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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
S. Rocchi, E. Scherer, PL. White, A. Guitton, A. Alanio, F. Botterel, ME. Bougnoux, MJ. Buitrago, M. Cogliati, M. Cornu, C. Damiani, J. Denis, D. Dupont, S. Fuchs, R. Gorton, P-J. Haas, F. Hagen, R. Hare, X. Iriart, CHW. Klaassen, M. Lackner, M....
Jazyk angličtina Země Spojené státy americké
Typ dokumentu časopisecké články, hodnotící studie
NLK
Free Medical Journals
od 1975 do Před 6 měsíci
Freely Accessible Science Journals
od 1995 do Před 6 měsíci
Europe PubMed Central
od 1975 do Před 6 měsíci
Open Access Digital Library
od 1975-01-01
Open Access Digital Library
od 1975-01-01
PubMed
39745482
DOI
10.1128/jcm.01525-24
Knihovny.cz E-zdroje
- MeSH
- diagnostické techniky molekulární normy metody MeSH
- DNA fungální * krev genetika MeSH
- kvantitativní polymerázová řetězová reakce * normy metody MeSH
- lidé MeSH
- Mucorales * genetika izolace a purifikace MeSH
- mukormykóza * diagnóza mikrobiologie krev MeSH
- senzitivita a specificita * MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- časopisecké články MeSH
- hodnotící studie MeSH
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.
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 Medical Microbiology University Medical Center Utrecht Utrecht the Netherlands
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
IRCCS Sacro Cuore Don Calabria Hospital Negrar di Valpolicella Verona Italy
Laboratoire de parasitologie mycologie AP HP Hôpital Saint Louis Paris Île de France France
Laboratoire de Parasitologie Mycologie CHU Clermont Ferrand 3IHP Paris France
Nantes Université CHU Nantes Cibles et Médicaments des Infections et de l'Immunité Nantes France
Parasitology Mycology Unit Necker Enfants Malades Hospital APHP Paris Île de France France
Service de Parasitologie Mycologie CHU Toulouse Toulouse France
Westerdijk Fungal Biodiversity Institute Utrecht the Netherlands
Citace poskytuje Crossref.org
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