Detection of ganciclovir resistance mutations by pyrosequencing in HCMV-infected pediatric patients

Fabio Benzi, Irene Vanni, Giulia Cassina, Elisabetta Ugolotti, Eddi Di Marco, Carmela Cirillo, Emilio Cristina, Giuseppe Morreale, Giovanni Melioli, Mauro Malnati, Roberto Biassoni

Research output: Contribution to journalArticle

Abstract

Background: Human cytomegalovirus (HCMV) is an opportunistic pathogen especially for immuno-suppressed subjects that might develop pharmacological resistance in patients undergoing prolonged antiviral treatment. Ganciclovir (GCV) is the drug used as first choice therapy in affected children and a GCV-resistant phenotype is mainly linked to mutations of the viral protein kinase UL97. Objectives: Here a new quantitative pyrosequence (PSQ) method is presented that allows detection and quantification of the viral species carrying the more frequent UL97 mutations responsible for GCV resistance in clinical samples (>80% of known cases). Study design: The system has been validated using two independent approaches (cloning and sequencing of UL-97 gene fragments and real-time PCR) and clinical samples derived from 3 pediatric patients. Results: The UL97 pyrosequencing analysis has indicated a significant increase of mutant viruses carrying the H520Q and C592G mutations.In particular, the H520Q viral mutation, known to increase GCV resistance (IC50 = 10) increased around 5 times during hospitalization. In addition, C592G (known to have IC50 = 2.9) also increased 3 times. Conclusions: PSQ is a quick, cheap, high throughput and sensitive analysis method to detect GCV-associated resistance mutation useful to follow antiviral therapy in perinatal CMV-infection as well as in immune-suppressed patients.

Original languageEnglish
Pages (from-to)48-55
Number of pages8
JournalJournal of Clinical Virology
Volume54
Issue number1
DOIs
Publication statusPublished - May 2012

Keywords

  • Drug-resistance-associated mutation
  • HCMV
  • Pyrosequencing
  • QPCR
  • SNPs analysis
  • UL97 mutations

ASJC Scopus subject areas

  • Virology
  • Infectious Diseases

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  • Cite this

    Benzi, F., Vanni, I., Cassina, G., Ugolotti, E., Di Marco, E., Cirillo, C., Cristina, E., Morreale, G., Melioli, G., Malnati, M., & Biassoni, R. (2012). Detection of ganciclovir resistance mutations by pyrosequencing in HCMV-infected pediatric patients. Journal of Clinical Virology, 54(1), 48-55. https://doi.org/10.1016/j.jcv.2012.01.006