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Journal Articles Medical Mycology Year : 2020

The Fungal PCR Initiative's evaluation of in-house and commercial Pneumocystis jirovecii qPCR assays: Toward a standard for a diagnostics assay

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P Lewis White
  • Function : Author
Carlo Mengoli
  • Function : Author
Sharon Chen
  • Function : Author
Brendan Crowley
  • Function : Author
Gijs Dingemans
  • Function : Author
Emilie Fréalle
Rebecca L Gorton
  • Function : Author
Malcom Guiver
  • Function : Author
Ferry Hagen
  • Function : Author
Catriona Halliday
  • Function : Author
Gemma Johnson
  • Function : Author
Katrien Lagrou
  • Function : Author
Martina Lengerova
  • Function : Author
Willem Melchers
  • Function : Author
Lily Novak-Frazer
  • Function : Author
Riina Rautemaa-Richardson
  • Function : Author
Joerg Steinmann
  • Function : Author
Mario Cruciani
  • Function : Author
Rosemary Barnes
  • Function : Author
J Peter Donnelly
  • Function : Author
Juergen Loeffler
  • Function : Author
Stéphane Bretagne
Alexandre Alanio

Abstract

Abstract Quantitative real-time PCR (qPCR) is increasingly used to detect Pneumocystis jirovecii for the diagnosis of Pneumocystis pneumonia (PCP), but there are differences in the nucleic acids targeted, DNA only versus whole nucleic acid (WNA), and also the target genes for amplification. Through the Fungal PCR Initiative, a working group of the International Society for Human and Animal Mycology, a multicenter and monocenter evaluation of PCP qPCR assays was performed. For the multicenter study, 16 reference laboratories from eight different countries, performing 20 assays analyzed a panel consisting of two negative and three PCP positive samples. Aliquots were prepared by pooling residual material from 20 negative or positive- P. jirovecii bronchoalveolar lavage fluids (BALFs). The positive pool was diluted to obtain three concentrations (pure 1:1; 1:100; and 1:1000 to mimic high, medium, and low fungal loads, respectively). The monocenter study compared five in-house and five commercial qPCR assays testing 19 individual BALFs on the same amplification platform. Across both evaluations and for all fungal loads, targeting WNA and the mitochondrial small sub-unit (mtSSU) provided the earliest Cq values, compared to only targeting DNA and the mitochondrial large subunit, the major surface glycoprotein or the beta-tubulin genes. Thus, reverse transcriptase-qPCR targeting the mtSSU gene could serve as a basis for standardizing the P. jirovecii load, which is essential if qPCR is to be incorporated into clinical care pathways as the reference method, accepting that additional parameters such as amplification platforms still need evaluation.

Dates and versions

hal-03529754 , version 1 (17-01-2022)

Identifiers

Cite

Maud Gits-Muselli, P Lewis White, Carlo Mengoli, Sharon Chen, Brendan Crowley, et al.. The Fungal PCR Initiative's evaluation of in-house and commercial Pneumocystis jirovecii qPCR assays: Toward a standard for a diagnostics assay. Medical Mycology, 2020, 58 (6), pp.779-788. ⟨10.1093/mmy/myz115⟩. ⟨hal-03529754⟩
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