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.

Cardiff University School of Medicine Cardiff UK

CHU Lille Laboratoire de Parasitologie Mycologie F 59000 Lille France and Univ Lille CNRS Inserm CHU Lille Institut Pasteur de Lille U1019 UMR8204 CIIL Centre for Infection and Immunity of Lille F 59000 Lille France

Clinical Mycology reference Laboratory Centre for Infectious Diseases and Microbiology Laboratory Services Institute of Clinical Pathology and Medical Research New South Wales Health Pathology Westmead Hospital and the University of Sydney Australia

Department of Infectious Diseases and the Mycology Reference Centre Manchester Manchester University NHS Foundation Trust; and Division of Infection Immunity and Respiratory Medicine Faculty of Biology Medicine and Health University of Manchester UK

Department of Internal Medicine Hematology and Oncology University Hospital Brno Brno Czech Republic

Department of Medical Microbiology University Medical Centre Utrecht Utrecht The Netherlands

Department of Microbiology Immunology and Transplantation KU Leuven and Department of Laboratory Medicine and National Reference Centre for Mycosis Excellence Centre for Medical Mycology University Hospitals Leuven Leuven Belgium

Department of Parasitology Mycology University Hospital of Besançon Besançon France

Department of Virology St James's Hospital Dublin Ireland

Infectious Diseases Unit San Bonifacio Hospital Verona Italy

Institut Pasteur Molecular Mycology Unit CNRS UMR2000 Paris France

Institute of Clinical Hygiene Medical Microbiology and Infectiology Klinikum Nürnberg Paracelsus Medical University Nuremberg Germany

Institute of Medical Microbiology University Hospital Essen University of Duisburg Essen Essen Germany

Laboratoire de Parasitologie Mycologie Hôpital Saint Louis Groupe Hospitalier Lariboisière Saint Louis Fernand Widal Assistance Publique Hôpitaux de Paris Paris France

Laboratory of Medical Mycology Jining No 1 People's Hospital Jining China

Mycology Reference Centre Manchester Manchester University NHS Foundation Trust; and Division of Infection Immunity and Respiratory Medicine Faculty of Biology Medicine and Health University of Manchester UK

Nijmegen The Netherlands

OLM Diagnostics Newcastle upon Tyne UK

PathoNostics B 5 Maastricht The Netherlands

Public Health Laboratory National Infection Service Public Health England Manchester University NHS Foundation Trust Manchester UK

Public Health Wales Microbiology Cardiff UHW Heath Park Cardiff UK

Radboud University Medical Centre Department of Medical Microbiology Nijmegen The Netherlands

Regional UK Clinical Mycology Network LLP London UK

Université de Paris Paris France

University Hospital Wuerzburg Medical Hospital 2 C11 Wuerzburg Germany

University of Padua Padua Italy

Westerdijk Fungal Biodiversity Institute Utrecht The Netherlands

Citace poskytuje Crossref.org