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

• Published in: Journal of clinical virology Vol. 54; no. 1; pp. 48 - 55
• Main Authors: Benzi, Fabio, Vanni, Irene, Cassina, Giulia, Ugolotti, Elisabetta, Di Marco, Eddi, Cirillo, Carmela, Cristina, Emilio, Morreale, Giuseppe, Melioli, Giovanni, Malnati, Mauro, Biassoni, Roberto
• Format: Journal Article
• Language: English
• Published: Amsterdam Elsevier B.V 01.05.2012; Elsevier
• Subjects: Allergy and Immunology; Antiviral Agents - pharmacology; Biological and medical sciences; Cytomegalovirus - drug effects; Cytomegalovirus - genetics; Cytomegalovirus - isolation & purification; Cytomegalovirus Infections - virology; DNA, Viral - chemistry; DNA, Viral - genetics; Drug-resistance-associated mutation; Fundamental and applied biological sciences. Psychology; Ganciclovir - pharmacology; HCMV; Human viral diseases; Humans; Infant; Infant, Newborn; Infectious Disease; Infectious diseases; Medical sciences; Microbial Sensitivity Tests - methods; Microbiology; Miscellaneous; Molecular Sequence Data; Mutation, Missense; Phosphotransferases (Alcohol Group Acceptor) - genetics; Pyrosequencing; qPCR; Sequence Analysis, DNA - methods; SNPs analysis; UL97 mutations; Viral diseases; Virology; SNPs analysis; qPCR; Drug-resistance-associated mutation; HCMV; UL97 mutations; Pyrosequencing; Human; Resistance; Ganciclovir; Antiviral; Mutation; Detection
• ISSN: 1386-6532; 1873-5967; 1873-5967
• DOI: 10.1016/j.jcv.2012.01.006

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. 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). 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. 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. 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.