title: Analytical Comparison of In Vitro-Spiked Human Serum and Plasma for PCR-Based Detection of Aspergillus fumigatus DNA: a Study by the European Aspergillus PCR Initiative.
abstract: The use of serum or plasma for Aspergillus PCR testing facilitates automated and standardized technology. Recommendations for serum testing are available, and while serum and plasma are regularly considered interchangeable for use in fungal diagnostics, differences in galactomannan enzyme immunoassay (GM-EIA) performance have been reported and are attributed to clot formation. Therefore, it is important to assess plasma PCR testing to determine if previous recommendations for serum are applicable and also to compare analytical performance with that of serum PCR. Molecular methods testing serum and plasma were compared through multicenter distribution of quality control panels, with additional studies to investigate the effect of clot formation and blood fractionation on DNA availability. Analytical sensitivity and time to positivity (TTP) were compared, and a regression analysis was performed to identify variables that enhanced plasma PCR performance. When testing plasma, sample volume, preextraction-to-postextraction volume ratio, PCR volume, duplicate testing, and the use of an internal control for PCR were positively associated with performance. When whole-blood samples were spiked and then fractionated, the analytical sensitivity and TTP were superior when testing plasma. Centrifugation had no effect on DNA availability, whereas the presence of clot material significantly lowered the concentration (P = 0.028). Technically, there are no major differences in the molecular processing of serum and plasma, but the formation of clot material potentially reduces available DNA in serum. During disease, Aspergillus DNA burdens in blood are often at the limits of PCR performance. Using plasma might improve performance while maintaining the methodological simplicity of serum testing.
authors.affiliation: Würzburg University, Würzburg, Germany loeffler_j@ukw.de.
authors.forename: Juergen
authors.initials: J
authors.lastname: Loeffler
authors.affiliation: University of Padua, Padua, Italy.
authors.forename: Carlo
authors.initials: C
authors.lastname: Mengoli
authors.affiliation: Würzburg University, Würzburg, Germany.
authors.forename: Jan
authors.initials: J
authors.lastname: Springer
authors.affiliation: Paris Diderot University, Saint Louis Hospital-APHP, Paris, France.
authors.forename: Stéphane
authors.initials: S
authors.lastname: Bretagne
authors.affiliation: Spanish National Centre for Microbiology, Instituto de Salud Carlos III, Madrid, Spain.
authors.forename: Manuel
authors.initials: M
authors.lastname: Cuenca-Estrella
authors.affiliation: Karolinska University Hospital, Stockholm, Sweden.
authors.forename: Lena
authors.initials: L
authors.lastname: Klingspor
authors.affiliation: National Reference Center for Mycosis, University Hospitals Leuven, Leuven, Belgium Department of Microbiology and Immunology, KU Leuven, Leuven, Belgium.
authors.forename: Katrien
authors.initials: K
authors.lastname: Lagrou
authors.affiliation: Radboud University Medical Centre, Nijmegen, The Netherlands.
authors.forename: Willem J G
authors.initials: WJ
authors.lastname: Melchers
authors.affiliation: University of Western Sydney, Sydney, NSW, Australia.
authors.forename: C Oliver
authors.initials: CO
authors.lastname: Morton
authors.affiliation: Cardiff University, UHW, Cardiff, United Kingdom.
authors.forename: Rosemary A
authors.initials: RA
authors.lastname: Barnes
authors.affiliation: Radboud University Medical Centre, Nijmegen, The Netherlands.
authors.forename: J Peter
authors.initials: JP
authors.lastname: Donnelly
authors.affiliation: Public Health Wales Microbiology Cardiff, Cardiff, United Kingdom.
authors.forename: P Lewis
authors.initials: PL
authors.lastname: White
mesh_terms: D001228:Aspergillosis / Q000175:diagnosis* / Q000382:microbiology; D001232:Aspergillus fumigatus / Q000235:genetics / Q000302:isolation & purification*; D004271:DNA, Fungal / Q000097:blood*; D006801:Humans; D008954:Models, Biological; D025202:Molecular Diagnostic Techniques / Q000379:methods*; D010949:Plasma / Q000382:microbiology*; D016133:Polymerase Chain Reaction / Q000379:methods*; D012680:Sensitivity and Specificity; D013048:Specimen Handling / Q000379:methods*; D013997:Time Factors
chemical_list: D004271:DNA, Fungal