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. © 2012 Elsevier B.V.

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

Vanni I.;
2012-01-01

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. © 2012 Elsevier B.V.
File in questo prodotto:
Non ci sono file associati a questo prodotto.

I documenti in IRIS sono protetti da copyright e tutti i diritti sono riservati, salvo diversa indicazione.

Utilizza questo identificativo per citare o creare un link a questo documento: https://hdl.handle.net/11567/1035686
Citazioni
  • ???jsp.display-item.citation.pmc??? 6
  • Scopus 13
  • ???jsp.display-item.citation.isi??? 14
social impact