title: New LightCycler PCR for rapid and sensitive quantification of parvovirus B19 DNA guides therapeutic decision-making in relapsing infections.
abstract: Detection of parvovirus B19 DNA offers diagnostic advantages over serology, particularly in persistent infections of immunocompromised patients. A rapid, novel method of B19 DNA detection and quantification is introduced. This method, a quantitative PCR assay, is based on real-time glass capillary thermocycling (LightCycler [LC]) and fluorescence resonance energy transfer (FRET). The PCR assay allowed quantification over a dynamic range of over 7 logs and could quantify as little as 250 B19 genome equivalents (geq) per ml as calculated for plasmid DNA (i.e., theoretically >or=5 geq per assay). Interrater agreement analysis demonstrated equivalence of LC-FRET PCR and conventional nested PCR in the diagnosis of an active B19 infection (kappa coefficient = 0.83). The benefit of the new method was demonstrated in an immunocompromised child with a relapsing infection, who required an attenuation of the immunosuppressive therapy in addition to repeated doses of immunoglobulin to eliminate the virus.
authors.affiliation: Department of Medical Microbiology and Virology, Christian-Albrechts University, Kiel, Germany. timm.harder@lvua-sh.de
authors.forename: T C
authors.initials: TC
authors.lastname: Harder
authors.forename: M
authors.initials: M
authors.lastname: Hufnagel
authors.forename: K
authors.initials: K
authors.lastname: Zahn
authors.forename: K
authors.initials: K
authors.lastname: Beutel
authors.forename: H J
authors.initials: HJ
authors.lastname: Schmitt
authors.forename: U
authors.initials: U
authors.lastname: Ullmann
authors.forename: P
authors.initials: P
authors.lastname: Rautenberg
mesh_terms: D002648:Child; D004279:DNA, Viral / Q000097:blood*; D004735:Energy Transfer; D005260:Female; D005453:Fluorescence; D006801:Humans; D007121:Immunocompetence; D016867:Immunocompromised Host; D010322:Parvoviridae Infections / Q000517:prevention & control / Q000821:virology*; D016732:Parvovirus B19, Human / Q000235:genetics / Q000302:isolation & purification* / Q000502:physiology*; D016133:Polymerase Chain Reaction / Q000295:instrumentation / Q000379:methods; D012008:Recurrence; D012680:Sensitivity and Specificity; D013050:Spectrometry, Fluorescence; D013997:Time Factors
chemical_list: D004279:DNA, Viral