Sensitive and rapid detection of ganciclovir resistance by PCR based MALDI-TOF analysis

• Published in: Journal of clinical virology 2012-08, Vol.54 (4), p.359-363
• Main Authors: Zürcher, Samuel, Mooser, Catherine, Lüthi, Alexander Urs, Mühlemann, Kathrin, Barbani, Maria Teresa, Mohacsi, Paul, Garzoni, Christian, Gorgievski-Hrisoho, Meri, Schaller, André, Flatz, Lukas
• Format: Article
• Language: English
• Subjects: Allergy and Immunology; Antiviral Agents - pharmacology; Biological and medical sciences; CMV; Cytomegalovirus - drug effects; Cytomegalovirus - genetics; Cytomegalovirus Infections - diagnosis; Cytomegalovirus Infections - virology; DNA Primers; Drug resistance; Drug Resistance, Viral - genetics; Female; Fundamental and applied biological sciences. Psychology; Ganciclovir; Ganciclovir - pharmacology; Heart Transplantation - adverse effects; Human viral diseases; Humans; Infectious Disease; Infectious diseases; MALDI-TOF; Medical sciences; Microbiology; Middle Aged; Miscellaneous; Mutation; PEX; Real-Time Polymerase Chain Reaction - economics; Real-Time Polymerase Chain Reaction - methods; Sensitivity and Specificity; Sequence Analysis, DNA; Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization - economics; Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization - methods; Stem Cell Transplantation - adverse effects; Time Factors; Transplantation; Viral diseases; Virology
• ISSN: 1386-6532; 1873-5967
• DOI: 10.1016/j.jcv.2012.04.019

Abstract 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. Objectives 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. Study design 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. Conclusion 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.