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

• Published in: Journal of clinical microbiology 2015-09, Vol.53 (9), p.2838-2845
• Main Authors: Loeffler, Juergen, Mengoli, Carlo, Springer, Jan, Bretagne, Stéphane, Cuenca-Estrella, Manuel, Klingspor, Lena, Lagrou, Katrien, Melchers, Willem J G, Morton, C Oliver, Barnes, Rosemary A, Donnelly, J Peter, White, P Lewis
• Format: Article
• Language: English
• Subjects: Aspergillosis - diagnosis; Aspergillosis - microbiology; Aspergillus fumigatus - genetics; Aspergillus fumigatus - isolation & purification; DNA, Fungal - blood; Humans; Medicin och hälsovetenskap; Models, Biological; Molecular Diagnostic Techniques - methods; Mycology; Plasma - microbiology; Polymerase Chain Reaction - methods; Sensitivity and Specificity; Specimen Handling - methods; Time Factors
• ISSN: 0095-1137; 1098-660X; 1098-660X
• DOI: 10.1128/JCM.00906-15

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.