Faculty Collaboration Database

Detection of cytomegalovirus drug resistance mutations by next-generation sequencing. J Clin Microbiol 2013 Nov;51(11):3700-10

Date

08/30/2013

Pubmed ID

23985916

Pubmed Central ID

PMC3889754

DOI

10.1128/JCM.01605-13

Scopus ID

2-s2.0-84886474257 (requires institutional sign-in at Scopus site)   78 Citations

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.

Author List

Sahoo MK, Lefterova MI, Yamamoto F, Waggoner JJ, Chou S, Holmes SP, Anderson MW, Pinsky BA

Author

Matthew W. Anderson MD, PhD Assistant Professor in the Pathology department at Medical College of Wisconsin

MESH terms used to index this publication - Major topics in bold

Adolescent
Adult
Aged
Antiviral Agents
Cytomegalovirus
Cytomegalovirus Infections
DNA, Viral
DNA-Directed DNA Polymerase
Drug Resistance, Viral
Female
High-Throughput Nucleotide Sequencing
Humans
Male
Microbial Sensitivity Tests
Middle Aged
Mutation, Missense
Phosphotransferases (Alcohol Group Acceptor)
Sensitivity and Specificity
Viral Proteins
Young Adult