Determining the analytical specificity of PCR-based assays for the diagnosis of IA: What is Aspergillus?

C. Oliver Morton, P. Lewis White, Rosemary A. Barnes, Lena Klingspor, Manuel Cuenca-Estrella, Katrien Lagrou, Stéphane Bretagne, Willem Melchers, Carlo Mengoli, Angela M. Caliendo, Massimo Cogliati, Yvette Debets-Ossenkopp, Rebecca Gorton, Ferry Hagen, Catriona Halliday, Petr Hamal, Kathleen Harvey-Wood, Katia Jaton, Gemma Johnson, Sarah KiddMartina Lengerova, Cornelia Lass-Florl, Chris Linton, Laurence Millon, C. Orla Morrissey, Melinda Paholcsek, Alida Fe Talento, Markus Ruhnke, Birgit Willinger, J. Peter Donnelly, Juergen Loeffler, on behalf of the EAPCRI

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21 Citations (Scopus)

Abstract

A wide array of PCR tests has been developed to aid the diagnosis of invasive aspergillosis (IA), providing technical diversity but limiting standardisation and acceptance. Methodological recommendations for testing blood samples using PCR exist, based on achieving optimal assay sensitivity to help exclude IA. Conversely, when testing more invasive samples (BAL, biopsy, CSF) emphasis is placed on confirming disease, so analytical specificity is paramount. This multicenter study examined the analytical specificity of PCR methods for detecting IA by blind testing a panel of DNA extracted from a various fungal species to explore the range of Aspergillus species that could be detected, but also potential cross reactivity with other fungal species. Positivity rates were calculated and regression analysis was performed to determine any associations between technical specifications and performance. The accuracy of Aspergillus genus specific assays was 71.8%, significantly greater (P < .0001) than assays specific for individual Aspergillus species (47.2%). For genus specific assays the most often missed species were A. lentulus (25.0%), A. versicolor (24.1%), A. terreus (16.1%), A. flavus (15.2%), A. niger (13.4%), and A. fumigatus (6.2%). There was a significant positive association between accuracy and using an Aspergillus genus PCR assay targeting the rRNA genes (P = .0011). Conversely, there was a significant association between rRNA PCR targets and false positivity (P = .0032). To conclude current Aspergillus PCR assays are better suited for detecting A. fumigatus, with inferior detection of most other Aspergillus species. The use of an Aspergillus genus specific PCR assay targeting the rRNA genes is preferential.

Original languageEnglish
Pages (from-to)402-413
Number of pages12
JournalMedical Mycology
Volume55
Issue number4
DOIs
Publication statusPublished - 1 Jun 2017

Keywords

  • analytical specificity
  • Aspergillus PCR
  • cross reactivity
  • detection range

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