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

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• 作者：Fabio Benzi^a ; Irene Vanni^b ; Giulia Cassina^d ; Elisabetta Ugolotti^b ; Eddi Di Marco^a ; Carmela Cirillo^a ; Emilio Cristina^c ; Giuseppe Morreale^e ; Giovanni Melioli^a ; Mauro Malnati^d ; Roberto Biassoni^b ; ^{robertobiassoni@ospedale-gaslini.ge.it}
• 关键词：HCMV ; Pyrosequencing ; qPCR ; Drug-resistance-associated mutation ; UL97 mutations ; SNPs analysis
• 刊名：Journal of Clinical Virology
• 出版年：2012
• 期刊代码：150_13866532
• 类别：med
• 出版时间：May, 2012
• 卷：54
• 期：1
• 页码：48-55
• 文件大小：1045 K

摘要

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.