasensitive plasma-based PCR assay for renal transplant recipients. The pp65 CMV antigen (pp65 Ag) assay using leukocytes was employed as a routine test for the monitoring of CMV in 23 transplant recipients. We compared the pp65 antigenemia with the CMV load quantified by an ultrasensitive PCR (US-PCR) with a limit of detection of 20 CMV DNA copies/ml of plasma. CMV infection was detected in 215 (67%) of 321 plasma samples by the US-PCR compared with 124 (39%) of 321 samples by the pp65 Ag assay. The US-PCR assay permitted the detection of CMV infection episodes following transplantation a median of 12 days earlier than the pp65 Ag assay. Moreover, during CMV infection episodes, DNA detection by the US-PCR was consistently positive, whereas false negative results were frequently observed with the pp65 Ag assay. We found a good correlation between the two assays, and the peak viral loads were significantly higher in patients with CMV-related complications (median, 5,000 DNA copies/ml) than in those without symptoms (1,160 DNA copies/ml) (P ‫؍‬ 0.048). In addition, patients that did not require preemptive therapy based on the results of the pp65 assay had CMV loads significantly lower (median, 36 DNA copies/ml) than those that needed treatment (median, 4,703 DNA copies/ml) (P < 0.001). These observations provided cutoff levels that could be applied in clinical practice. The ultrasensitive plasmabased PCR detected CMV infection episodes earlier and provided more consistent results than the pp65 Ag assay. This test could improve the monitoring of CMV infection or reactivation in renal transplant recipients.

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Section: Discussionmentioning

confidence: 99%

Monitoring of Cytomegalovirus Infection in Solid-Organ Transplant Recipients by an Ultrasensitive Plasma PCR Assay

Hadaya¹,

Wunderli²,

Deffernez³

et al. 2003

J Clin Microbiol

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“…However, because the primary use of these drugs is likely to be for prophylaxis of KS in high-risk groups or for preemptive therapy at a time of active viral replication but before the development of HHV-8-associated diseases (8,11,13,21,26), the convenience of administration and the absence of toxicity are two key factors (in addition to IC 50 values) that should be considered in the selection of an optimal antiviral agent. In that context, valganciclovir, which is a new bioavailable prodrug of ganciclovir, appears particularly interesting (2,19). Accordingly, the protective effect of oral ganciclovir (which is 10-fold less bioavailable than valganciclovir) for the development of KS has been previously reported in a CMV study in HIV-infected subjects (18).…”

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confidence: 93%

Evaluation of Susceptibility of Human Herpesvirus 8 to Antiviral Drugs by Quantitative Real-Time PCR

Sergerie

Boivin

2003

J Clin Microbiol

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A new in vitro system based on real-time PCR was developed for evaluation of human herpesvirus 8 susceptibility to antiviral agents. Cidofovir had the greatest inhibitory activity against HHV-8 (50% inhibitory concentration [IC 50 ], 0.43 M) followed by ganciclovir (2.61 M), adefovir (18.00 M), acyclovir (31.00 M), and foscarnet (34.15 M). The potential therapeutic efficacy for HHV-8 (i.e., peak serum drug level/IC 50 ) is highest for cidofovir (167) and foscarnet (22).The human herpesvirus 8 (HHV-8), also known as Kaposi's sarcoma-associated herpesvirus, is a new member of the ␥-herpesvirinae subfamily that has been associated with human immunodeficiency virus (HIV)-and non-HIV-related Kaposi's sarcoma (KS) as well as with multicentric Castleman's disease and primary effusion lymphoma (5,6,17,25). Despite the decrease in HIV-related KS with the advent of highly active antiretroviral therapy, there is still an interest in evaluating strategies to inhibit the early stages of HHV-8 replication.Several in vitro systems have been reported for determination of HHV-8 susceptibility to antiviral drugs (10,16,20,22). Because most B cells are latently infected by the virus, inducing agents such as 12-O-tetradecanoylphorbol-13-acetate (TPA) or sodium butyrate have been used to promote lytic viral replication (23). Also, due to the absence of viral plaques in infected lymphoma cell lines, most susceptibility systems were designed to measure viral DNA synthesis. However, the existing assays are very cumbersome, as they require gel electrophoresis and isotopic hybridization. In this report, we describe the development of a rapid and objective in vitro susceptibility system for HHV-8 based on real-time quantitative PCR.The BCBL-1 cell line, which is a B-cell line latently infected by HHV-8 (23), was kindly provided by Benoît Barbeau (Centre de Recherche en Infectiologie, Ste-Foy, Québec, Canada). The cells were maintained as described previously (16). On day 1, 10 ml of BCBL-1 cells at 2 ϫ 10 5 cells/ml (in RPMI 1640 medium [Life Technologies, Burlington, Ontario, Canada] supplemented with 10% heat-inactivated fetal bovine serum) were pelleted at 250 ϫ g for 10 min and then washed with 2 ml of phosphate-buffered saline. The cell pellet was then resuspended in an equal volume of medium with TPA (without TPA for the negative control) at a final concentration of 20 ng/ml in 25-cm 2 flasks (BD Biosciences, Oakville, Ontario, Canada). Serial concentrations of antivirals, i.e., acyclovir (zovirax; GlaxoSmithKline), foscarnet (Sigma, Oakville, Ontario, Canada), ganciclovir (cytovene; Hoffman La Roche), cidofovir (vistide; Gilead Sciences, Foster City, Calif.), and adefovir (kindly provided by Tomas Cihlar; Gilead Sciences) were made in triplicate and added to culture medium. On day 2, 20 h after TPA stimulation, the cells were repelleted as described above, washed with phosphate-buffered saline, and resuspended in 10 ml of fresh medium containing the same antiviral drugs but without TPA. On day 4 (3 days after the addition of TPA...

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“…In HIV-infected patients, GCV resistance has been reported in Ͼ20% of patients before the availability of highly active antiretroviral therapy (HAART) and in 9% after and is associated with CD4 ϩ T-cell counts of Ͻ50 cells/l (18)(19)(20)(21). Continued administration of a drug to which resistance has developed can lead to an accumulation of multiple drug resistance mutations (6), increased morbidity, and, in SOT recipients, shortened graft survival (15,16).…”

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confidence: 99%

Detection of Cytomegalovirus Drug Resistance Mutations by Next-Generation Sequencing

et al. 2013

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Antiviral therapy for cytomegalovirus (CMV) plays an important role in the clinical management of solid organ and hematopoietic stem cell transplant recipients. However, CMV antiviral therapy can be complicated by drug resistance associated with mutations in the phosphotransferase UL97 and the DNA polymerase UL54. We have developed an amplicon-based high-throughput sequencing strategy for detecting CMV drug resistance mutations in clinical plasma specimens using a microfluidics PCR platform for multiplexed library preparation and a benchtop next-generation sequencing instrument. Plasmid clones of the UL97 and UL54 genes were used to demonstrate the low overall empirical error rate of the assay (0.189%) and to develop a statistical algorithm for identifying authentic low-abundance variants. The ability of the assay to detect resistance mutations was tested with mixes of wild-type and mutant plasmids, as well as clinical CMV isolates and plasma samples that were known to contain mutations that confer resistance. Finally, 48 clinical plasma specimens with a range of viral loads (394 to 2,191,011 copies/ml plasma) were sequenced using multiplexing of up to 24 specimens per run. This led to the identification of seven resistance mutations, three of which were present in <20% of the sequenced population. Thus, this assay offers more sensitive detection of minor variants and a higher multiplexing capacity than current methods for the genotypic detection of CMV drug resistance mutations. H uman cytomegalovirus (CMV)is an important cause of severe systemic and tissue-invasive disease in immunocompromised patients (reviewed in reference 1), such as solid organ transplant (SOT) and hematopoietic stem cell transplant (HSCT) recipients. In these patients, prophylactic or preemptive treatment with antiviral agents for CMV significantly reduces the rates of CMV disease, the risk of other viral and bacterial infections, and all-cause mortality (2-5), but these treatments place patients at risk for the development of CMV drug resistance (6). In addition to anti-CMV therapy lasting for Ն3 months, other risk factors for drug resistance are intensive immunosuppression, multiple episodes of CMV reactivation, high viral counts, and suboptimal antiviral concentrations due to poor compliance or low drug absorption (7).The number of antiviral drugs approved for CMV treatment is limited and includes ganciclovir (GCV) and its prodrug valganciclovir (VCV), foscarnet (FOS), and cidofovir (CDV). Intravenous GCV and oral VCV are used as first-line agents for both prophylaxis and treatment, largely because of their lower toxicities than those of FOS and CDV (8-13). All of the available CMV drugs ultimately target the viral DNA polymerase UL54; however, GCV and VCV also require phosphorylation by the phosphotransferase UL97 for their antiviral activity (6). Thus, mutations in the UL97 and UL54 genes can confer resistance to GCV and VCV, while CDV and FOS resistance is only associated with mutations in UL54. Large numbers of sequence variants ha...

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Rate of Emergence of Cytomegalovirus (CMV) Mutations in Leukocytes of Patients with Acquired Immunodeficiency Syndrome Who Are Receiving Valganciclovir as Induction and Maintenance Therapy for CMV Retinitis

Cited by 119 publications

References 15 publications

Monitoring of Cytomegalovirus Infection in Solid-Organ Transplant Recipients by an Ultrasensitive Plasma PCR Assay

Monitoring of Cytomegalovirus Infection in Solid-Organ Transplant Recipients by an Ultrasensitive Plasma PCR Assay

Evaluation of Susceptibility of Human Herpesvirus 8 to Antiviral Drugs by Quantitative Real-Time PCR

Detection of Cytomegalovirus Drug Resistance Mutations by Next-Generation Sequencing

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