Clinical next-generation sequencing assay combining full-length gene amplification and shotgun sequencing for the detection of CMV drug resistance mutations

Human Cytomegalovirus (HCMV) Genetic Diversity, Drug Resistance Testing and Prevalence of the Resistance Mutations: A Literature Review

“…Moreover, NGS-improved sensitivity helps in studying viral abundance, dynamics and diversity, which are less approachable with Sanger sequencing [ 63 ]. A dual-step NGS-based clinical assay that utilizes full-length gene amplification with a long-range PCR followed by shotgun sequencing for mutation analysis was developed by von Bredow et al Their test achieved satisfactory performance with 96.4% accuracy, 100% precision and an analytical sensitivity of 300 IU/mL with a 20% allele frequency, showing that the implementation of a robust NGS LDT offers greater testing flexibility and sensitivity, accommodating a more diverse patient population [ 64 ]. Streck et al compared NGS to Sanger sequencing and demonstrated two-test agreement for determining antiviral resistance/susceptibility and 88% (22/25) agreement at the level of resistance-associated mutations.…”

Next-generation sequencing for cytomegalovirus genotypic antiviral resistance testing

“…Previous studies have characterized the performance of next-generation sequencing (NGS) for CMV resistance testing and emphasized its increased sensitivity and improved detection of low-frequency mutations ( 8 – 11 ). NGS-based approaches offer several advantages including: (i) improved analytical performance by increasing sensitivity for identifying minor variants and (ii) enhanced efficiency in the clinical laboratory and cost-effectiveness by multiplexing targets and samples.…”

“…A significant challenge in applying NGS for clinical testing is standardization, primarily due to differences in assay design, sequencing platforms, and, most importantly, analysis pipelines. CMV resistance testing by NGS has been evaluated using various sequencing chemistries, including Ion Torrent, Illumina, and Oxford Nanopore Technologies, and resistance mutation analyses were performed using different bioinformatics pipelines ( 8 – 11 ). Quality metrics and thresholds for calling resistance mutations can contribute to sequencing artifacts and false positives, and the quality of data and the rigor of bioinformatics analysis are key factors in ensuring a high-quality NGS-based diagnostic method.…”

“…In conclusion, given the burden of CMV infections and their associated poor outcomes in immunocompromised patients, it is crucial to rapidly identify mutations associated with antiviral resistance and understand their clinical implications. The analytical sensitivity of NGS is reportedly consistently higher than that of Sanger-based methods, which can have a significant clinical utility ( 11 ). NGS is currently the most sensitive and inclusive option; however, due to the high complexity, sequencing-based assays are currently primarily offered by reference laboratories, and thorough analytical and clinical validations are essential before their clinical implementation.…”