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

J Clin Virol. 2023 Aug:165:105520. doi: 10.1016/j.jcv.2023.105520. Epub 2023 Jun 8.

Abstract

Cytomegalovirus (CMV) causes severe systemic and tissue-invasive disease in immunocompromised patients, particularly solid organ and hematopoietic stem cell transplant recipients. While antiviral drugs offer promising efficacy, clinical management is complicated by the high frequency of drug resistance-associated mutations. The most commonly encountered mutations occur in the genes encoding for the drug targets: UL54 (DNA polymerase), UL56 (terminase complex), and UL97 (phosphotransferase), conferring resistance to ganciclovir/cidofovir/foscarnet, letermovir, and ganciclovir/maribavir, respectively. Currently, standard practice for detecting drug resistance is sequencing-based genotypic analysis by commercial reference laboratories with strictly prescribed sample requirements and reporting parameters that can often restrict testing in a highly vulnerable population. In order to circumvent these limitations, we developed a dual-step next-generation sequencing (NGS)-based clinical assay that utilizes full-length gene amplification by long-range PCR followed by shotgun sequencing for mutation analysis. This laboratory-developed test (LDT) achieved satisfactory performance with 96.4% accuracy, 100% precision, and an analytical sensitivity of 300IU/mL with 20% allele frequency. Highlighted by two clinical cases, our NGS LDT was able to provide critical results from patient specimens with viral loads <500IU/mL and volumes <0.5 mL - conditions otherwise unacceptable by reference laboratories. Here, we describe the development and implementation of a robust NGS LDT that offers greater testing flexibility and sensitivity to accommodate a more diverse patient population.

Keywords: CMV; Drug resistance; Genotyping; Mutation analysis; NGS; Transplant.

MeSH terms

  • Antiviral Agents / pharmacology
  • Antiviral Agents / therapeutic use
  • Cytomegalovirus Infections* / diagnosis
  • Cytomegalovirus Infections* / drug therapy
  • Cytomegalovirus* / genetics
  • Drug Resistance, Viral / genetics
  • Ganciclovir / therapeutic use
  • Gene Amplification
  • High-Throughput Nucleotide Sequencing / methods
  • Humans
  • Mutation
  • Phosphotransferases (Alcohol Group Acceptor) / genetics
  • Phosphotransferases (Alcohol Group Acceptor) / therapeutic use

Substances

  • Antiviral Agents
  • Ganciclovir
  • Phosphotransferases (Alcohol Group Acceptor)