title: Contribution of next generation sequencing to early detection of cytomegalovirus UL97 emerging mutants and viral subpopulations analysis in kidney transplant recipients.
abstract: Cytomegalovirus (CMV) is the major opportunistic virus encountered after transplantation, and resistant variants challenge antiviral treatment. We studied the emergence and evolution of the canonical UL97 L595S mutation in four kidney recipients by comparing Sanger sequencing with a specific next-generation sequencing (NGS) assay, and assessed the global evolution of UL97 gene variability.
  
  
  
  Plasmids harbouring wild-type and/or L595S mutated UL97 genes were used to assess the analytical performances of NGS assay. UL97 gene was retrospectively analysed in patients' samples drawn during CMV infection follow-up, Shannon entropy (Sn) was calculated and phylogenetic analyses were performed.
  
  
  
  Wild-type and L595S plasmids PCR products were mixed to obtain L595S concentrations of 0, 1, 2, 5, 10, 20 and 100%. Mean triplicate NGS results were 0, 0.71, 1.79, 5.30, 13.17, 17 and 100%, respectively, while Sanger sequencing only detected L595S when above 20%. The NGS mean error rate was 0.196±0.07%. In the four patients, emergence of L595S mutation under ganciclovir treatment was followed-up. After foscarnet rescue therapy, leading to undetectable CMV viral load, in two patients, L595S mutant re-emerged, but was only detected by NGS technology (14% and 9.6%). Using NGS data, phylogenetic trees and Sn showed a complex evolution of concomitant viral subpopulations.
  
  
  
  NGS technology allowed a deeper discrimination of the emergence and persistence of a drug resistance mutation, which could be pertinent to investigate when routine Sanger sequencing detects only wild-type strains. Moreover, NGS improved sensitivity helps in studying viral abundance, dynamics and diversity, less approachable with Sanger sequencing.
authors.affiliation: CHU Bordeaux, Laboratoire de Virologie, et Pôle de Biologie, Bordeaux, France; Université de Bordeaux, Microbiologie Fondamentale et Pathogénicité, UMR 5234, Bordeaux, France; Centre National de la Recherche Scientifique, Microbiologie Fondamentale et Pathogénicité, UMR 5234, Bordeaux, France. Electronic address: isabelle.garrigue@chu-bordeaux.fr.
authors.forename: Isabelle
authors.initials: I
authors.lastname: Garrigue
authors.affiliation: CHU Limoges, Laboratoire de Bactériologie-Virologie, Centre National de Référence des Cytomégalovirus, Limoges, France; Université de Limoges, UMR-S1092, Limoges, France; INSERM, U1092, Limoges, France; GenoLim, Genomic Analysis Platform, Université de Limoges, France.
authors.forename: Rémi
authors.initials: R
authors.lastname: Moulinas
authors.affiliation: Université de Bordeaux, Microbiologie Fondamentale et Pathogénicité, UMR 5234, Bordeaux, France; Centre National de la Recherche Scientifique, Microbiologie Fondamentale et Pathogénicité, UMR 5234, Bordeaux, France.
authors.forename: Patricia
authors.initials: P
authors.lastname: Recordon-Pinson
authors.affiliation: Université de Limoges, UMR-S1092, Limoges, France; INSERM, U1092, Limoges, France.
authors.forename: Marie-Laure
authors.initials: ML
authors.lastname: Delacour
authors.affiliation: CHU Limoges, Service de Néphrologie, Limoges, France.
authors.forename: Marie
authors.initials: M
authors.lastname: Essig
authors.affiliation: CHU Bordeaux, Unité de Transplantation Rénale, Bordeaux, France.
authors.forename: Hannah
authors.initials: H
authors.lastname: Kaminski
authors.affiliation: CHU Limoges, Service de Néphrologie, Limoges, France.
authors.forename: Jean-Philippe
authors.initials: JP
authors.lastname: Rerolle
authors.affiliation: CHU Bordeaux, Unité de Transplantation Rénale, Bordeaux, France.
authors.forename: Pierre
authors.initials: P
authors.lastname: Merville
authors.affiliation: CHU Bordeaux, Laboratoire de Virologie, et Pôle de Biologie, Bordeaux, France; Université de Bordeaux, Microbiologie Fondamentale et Pathogénicité, UMR 5234, Bordeaux, France; Centre National de la Recherche Scientifique, Microbiologie Fondamentale et Pathogénicité, UMR 5234, Bordeaux, France.
authors.forename: Hervé
authors.initials: H
authors.lastname: Fleury
authors.affiliation: CHU Limoges, Laboratoire de Bactériologie-Virologie, Centre National de Référence des Cytomégalovirus, Limoges, France; Université de Limoges, UMR-S1092, Limoges, France; INSERM, U1092, Limoges, France; GenoLim, Genomic Analysis Platform, Université de Limoges, France. Electronic address: sophie.alain@unilim.fr.
authors.forename: Sophie
authors.initials: S
authors.lastname: Alain
mesh_terms: D000368:Aged; D000998:Antiviral Agents / Q000627:therapeutic use; D003587:Cytomegalovirus / Q000145:classification / Q000235:genetics*; D003586:Cytomegalovirus Infections / Q000175:diagnosis* / Q000517:prevention & control; D024882:Drug Resistance, Viral; D005260:Female; D017245:Foscarnet / Q000627:therapeutic use; D015774:Ganciclovir / Q000627:therapeutic use; D059014:High-Throughput Nucleotide Sequencing / Q000379:methods*; D006801:Humans; D016030:Kidney Transplantation / Q000009:adverse effects*; D008297:Male; D008875:Middle Aged; D009154:Mutation; D010802:Phylogeny; D012189:Retrospective Studies; D017422:Sequence Analysis, DNA / Q000379:methods*; D019562:Viral Load / Q000187:drug effects
chemical_list: D000998:Antiviral Agents; D017245:Foscarnet; D015774:Ganciclovir
keywords: Antiviral resistance mutation; Cytomegalovirus; L595S UL97 mutation; Next generation sequencing; Shannon entropy