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


OBJECTIVE
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).


METHODS
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.
Clinical Pathology, Istituto Giannina Gaslini, Genova, Italy.
Fabio
F
Benzi
Irene
I
Vanni
Giulia
G
Cassina
Elisabetta
E
Ugolotti
Eddi
E
Di Marco
Carmela
C
Cirillo
Emilio
E
Cristina
Giuseppe
G
Morreale
Giovanni
G
Melioli
Mauro
M
Malnati
Roberto
R
Biassoni
D000998:Antiviral Agents / Q000494:pharmacology*; D003587:Cytomegalovirus / Q000187:drug effects* / Q000235:genetics / Q000302:isolation & purification; D003586:Cytomegalovirus Infections / Q000821:virology*; D004279:DNA, Viral / Q000737:chemistry / Q000235:genetics; D015774:Ganciclovir / Q000494:pharmacology*; D006801:Humans; D007223:Infant; D007231:Infant, Newborn; D008826:Microbial Sensitivity Tests / Q000379:methods; D008969:Molecular Sequence Data; D020125:Mutation, Missense; D017853:Phosphotransferases (Alcohol Group Acceptor) / Q000235:genetics*; D017422:Sequence Analysis, DNA / Q000379:methods*
D000998:Antiviral Agents; D004279:DNA, Viral; D017853:Phosphotransferases (Alcohol Group Acceptor); C075365:ganciclovir kinase; D015774:Ganciclovir