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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

Identifieur interne : 003167 ( Main/Exploration ); précédent : 003166; suivant : 003168

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

Auteurs : Juergen Loeffler [Allemagne] ; Carlo Mengoli [Italie] ; Jan Springer [Allemagne] ; Stéphane Bretagne [France] ; Manuel Cuenca-Estrella [Espagne] ; Lena Klingspor [Suède] ; Katrien Lagrou [Belgique] ; Willem J. G. Melchers [Pays-Bas] ; C. Oliver Morton [Australie] ; Rosemary A. Barnes [Royaume-Uni] ; J. Peter Donnelly [Pays-Bas] ; P. Lewis White [Royaume-Uni]

Source :

RBID : PMC:4540929

Descripteurs français

English descriptors

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.


Url:
DOI: 10.1128/JCM.00906-15
PubMed: 26085614
PubMed Central: 4540929


Affiliations:


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Le document en format XML

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<p>The use of serum or plasma for
<named-content content-type="genus-species">Aspergillus</named-content>
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 (
<italic>P</italic>
= 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,
<named-content content-type="genus-species">Aspergillus</named-content>
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.</p>
</div>
</front>
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<li>Allemagne</li>
<li>Australie</li>
<li>Belgique</li>
<li>Espagne</li>
<li>France</li>
<li>Italie</li>
<li>Pays-Bas</li>
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<li>Suède</li>
</country>
<region>
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<li>District de Basse-Franconie</li>
<li>Gueldre</li>
<li>Svealand</li>
<li>Île-de-France</li>
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<settlement>
<li>Madrid</li>
<li>Nimègue</li>
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<country name="France">
<region name="Île-de-France">
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</country>
<country name="Espagne">
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</country>
<country name="Suède">
<region name="Svealand">
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</country>
<country name="Belgique">
<noRegion>
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</noRegion>
<name sortKey="Lagrou, Katrien" sort="Lagrou, Katrien" uniqKey="Lagrou K" first="Katrien" last="Lagrou">Katrien Lagrou</name>
</country>
<country name="Pays-Bas">
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<country name="Australie">
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<name sortKey="White, P Lewis" sort="White, P Lewis" uniqKey="White P" first="P. Lewis" last="White">P. Lewis White</name>
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</record>

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