Mass spectrometry-based comparative sequencing to detect ganciclovir resistance in the UL97 gene of human cytomegalovirus.

Abstract

BACKGROUND:

Persistent infections with herpesviruses such as human cytomegalovirus (HCMV) frequently occur after solid organ or stem cell transplantation, and are due to either failure of the host to immunologically control the virus or emerging resistance of the virus to the antiviral drug(s) used. Antiviral therapy can be guided by viral drug susceptibility testing based on screening for known resistance-inducing mutations in the viral genome. Mass spectrometry-based comparative sequence analysis (MSCSA) might be advantageous for this purpose because of its suitability for semi-automation.

OBJECTIVES:

The applicability of MSCSA to detect sequence polymorphisms and drug resistance-inducing mutations in the HCMV genome was investigated.

STUDY DESIGN:

We analyzed the 3' part of the HCMV UL97 gene, which encodes the kinase that is activated by the commonly used anti-HCMV drug ganciclovir. Sequences obtained by MSCSA of material from HCMV-infected patients (43 samples) and the HCMV type strain were compared to conventional cycle sequencing results.

RESULTS:

In 94.1% of all samples the results obtained by MSCSA of the UL97 gene were identical to those from conventional cycle sequencing. The threshold to detect mutant sequences in a mixture with wild-type material was 20% using either technique. Furthermore, MSCSA was successfully applied to study the development of drug resistance in a patient who developed encephalitis due to ganciclovir-resistant HCMV.

CONCLUSIONS:

MSCSA was found to be equally accurate compared to conventional cycle sequencing in the analysis of UL97 of HCMV.

• Authors: Clara C Posthuma, Martha T van der Beek, Caroline S van der Blij-de Brouwer, Pim L J van der Heiden, Erik W A Marijt, Willy J M Spaan, Eric C J Claas, Christa Nederstigt, Ann C T M Vossen, Eric J Snijder, Aloys C M Kroes
• Journal: Journal of clinical virology : the official publication of the Pan American Society for Clinical Virology (J Clin Virol) Vol. 51 Issue 1 Pg. 25-30 (May 2011) ISSN: 1873-5967 [Electronic] Netherlands
• PMID: 21388868 (Publication Type: Case Reports, Journal Article)
• Copyright: Copyright © 2011 Elsevier B.V. All rights reserved.

Chemical References

• Antiviral Agents
• DNA, Viral
• Phosphotransferases (Alcohol Group Acceptor)
• ganciclovir kinase
• Ganciclovir

Topics

• Antiviral Agents (pharmacology, therapeutic use)
• Base Sequence
• Cytomegalovirus (drug effects, genetics, pathogenicity)
• Cytomegalovirus Infections (drug therapy, virology)
• DNA Mutational Analysis (methods)
• DNA, Viral (analysis, genetics)
• Drug Resistance, Viral (genetics)
• Female
• Ganciclovir (pharmacology, therapeutic use)
• Genotype
• Humans
• Middle Aged
• Molecular Sequence Data
• Molecular Typing
• Mutation
• Phosphotransferases (Alcohol Group Acceptor) (genetics)
• Polymorphism, Single Nucleotide
• Reproducibility of Results
• Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization (methods)