Cytomegalovirus sequence variability, amplicon length, and DNase-sensitive non-encapsidated genomes are obstacles to standardization and commutability of plasma viral load results

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Naegele , K , Lautenschlager , I , Gosert , R , Loginov , R , Bir , K , Helanterä , I , Schaub , S , Khanna , N & Hirsch , H H 2018 , ' Cytomegalovirus sequence variability, amplicon length, and DNase-sensitive non-encapsidated genomes are obstacles to standardization and commutability of plasma viral load results ' , Journal of Clinical Virology , vol. 104 , pp. 39-47 . https://doi.org/10.1016/j.jcv.2018.04.013

Title: Cytomegalovirus sequence variability, amplicon length, and DNase-sensitive non-encapsidated genomes are obstacles to standardization and commutability of plasma viral load results
Author: Naegele, Klaudia; Lautenschlager, Irmeli; Gosert, Rainer; Loginov, Raisa; Bir, Katia; Helanterä, Ilkka; Schaub, Stefan; Khanna, Nina; Hirsch, Hans H.
Contributor: University of Helsinki, Medicum
University of Helsinki, Department of Virology
University of Helsinki, Clinicum
Date: 2018-07
Language: eng
Number of pages: 9
Belongs to series: Journal of Clinical Virology
ISSN: 1386-6532
URI: http://hdl.handle.net/10138/301105
Abstract: Background: Cytomegalovirus (CMV) management post-transplantation relies on quantification in blood, but inter-laboratory and inter-assay variability impairs commutability. An international multicenter study demonstrated that variability is mitigated by standardizing plasma volumes, automating DNA extraction and amplification, and calibration to the 1st-CMV-WHO-International-Standard as in the FDA-approved Roche-CAP/CTMCMV. However, Roche-CAP/CTM-CMV showed under-quantification and false-negative results in a quality assurance program (UK-NEQAS-2014). Objectives: To evaluate factors contributing to quantification variability of CMV viral load and to develop optimized CMV-UL54-QNAT. Study design: The UL54 target of the UK-NEQAS-2014 variant was sequenced and compared to 329 available CMV GenBank sequences. Four Basel-CMV-UL54-QNAT assays of 361 bp, 254 bp, 151 bp, and 95 bp amplicons were developed that only differed in reverse primer positions. The assays were validated using plasmid dilutions, UK-NEQAS-2014 sample, as well as 107 frozen and 69 prospectively collected plasma samples from transplant patients submitted for CMV QNAT, with and without DNase-digestion prior to nucleic acid extraction. Results: Eight of 43 mutations were identified as relevant in the UK-NEQAS-2014 target. All Basel-CMV-UL54 QNATs quantified the UK-NEQAS-2014 but revealed 10-fold increasing CMV loads as amplicon size decreased. The inverse correlation of amplicon size and viral loads was confirmed using 1st-WHO-International-Standard and patient samples. DNase pre-treatment reduced plasma CMV loads by > 90% indicating the presence of unprotected CMV genomic DNA. Conclusions: Sequence variability, amplicon length, and non-encapsidated genomes obstruct standardization and commutability of CMV loads needed to develop thresholds for clinical research and management. Besides regular sequence surveys, matrix and extraction standardization, we propose developing reference calibrators using 100 bp amplicons.
Subject: 3111 Biomedicine
1184 Genetics, developmental biology, physiology
Cytomegalovirus
transplantation
plasma
DNAemia
DNA loads
standardization
SOLID-ORGAN TRANSPLANTATION
Plasma
QUANTITATIVE DETECTION
MANAGEMENT
Standardization
Transplantation
RECIPIENTS
BRONCHOALVEOLAR LAVAGE
REAL-TIME PCR
CMV PNEUMONIA
DISEASE
ORGANIZATION INTERNATIONAL STANDARD
INFECTION
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