Interlaboratory assays from the fungal PCR Initiative and the Modimucor Study Group to improve qPCR detection of Mucorales DNA in serum: one more step toward standardization - Université de Picardie Jules Verne
Article Dans Une Revue Journal of Clinical Microbiology Année : 2024

Interlaboratory assays from the fungal PCR Initiative and the Modimucor Study Group to improve qPCR detection of Mucorales DNA in serum: one more step toward standardization

1 LCE - Laboratoire Chrono-environnement (UMR 6249)
2 Service de parasitologie et mycologie [CHRU de Besançon]
3 UHW - University Hospital of Wales
4 Cardiff University
5 AP-HP - Hopital Saint-Louis [AP-HP]
6 Département de Mycologie - Department of Mycology
7 CNRMA - Centre National de Référence Mycoses Invasives et Antifongiques - National Reference Center Invasive Mycoses & Antifungals
8 Mycologie translationnelle - Translational Mycology
9 Hôpital Henri Mondor
10 Unité de Parasitologie-Mycologie, Service de Microbiologie [Hôpital Necker-Enfants-Malades, Paris]
11 ISCIII - Instituto de Salud Carlos III [Madrid]
12 UNIMI - Università degli Studi di Milano = University of Milan
13 Service de Parasitologie-Mycologie [CHRU LIlle]
14 UGSF - Unité de Glycobiologie Structurale et Fonctionnelle - UMR 8576
15 AGIR - Agents infectieux, résistance et chimiothérapie - UR UPJV 4294
16 HUS - Les Hôpitaux Universitaires de Strasbourg
17 IAI - Institut des Agents Infectieux [Lyon]
18 Leopold Franzens Universität Innsbruck - University of Innsbruck
19 Health Services Laboratories [London]
20 UMCU - University Medical Center [Utrecht]
21 WI - Westerdijk Fungal Biodiversity Institute [Utrecht]
22 Statens Serum Institut [Copenhagen]
23 Service de Parasitologie et Mycologie [CHU Toulouse]
24 Infinity - Institut Toulousain des Maladies Infectieuses et Inflammatoires
25 Erasmus MC - Erasmus University Medical Center [Rotterdam]
26 IMU - Innsbruck Medical University = Medizinische Universität Innsbruck
27 University Hospital Brno
28 Radboud University Medical Center [Nijmegen]
29 IICiMed - Cibles et Médicaments des Infections et de l'Immunité - UR 1155
30 CHU Nantes - Centre Hospitalier Universitaire de Nantes = Nantes University Hospital
31 3IHP - Infection Inflammation et Interaction Hôtes Pathogènes [CHU Clermont-Ferrand]
32 University Hospital Wuerzburg / Universitäts­klinikum Würzburg
33 CHU Dijon
34 Medizinische Universität Wien = Medical University of Vienna
35 IRCCS Sacro Cuore Don Calabria Hospital [Vérone, Italie]
36 FPCRI - Fungal PCR Initiative
37 CHRU Besançon - Centre Hospitalier Régional Universitaire de Besançon
Steffi Rocchi
Julie Denis
Damien Dupont
Pieter-Jan Haas
Rasmus Hare
Stephane Valot
  • Fonction : Auteur

Résumé

The aim of this study was to identify parameters influencing DNA extraction and PCR amplification efficiencies in an attempt to standardize Mucorales qPCR. The Fungal PCR Initiative Mucorales Laboratory Working Group distributed two panels of simulated samples to 26 laboratories: Panel A (six sera spiked with Mucorales DNA and one negative control serum) and Panel B (six Mucorales DNA extracts). Panel A underwent DNA extraction in each laboratory according to the local procedure and were sent to a central laboratory for testing using three different qPCR techniques: one in-house qPCR assay and two commercial assays (MucorGenius and Fungiplex). Panel B DNA extracts were PCR amplified in each laboratory using local procedures: nine in-house qPCR assays and two commercial kits (MucorGenius and MycoGENIE). All data were compiled and anonymously analyzed at the central laboratory. For Panel A, a total of six different automated platforms and five manual extraction methods were used. Positive rates were 64%, 70%, and 89%, for the MucorGenius, Fungiplex, and the in-house qPCR assay, respectively. Using a large volume of serum for DNA extraction provided the highest analytical sensitivity (82.5% for 1 mL compared with 62.7% for smaller volumes, P < 0.01). For Panel B, five in-house qPCR assays and two commercial kits had >78% positivity. Using larger PCR input volumes (≥7 µL) was associated with the highest sensitivity at 95.5% compared to 58.3% when lower input volumes were used ( P < 0.01). Using larger sample volumes for nucleic acid extraction and DNA template volumes for PCR amplification significantly improves the performance of Mucorales qPCR when testing serum. IMPORTANCE Mucormycosis is a life-threatening mold infection affecting immunosuppressed patients but also other patients with diabetes or trauma. Better survival is linked to shorter delays in diagnosis and treatment initiation. Detection of Mucorales-free DNA in serum or plasma using quantitative PCR allows a prompt diagnosis and earlier treatment. Several techniques and protocols of quantitative Mucorales PCR are used in Europe, and improving performance remains a common objective of laboratories participating in the fungal PCR Initiative Working Group. This study, which combined results from 26 laboratories in Europe, showed that the main parameters underpinning sensitivity are the preanalytical variables (volume of serum used for DNA extraction and DNA template volume), irrespective of the extraction platforms and qPCR assay/platform.
Fichier principal
Vignette du fichier
rocchi-et-al-2024-interlaboratory-assays-from-the-fungal-pcr-initiative-and-the-modimucor-study-group-to-improve-qpcr.pdf (767.56 Ko) Télécharger le fichier
Origine Fichiers produits par l'(les) auteur(s)
Licence

Dates et versions

hal-04862485 , version 1 (03-01-2025)

Licence

Identifiants

Citer

Steffi Rocchi, Emeline Scherer, P. Lewis White, Audrey Guitton, Alexandre Alanio, et al.. Interlaboratory assays from the fungal PCR Initiative and the Modimucor Study Group to improve qPCR detection of Mucorales DNA in serum: one more step toward standardization. Journal of Clinical Microbiology, In press, pp.e0152524. ⟨10.1128/jcm.01525-24⟩. ⟨hal-04862485⟩
0 Consultations
0 Téléchargements

Altmetric

Partager

More