title: Efficient identification of inherited chromosomally integrated human herpesvirus 6 using specimen pooling.
abstract: BACKGROUND
  Human herpesvirus 6 (HHV-6) has a unique ability to integrate into chromosomal telomeres. Vertical transmission via germ cell integration results in offspring with inherited chromosomally integrated (ci)HHV-6 in all nucleated cells, affecting ∼1% of the population.
  
  
  OBJECTIVE
  Inherited ciHHV-6 may be a direct or indirect mediator of human disease, but efficient identification of affected individuals is a fundamental roadblock to larger studies exploring the clinical importance of this condition.
  
  
  METHODS
  A group testing strategy was designed to efficiently identify individuals with inherited ciHHV-6. DNA was extracted from 2496 cellular samples from hematopoietic cell transplant (HCT) donor-recipient pairs. Pools of 12 samples were screened for HHV-6 DNA with quantitative (q)PCR. Individual samples from high positive pools were tested with qPCR, and high positive individual samples were tested for inherited ciHHV-6 using droplet digital (dd)PCR to determine HHV-6 DNA copies/cellular genome.
  
  
  RESULTS
  Thirty-one pools had high positive HHV-6 DNA detection with >10(3) HHV-6 DNA copies/μg. Each pool had one sample with >10(4) copies/μg HHV-6 DNA. Inherited ciHHV-6 was confirmed by ddPCR in every high positive sample (>10(3) HHV-6 DNA copies/μg), yielding a prevalence of 1.5% in HCT recipients and 0.96% in donors. We performed 580 qPCR tests to screen 2496 samples for inherited ciHHV-6, a 77% reduction in testing.
  
  
  CONCLUSIONS
  Inherited ciHHV-6 can be efficiently identified by specimen pooling coupled with modern molecular techniques. This algorithm can be used to facilitate cost-effective identification of patients with inherited ciHHV-6, thereby removing a major hurdle for large-scale study of its clinical impact.
authors.affiliation: Department of Medicine, University of Washington, United States; Vaccine and Infectious Disease Division, Fred Hutchinson Cancer Research Center, United States. Electronic address: jahill3@fredhutch.org.
authors.forename: Joshua A
authors.initials: JA
authors.lastname: Hill
authors.affiliation: Department of Laboratory Medicine, University of Washington, United States.
authors.forename: Ruth
authors.initials: R
authors.lastname: HallSedlak
authors.affiliation: Vaccine and Infectious Disease Division, Fred Hutchinson Cancer Research Center, United States; Department of Laboratory Medicine, University of Washington, United States.
authors.forename: Amalia
authors.initials: A
authors.lastname: Magaret
authors.affiliation: Department of Laboratory Medicine, University of Washington, United States.
authors.forename: Meei-Li
authors.initials: ML
authors.lastname: Huang
authors.affiliation: Department of Medicine, University of Washington, United States; Seattle Children's Research Institute, Seattle, WA, United States.
authors.forename: Danielle M
authors.initials: DM
authors.lastname: Zerr
authors.affiliation: Vaccine and Infectious Disease Division, Fred Hutchinson Cancer Research Center, United States; Department of Laboratory Medicine, University of Washington, United States.
authors.forename: Keith R
authors.initials: KR
authors.lastname: Jerome
authors.affiliation: Department of Medicine, University of Washington, United States; Vaccine and Infectious Disease Division, Fred Hutchinson Cancer Research Center, United States.
authors.forename: Michael
authors.initials: M
authors.lastname: Boeckh
mesh_terms: D000465:Algorithms; D004279:DNA, Viral; D005854:Germ Cells / Q000821:virology; D015654:Herpesvirus 6, Human / Q000502:physiology*; D006801:Humans; D018445:Infectious Disease Transmission, Vertical; D008403:Mass Screening; D060888:Real-Time Polymerase Chain Reaction; D015203:Reproducibility of Results; D019349:Roseolovirus Infections / Q000175:diagnosis / Q000453:epidemiology / Q000635:transmission* / Q000821:virology*; D012680:Sensitivity and Specificity; D016662:Virus Integration
chemical_list: D004279:DNA, Viral
keywords: Chromosomally integrated; Diagnostic; HHV-6; Herpes; PCR