UNLABELLED: 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.

Chrono environnement UMR6249 CNRS University of Franche Comté Besançon Bourgogne Franche Comté France

CIBERINFEC ISCIII CIBER de Enfermedades Infecciosas Instituto de Salud Carlos 3 Madrid Community of Madrid Spain

Department of Biomedical Sciences for Health Università degli Studi di Milano Milan Italy

Department of Internal Medicine 2 WÜ4i University Hospital Wuerzburg Wuerzburg Germany

Department of Internal Medicine Hematology and Oncology University Hospital Brno Brno South Moravian Region Czechia

Department of Medical Microbiology and Infectious Diseases Erasmus MC University Medical Center Rotterdam Rotterdam South Holland The Netherlands

Department of Medical Microbiology Radboud University Medical Center Nijmegen Gelderland the Netherlands

Department of Medical Microbiology University Medical Center Utrecht Utrecht the Netherlands

Division of Clinical Microbiology Department of Laboratory Medicine Medical University of Vienna Vienna Austria

Division of Infection and Immunity Centre for Trials Research Cardiff Wales United Kingdom

Fungal PCR Initiative Nijmegen the Netherlands

Fungal PCR Initiative Verona Italy

Health Services Laboratories London United Kingdom

Institut des Agents Infectieux Service de Parasitologie et Mycologie Médicale Hospices Civils de Lyon Hôpital Croix Rousse Lyon Auvergne Rhône Alpes France

Institut Toulousain des Maladies Infectieuses et Inflammatoires Université de Toulouse CNRS UMR5051 INSERM UMR1291 Universite Paul Sabatier Toulouse Occitanie France

IRCCS Sacro Cuore Don Calabria Hospital Negrar di Valpolicella Verona Italy

Laboratoire de Parasitologie et de Mycologie Médicale Hôpitaux Universitaires de Strasbourg Strasbourg Grand Est France

Laboratoire de Parasitologie et Mycologie Médicales Centre de Biologie Humaine CHU Amiens Picardie Agents Infectieux Résistance et Chimiothérapie UR 4294 Université de Picardie Jules Verne Amiens Hauts de France France

Laboratoire de parasitologie mycologie AP HP Hôpital Saint Louis Paris Île de France France

Laboratoire de Parasitologie Mycologie CHU Clermont Ferrand 3IHP Paris France

Laboratoire de Parasitologie Mycologie Plateforme de Biologie Hospitalo Universitaire Gérard Mack Dijon France

Medical Microbiology and Infectious Diseases Cardiff University School of Medicine Cardiff United Kingdom

Molecular Diagnostics Institute of Hygiene and Medical Microbiology Medical University of Innsbruck Innsbruck Tyrol Austria

Mycology Department Institut Pasteur Université Paris Cité National Reference Center for Invasive Mycoses and Antifungals Translational Mycology research group Paris Île de France France

Mycology Reference Laboratory Centro Nacional de Microbiologia Instituto de Salud Carlos 3 Madrid Community of Madrid Spain

Mycology Research Group Institute for Hygiene and Medical Microbiology Medical University of Innsbruck Innsbruck Tyrol Austria

Mycology Unit Department for Bacteria Parasites and Fungi Statens Serum Institut Copenhagen Capital Region of Denmark Denmark

Nantes Université CHU Nantes Cibles et Médicaments des Infections et de l'Immunité Nantes France

Parasitology Mycology Department Besançon University Hospital Besançon Bourgogne Franche Comté France

Parasitology Mycology Unit Necker Enfants Malades Hospital APHP Paris Île de France France

Public Health Wales Mycology Reference Laboratory University Hospital of Wales Cardiff Wales United Kingdom

Service de Parasitologie Mycologie CHU Toulouse Toulouse France

Unité de Parasitologie Mycologie Département de Prévention Diagnostic et Traitement des Infections CHU Henri Mondor Assistance Publique des Hôpitaux de Paris Créteil France

Université de Lille Inserm U1285 CHU Lille Laboratoire de Parasitologie Mycologie CNRS UMR 8576 UGSF Unité de Glycobiologie Structurale et Fonctionnelle Lille Hauts de France France

Westerdijk Fungal Biodiversity Institute Utrecht the Netherlands

Zobrazit více v PubMed

Cornely OA, Alastruey-Izquierdo A, Arenz D, Chen SCA, Dannaoui E, Hochhegger B, Hoenigl M, Jensen HE, Lagrou K, Lewis RE, et al. . 2019. Global guideline for the diagnosis and management of mucormycosis: an initiative of the European Confederation of Medical Mycology in cooperation with the Mycoses Study Group Education and Research Consortium. Lancet Infect Dis 19:e405–e421. doi:10.1016/S1473-3099(19)30312-3 PubMed DOI PMC

Millon L, Herbrecht R, Grenouillet F, Morio F, Alanio A, Letscher-Bru V, Cassaing S, Chouaki T, Kauffmann-Lacroix C, Poirier P, Toubas D, Augereau O, Rocchi S, Garcia-Hermoso D, Bretagne S, French Mycosis Study Group . 2016. Early diagnosis and monitoring of mucormycosis by detection of circulating DNA in serum: retrospective analysis of 44 cases collected through the French Surveillance Network of Invasive Fungal Infections (RESSIF). Clin Microbiol Infect 22:810. doi:10.1016/j.cmi.2015.12.006 PubMed DOI

Ibrahim AS, Spellberg B, Walsh TJ, Kontoyiannis DP. 2012. Pathogenesis of mucormycosis. Clin Infect Dis 54 Suppl 1:S16–22. doi:10.1093/cid/cir865 PubMed DOI PMC

Millon L, Larosa F, Lepiller Q, Legrand F, Rocchi S, Daguindau E, Scherer E, Bellanger A-P, Leroy J, Grenouillet F. 2013. Quantitative polymerase chain reaction detection of circulating DNA in serum for early diagnosis of mucormycosis in immunocompromised patients. Clin Infect Dis 56:e95–e101. doi:10.1093/cid/cit094 PubMed DOI

Legrand M, Gits-Muselli M, Boutin L, Garcia-Hermoso D, Maurel V, Soussi S, Benyamina M, Ferry A, Chaussard M, Hamane S, Denis B, Touratier S, Guigue N, Fréalle E, Jeanne M, Shaal J-V, Soler C, Mimoun M, Chaouat M, Lafaurie M, Mebazaa A, Bretagne S, Alanio A. 2016. Detection of circulating Mucorales DNA in critically Ill burn patients: preliminary report of a screening strategy for early diagnosis and treatment. Clin Infect Dis 63:1312–1317. doi:10.1093/cid/ciw563 PubMed DOI

Springer J, Lackner M, Ensinger C, Risslegger B, Morton CO, Nachbaur D, Lass-Flörl C, Einsele H, Heinz WJ, Loeffler J. 2016. Clinical evaluation of a Mucorales-specific real-time PCR assay in tissue and serum samples. J Med Microbiol 65:1414–1421. doi:10.1099/jmm.0.000375 PubMed DOI

Senchyna F, Hogan CA, Murugesan K, Moreno A, Ho DY, Subramanian A, Schwenk HT, Budvytiene I, Costa HA, Gombar S, Banaei N. 2021. Clinical accuracy and impact of plasma cell-free DNA fungal polymerase chain reaction panel for noninvasive diagnosis of fungal infection. Clin Infect Dis 73:1677–1684. doi:10.1093/cid/ciab158 PubMed DOI

Millon L, Caillot D, Berceanu A, Bretagne S, Lanternier F, Morio F, Letscher-Bru V, Dalle F, Denis B, Alanio A, Boutoille D, Bougnoux ME, Botterel F, Chouaki T, Charbonnier A, Ader F, Dupont D, Bellanger AP, Rocchi S, Scherer E, Gbaguidi-Haore H, Herbrecht R. 2022. Evaluation of serum Mucorales polymerase chain reaction (PCR) for the diagnosis of mucormycoses: the MODIMUCOR prospective trial. Clin Infect Dis 75:777–785. doi:10.1093/cid/ciab1066 PubMed DOI

Ghelfenstein-Ferreira T, Verdurme L, Alanio A. 2022. Analytical performance of the commercial MucorGenius assay as compared to an in-house qPCR assay to detect Mucorales DNA in serum specimens. J Fungi (Basel) 8:786. doi:10.3390/jof8080786 PubMed DOI PMC

Caillot D, Valot S, Lafon I, Basmaciyan L, Chretien ML, Sautour M, Million L, Legouge C, Payssot A, Dalle F. 2016. Is it time to include CT “Reverse Halo Sign” and qPCR targeting Mucorales in serum to EORTC-MSG criteria for the diagnosis of pulmonary mucormycosis in leukemia patients? Open Forum Infect Dis 3:fw190. doi:10.1093/ofid/ofw190 PubMed DOI PMC

Imbert S, Portejoie L, Pfister E, Tauzin B, Revers M, Uthurriague J, Hernandez-Grande M, Lafon M-E, Jubert C, Issa N, Dumas P-Y, Delhaes L. 2023. A multiplex PCR and DNA-sequencing workflow on serum for the diagnosis and species identification for invasive aspergillosis and mucormycosis. J Clin Microbiol 61:e0140922. doi:10.1128/jcm.01409-22 PubMed DOI PMC

Mercier T, Reynders M, Beuselinck K, Guldentops E, Maertens J, Lagrou K. 2019. Serial detection of circulating mucorales DNA in invasive mucormycosis: a retrospective multicenter evaluation. J Fungi (Basel) 5:113. doi:10.3390/jof5040113 PubMed DOI PMC

Risum M, Helweg-Larsen J, Petersen SL, Kampmann P, Overgaard UM, El Fassi D, Nielsen OJ, Brabrand M, Rubek N, Munksgaard L, Severinsen MT, Nielsen B, Gertsen JB, Gylfe Å, Hjort U, Vourtsi A, Hare RK, Arendrup MC. 2020. Introduction of a comprehensive diagnostic and interdisciplinary management approach in haematological patients with mucormycosis: a pre and post-intervention analysis. J Fungi (Basel) 6:268. doi:10.3390/jof6040268 PubMed DOI PMC

Rocchi S, Scherer E, Mengoli C, Alanio A, Botterel F, Bougnoux ME, Bretagne S, Cogliati M, Cornu M, Dalle F, et al. . 2021. Interlaboratory evaluation of Mucorales PCR assays for testing serum specimens: a study by the fungal PCR initiative and the Modimucor study group. Med Mycol 59:126–138. doi:10.1093/mmy/myaa036 PubMed DOI

Gouzien L, Che D, Cassaing S, Lortholary O, Letscher-Bru V, Paccoud O, Obadia T, Morio F, Moniot M, Cateau E, Bougnoux ME, Chouaki T, Hasseine L, Desoubeaux G, Gautier C, Mahinc-Martin C, Huguenin A, Bonhomme J, Sitbon K, Durand J, Alanio A, Millon L, Garcia-Hermoso D, Lanternier F, French Mycoses Study Group . 2024. Epidemiology and prognostic factors of mucormycosis in France (2012-2022): a cross-sectional study nested in a prospective surveillance programme. Lancet Reg Health Eur 45:101010. doi:10.1016/j.lanepe.2024.101010 PubMed DOI PMC

White PL, Mengoli C, Bretagne S, Cuenca-Estrella M, Finnstrom N, Klingspor L, Melchers WJG, McCulloch E, Barnes RA, Donnelly JP, Loeffler J, European Aspergillus PCR Initiative (EAPCRI) . 2011. Evaluation of Aspergillus PCR protocols for testing serum specimens. J Clin Microbiol 49:3842–3848. doi:10.1128/JCM.05316-11 PubMed DOI PMC

Kuznetsova A, Brockhoff PB, Christensen RHB. 2017. lmerTest package: tests in linear mixed effects models. J Stat Soft 82:1–26. doi:10.18637/jss.v082.i13 DOI

Salehi E, Hedayati MT, Zoll J, Rafati H, Ghasemi M, Doroudinia A, Abastabar M, Tolooe A, Snelders E, van der Lee HA, Rijs AJMM, Verweij PE, Seyedmousavi S, Melchers WJG. 2016. Discrimination of Aspergillosis, Mucormycosis, Fusariosis, and Scedosporiosis in formalin-fixed paraffin-embedded tissue specimens by use of multiple real-time quantitative PCR assays. J Clin Microbiol 54:2798–2803. doi:10.1128/JCM.01185-16 PubMed DOI PMC

Lengerova M, Racil Z, Hrncirova K, Kocmanova I, Volfova P, Ricna D, Bejdak P, Moulis M, Pavlovsky Z, Weinbergerova B, Toskova M, Mayer J. 2014. Rapid detection and identification of mucormycetes in bronchoalveolar lavage samples from immunocompromised patients with pulmonary infiltrates by use of high-resolution melt analysis. J Clin Microbiol 52:2824–2828. doi:10.1128/JCM.00637-14 PubMed DOI PMC

Camp I, Manhart G, Schabereiter-Gurtner C, Spettel K, Selitsch B, Willinger B. 2020. Clinical evaluation of an in-house panfungal real-time PCR assay for the detection of fungal pathogens. Infection 48:345–355. doi:10.1007/s15010-020-01395-7 PubMed DOI PMC

Bernal-Martínez L, Buitrago MJ, Castelli MV, Rodriguez-Tudela JL, Cuenca-Estrella M. 2013. Development of a single tube multiplex real-time PCR to detect the most clinically relevant Mucormycetes species. Clin Microbiol Infect 19:E1–E7. doi:10.1111/j.1469-0691.2012.03976.x PubMed DOI

Loeffler J, Mengoli C, Springer J, Bretagne S, Cuenca-Estrella M, Klingspor L, Lagrou K, Melchers WJG, Morton CO, Barnes RA, Donnelly JP, White PL, European Aspergillus PCR Initiative . 2015. 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. J Clin Microbiol 53:2838–2845. doi:10.1128/JCM.00906-15 PubMed DOI PMC

White PL, Barnes RA, Springer J, Klingspor L, Cuenca-Estrella M, Morton CO, Lagrou K, Bretagne S, Melchers WJG, Mengoli C, Donnelly JP, Heinz WJ, Loeffler J, EAPCRI . 2015. Clinical performance of Aspergillus PCR for testing serum and plasma: a study by the European Aspergillus PCR initiative. J Clin Microbiol 53:2832–2837. doi:10.1128/JCM.00905-15 PubMed DOI PMC

Murugesan K, Hogan CA, Palmer Z, Reeve B, Theron G, Andama A, Somoskovi A, Steadman A, Madan D, Andrews J, Croda J, Sahoo MK, Cattamanchi A, Pinsky BA, Banaei N. 2019. Investigation of preanalytical variables impacting pathogen cell-free DNA in blood and urine. J Clin Microbiol 57:e00782-19. doi:10.1128/JCM.00782-19 PubMed DOI PMC

Mah J, Nicholas V, Tayyar R, Moreno A, Murugesan K, Budvytiene I, Banaei N. 2023. Superior accuracy of Aspergillus plasma cell-free DNA polymerase chain reaction over serum galactomannan for the diagnosis of invasive Aspergillosis. Clin Infect Dis 77:1282–1290. doi:10.1093/cid/ciad420 PubMed DOI

Heldman MR, Ahmed AA, Liu W, Vo A, Keane-Candib J, Stevens-Ayers T, Boeckh M, Blauwkamp TA, Fisher CE, Hill JA. 2024. Serial quantitation of plasma microbial cell-free DNA before and after diagnosis of pulmonary invasive mold infections in hematopoietic cell transplant recipients. J Infect Dis 229:576–587. doi:10.1093/infdis/jiad255 PubMed DOI

White PL, Bretagne S, Klingspor L, Melchers WJG, McCulloch E, Schulz B, Finnstrom N, Mengoli C, Barnes RA, Donnelly JP, Loeffler J, European Aspergillus PCR Initiative . 2010. Aspergillus PCR: one step closer to standardization. J Clin Microbiol 48:1231–1240. doi:10.1128/JCM.01767-09 PubMed DOI PMC