Sensitive and rapid detection of ganciclovir resistance by PCR based MALDI-TOF analysis.
BACKGROUND
Cytomegalovirus (CMV) infection is associated with significant morbidity and mortality in transplant recipients. Resistance against ganciclovir is increasingly observed. According to current guidelines, direct drug resistance testing is not always performed due to high costs and work effort, even when resistance is suspected.


OBJECTIVE
To develop a more sensitive, easy applicable and cost-effective assay as proof of concept for direct drug resistance testing in CMV surveillance of post-transplant patients.


METHODS
Five consecutive plasma samples from a heart transplant patient with a primary CMV infection were analyzed by quantitative real-time polymerase chain reaction (rtPCR) as a surrogate marker for therapy failure, and by direct drug resistance detection assays such as Sanger sequencing and the novel primer extension (PEX) reaction matrix-assisted laser desorption ionization time-of-flight (MALDI-TOF) based method.


RESULTS
This report demonstrates that PEX reaction followed by MALDI-TOF analysis detects the A594V mutation, encoding ganciclovir resistance, ten days earlier compared to Sanger sequencing and more than 30 days prior to an increase in viral load.


CONCLUSIONS
The greatly increased sensitivity and rapid turnaround-time combined with easy handling and moderate costs indicate that this procedure could make a major contribution to improve transplantation outcomes.
Institute for Infectious Diseases, University of Bern, 3010 Bern, Switzerland.
Samuel
S
Zürcher
Catherine
C
Mooser
Alexander Urs
AU
Lüthi
Kathrin
K
Mühlemann
Maria Teresa
MT
Barbani
Paul
P
Mohacsi
Christian
C
Garzoni
Meri
M
Gorgievski-Hrisoho
André
A
Schaller
Lukas
L
Flatz
D000998:Antiviral Agents / Q000494:pharmacology*; D003587:Cytomegalovirus / Q000187:drug effects* / Q000235:genetics; D003586:Cytomegalovirus Infections / Q000175:diagnosis / Q000821:virology; D017931:DNA Primers; D024882:Drug Resistance, Viral / Q000235:genetics*; D005260:Female; D015774:Ganciclovir / Q000494:pharmacology*; D016027:Heart Transplantation / Q000009:adverse effects*; D006801:Humans; D008875:Middle Aged; D009154:Mutation; D060888:Real-Time Polymerase Chain Reaction / Q000191:economics / Q000379:methods; D012680:Sensitivity and Specificity; D017422:Sequence Analysis, DNA; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization / Q000191:economics / Q000379:methods; D033581:Stem Cell Transplantation / Q000009:adverse effects*; D013997:Time Factors
D000998:Antiviral Agents; D017931:DNA Primers; D015774:Ganciclovir