TY - JOUR
T1 - Detection of ganciclovir resistance mutations by pyrosequencing in HCMV-infected pediatric patients
AU - Benzi, Fabio
AU - Vanni, Irene
AU - Cassina, Giulia
AU - Ugolotti, Elisabetta
AU - Di Marco, Eddi
AU - Cirillo, Carmela
AU - Cristina, Emilio
AU - Morreale, Giuseppe
AU - Melioli, Giovanni
AU - Malnati, Mauro
AU - Biassoni, Roberto
PY - 2012/5
Y1 - 2012/5
N2 - 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.
AB - 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.
KW - Drug-resistance-associated mutation
KW - HCMV
KW - Pyrosequencing
KW - QPCR
KW - SNPs analysis
KW - UL97 mutations
UR - http://www.scopus.com/inward/record.url?scp=84861911779&partnerID=8YFLogxK
UR - http://www.scopus.com/inward/citedby.url?scp=84861911779&partnerID=8YFLogxK
U2 - 10.1016/j.jcv.2012.01.006
DO - 10.1016/j.jcv.2012.01.006
M3 - Article
C2 - 22300656
AN - SCOPUS:84861911779
VL - 54
SP - 48
EP - 55
JO - Journal of Clinical Virology
JF - Journal of Clinical Virology
SN - 1386-6532
IS - 1
ER -