title: Identification of chromosomally integrated human herpesvirus 6 by droplet digital PCR.
abstract: BACKGROUND
  Human herpesvirus 6 (HHV-6) latently infects a majority of adults. In about 1% of the population HHV-6 exists in a chromosomally integrated form (ciHHV-6) that resides in every somatic and germ cell and can be transmitted through the germ line. Patients with ciHHV-6 have been misdiagnosed and unnecessarily treated for active HHV-6 infection, sometimes with important side effects, based on results from quantitative molecular HHV-6 tests.
  
  
  METHODS
  A droplet digital PCR (ddPCR) assay was developed to identify ciHHV-6 in cellular patient samples by precisely determining the ratio of HHV-6 to cellular DNA. We validated the assay on confirmed ciHHV-6 patient samples and a cell line derived from a ciHHV-6 patient, and we analyzed hematopoietic stem cell transplant patients suspected of having ciHHV-6. We additionally evaluated whether the assay could be applied to stored plasma samples from a study of clinical correlates of HHV-6.
  
  
  RESULTS
  The ddPCR assay accurately identified ciHHV-6 in cellular samples (buffy coat, peripheral blood mononuclear cells), giving a ratio very close to 1 HHV-6/cell [mean (SD), 1.02 (0.03)] in fluorescence in situ hybridization-confirmed sample). In stored plasma samples, the assay performance was set by design to have 100% sensitivity, which resulted in 82% specificity for ciHHV-6.
  
  
  CONCLUSIONS
  The possibility of ciHHV-6 is often overlooked in patients with detectable HHV-6 viral loads by quantitative PCR. Our ddPCR test provides rapid and accurate laboratory identification of ciHHV-6 from easily obtained cellular samples. In addition, the assay provides excellent sensitivity and specificity using stored plasma samples, facilitating retrospective analysis of the clinical significance of ciHHV-6.
authors.affiliation: Departments of Laboratory Medicine and.
authors.forename: Ruth Hall
authors.initials: RH
authors.lastname: Sedlak
authors.forename: Linda
authors.initials: L
authors.lastname: Cook
authors.forename: Meei-Li
authors.initials: ML
authors.lastname: Huang
authors.forename: Amalia
authors.initials: A
authors.lastname: Magaret
authors.forename: Danielle M
authors.initials: DM
authors.lastname: Zerr
authors.forename: Michael
authors.initials: M
authors.lastname: Boeckh
authors.forename: Keith R
authors.initials: KR
authors.lastname: Jerome
mesh_terms: D001402:B-Lymphocytes / Q000821:virology; D002460:Cell Line; D002877:Chromosomes, Human / Q000235:genetics / Q000821:virology; D018380:Hematopoietic Stem Cell Transplantation; D015654:Herpesvirus 6, Human / Q000235:genetics* / Q000302:isolation & purification*; D006801:Humans; D017404:In Situ Hybridization, Fluorescence; D060888:Real-Time Polymerase Chain Reaction / Q000379:methods*; D015203:Reproducibility of Results; D019349:Roseolovirus Infections / Q000821:virology; D012680:Sensitivity and Specificity; D016662:Virus Integration / Q000235:genetics*