title: Comparison of two multiplexed PCR assays for the detection of HSV-1, HSV-2, and VZV with extracted and unextracted cutaneous and mucosal specimens.
abstract: BACKGROUND
  Several analyte specific reagents (ASRs) are available for the detection and differentiation of HSV-1, HSV-2, and VZV in clinical specimens. However, there is limited data on the test performance of these reagents used in multiplexed PCR assays.
  
  
  OBJECTIVE
  This study compared the performance of two multiplexed ASR sets for detection of HSV-1, HSV-2, and VZV in dermal specimens.
  
  
  METHODS
  Two commercially available ASRs were combined to produce multiplexed PCR assays for simultaneous detection of HSV-1, HSV-2, and VZV. Seeded samples were used to determine the limit of detection (LOD) for each assay. Patient samples (n=156) were tested in duplicate and results for each method compared to the reference standard of culture. Both extracted and unextracted specimens were used in the study.
  
  
  RESULTS
  Both multiplexed PCR assays showed similar test performance, with minimal LOD differences observed. The LOD was 10(3) copies/mL for HSV-1 and HSV-2 using the Focus assay compared to 5×10(3) copies/mL and 2×10(4) copies/mL, respectively for the EraGen assay. Both assays showed equal performance for VZV with a LOD of 5×10(3) copies/mL. Analytical specificity testing showed no cross reactivity with other selected DNA viruses. Both assays showed similar performance when clinical samples were tested using both extracted and unextracted specimens.
  
  
  CONCLUSIONS
  Commercially available ASRs can be successfully multiplexed for the PCR detection of HSV-1, HSV-2, and VZV using dermal specimens. Either direct testing or nucleic acid extracted specimens can be used with similar performance in these assays.
authors.affiliation: Department of Pathology, St. Jude Children's Research Hospital, Memphis, TN 38105-2794, USA.
authors.forename: Daelynn R
authors.initials: DR
authors.lastname: Buelow
authors.forename: Matthew J
authors.initials: MJ
authors.lastname: Bankowski
authors.forename: Demba
authors.initials: D
authors.lastname: Fofana
authors.forename: Zhengming
authors.initials: Z
authors.lastname: Gu
authors.forename: Stanley
authors.initials: S
authors.lastname: Pounds
authors.forename: Randall T
authors.initials: RT
authors.lastname: Hayden
mesh_terms: D002644:Chickenpox / Q000175:diagnosis* / Q000821:virology; D006566:Herpesviridae Infections / Q000175:diagnosis* / Q000821:virology; D018259:Herpesvirus 1, Human / Q000302:isolation & purification; D018258:Herpesvirus 2, Human / Q000302:isolation & purification; D006801:Humans; D025202:Molecular Diagnostic Techniques / Q000379:methods*; D009092:Mucous Membrane / Q000821:virology*; D060885:Multiplex Polymerase Chain Reaction / Q000379:methods*; D011933:Reagent Kits, Diagnostic; D012680:Sensitivity and Specificity; D012867:Skin / Q000821:virology*; D018140:Varicellovirus / Q000302:isolation & purification; D014773:Virology / Q000379:methods
chemical_list: D011933:Reagent Kits, Diagnostic
keywords: Dermal; HSV; Herpes simplex; PCR; VZV; Varicella zoster