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

Maud Gits-Muselli (1, 2) , P. Lewis White (3) , Carlo Mengoli (4) , Sharon Chen (5) , Brendan Crowley (6) , Gijs Dingemans , Emilie Fréalle (7, 8) , Rebecca Gorton , Malcom Guiver (9) , Ferry Hagen (10) , Catriona Halliday (5) , Gemma Johnson , Katrien Lagrou (11) , Martina Lengerova (12) , Willem Jg Melchers (13) , Lily Novak-Frazer (9) , Riina Rautemaa-Richardson (9) , Emeline Scherer (14) , Joerg Steinmann (15) , Mario Cruciani (16) , Rosemary Barnes (17) , J. Peter Donnelly (18) , Juergen Loeffler (19) , Stéphane Bretagne (1, 2) , Alexandre Alanio (1, 2)

1 Mycologie moléculaire - Molecular Mycology
2 Laboratoire de Parasitologie-Mycologie [CHU Saint Louis, Paris]
3 IUHW Hospital - International University of Health and Welfare Hospital
4 Unipd - Università degli Studi di Padova = University of Padua
5 The University of Sydney
6 St James's University Hospital
7 CHRU Lille - Centre Hospitalier Régional Universitaire [CHU Lille]
8 CIIL - Centre d’Infection et d’Immunité de Lille - INSERM U 1019 - UMR 9017 - UMR 8204
9 MFT - Manchester University NHS Foundation Trust
10 University Medical Center [Utrecht]
11 University Hospitals Leuven [Leuven]
12 MENDELU - Mendel University in Brno
13 Radboud University Medical Center [Nijmegen]
14 CHRU Besançon - Centre Hospitalier Régional Universitaire de Besançon
15 Universität Duisburg-Essen = University of Duisburg-Essen [Essen]
16 Ospedale Fracastoro San Bonifacio [Verona]
17 Cardiff University
18 Radboud University [Nijmegen]
19 JMU - Julius-Maximilians-Universität Würzburg

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 qPCR Initiative, a working group of the International Society for Human and Animal Mycology, a multicentre and monocentre evaluation of PCP qPCR assays was performed. For the multicentre study, 16 reference laboratories from eight different countries, performing 20 assays analysed 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 monocentre 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

Domaines: Mycologie; Systématique, phylogénie et taxonomie; Maladies infectieuses

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: towards a standard for a diagnostics assay. 2019. ⟨pasteur-02337518⟩