Detection of Cytomegalovirus Drug Resistance Mutations by Next-Generation Sequencing

Abstract

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...

Limited Variation in BK Virus T-Cell Epitopes Revealed by Next-Generation Sequencing

“…At nucleotide positions where no variant was detected, the noise value was considered to be zero (20). Error rates for the plasmid clones were calculated as the mean percent noise reported for all sequenced positions (21).…”

“…The noise data were analyzed in R using packages vcd, ggplot2, and MASS. A generalized linear (Poisson) model was used to estimate the dependence of the number of noise reads on the covariates (20,22). The homopolymer covariate was defined as a repeat of 3 or more of the same nucleotide along with nonidentical flanking nucleotides (23).…”

Improved Detection of Emerging Drug-Resistant Mutant Cytomegalovirus Subpopulations by Deep Sequencing

“…Sequencing was performed using the Roche 454 GS Jr. pyrosequencing platform as recently published (6). After an initial long-range PCR to amplify ϳ2-kb and ϳ4-kb targets for UL97 and UL54, respectively, the PCR products were used as templates for further amplification using primers specific for overlapping ϳ350-bp segments of the CMV target sequence and incorporating 454 adapter sequences and barcodes specific to individual specimens.…”

“…The increasing adoption of massively parallel high-throughput sequencing technologies is enabling the acquisition of sufficient reads of individual DNA molecules to detail the sequence subpopulations that occur throughout the CMV genome in vivo (4,5). When focused on gene regions involved in drug resistance, it provides a cost-effective approach to deep sequencing whereby a targeted codon range is covered hundreds or thousands of times so that small variant subpopulations can be detected, as we recently showed for CMV (6). This may improve the detection of emerging drug resistance.…”

Treatment of CMV infection after allogeneic hematopoietic stem cell transplantation

“…UL97 kinase mutations, that alter the phosphorylation process, are the most represented cause of CMV-antiviral resistance, Seven most common UL97 mutations account for over 80% of GCVresistant CMV strains whereas UL54 mutations are generally associated with FOS and CDF-resistance though cross-resistance to GCV may be also observed [47]. Newer technologies such as next-generation sequencing are being explored for the detection of genotypic resistance [48]. CMV multidrug-resistance with mutations in both UL97 and UL54 genes usually results from UL54 cross-resistance [49,50].…”