Determining the analytical specificity of PCR-based assays for the diagnosis of IA: What is Aspergillus?
A wide array of PCR tests has been developed to aid the diagnosis of invasive aspergillosis (IA), providing technical diversity but limiting standardisation and acceptance. Methodological recommendations for testing blood samples using PCR exist, based on achieving optimal assay sensitivity to help exclude IA. Conversely, when testing more invasive samples (BAL, biopsy, CSF) emphasis is placed on confirming disease, so analytical specificity is paramount. This multicenter study examined the analytical specificity of PCR methods for detecting IA by blind testing a panel of DNA extracted from a various fungal species to explore the range of Aspergillus species that could be detected, but also potential cross reactivity with other fungal species. Positivity rates were calculated and regression analysis was performed to determine any associations between technical specifications and performance. The accuracy of Aspergillus genus specific assays was 71.8%, significantly greater (P < .0001) than assays specific for individual Aspergillus species (47.2%). For genus specific assays the most often missed species were A. lentulus (25.0%), A. versicolor (24.1%), A. terreus (16.1%), A. flavus (15.2%), A. niger (13.4%), and A. fumigatus (6.2%). There was a significant positive association between accuracy and using an Aspergillus genus PCR assay targeting the rRNA genes (P = .0011). Conversely, there was a significant association between rRNA PCR targets and false positivity (P = .0032). To conclude current Aspergillus PCR assays are better suited for detecting A. fumigatus, with inferior detection of most other Aspergillus species. The use of an Aspergillus genus specific PCR assay targeting the rRNA genes is preferential.
Western Sydney University, Sydney, Australia.
C Oliver
CO
Morton
Public Health Wales Microbiology Cardiff, UK.
P Lewis
PL
White
Cardiff University School of Medicine, Cardiff.
Rosemary A
RA
Barnes
Karolinska University Hospital, Stockholm, Sweden.
Lena
L
Klingspor
Spanish National Centre for Microbiology, Instituto de Salud Carlos III, Madrid, Spain.
Manuel
M
Cuenca-Estrella
University Hospitals Leuven, Department of Laboratory Medicine and National Reference Center for Mycosis, Leuven, Belgium, Belgium.
Katrien
K
Lagrou
Paris Diderot, Sorbonne Paris Cité University, Faculty of Medicine, Paris, France.
Stéphane
S
Bretagne
Radboud University Medical Centre, Nijmegen, Netherlands.
Willem
W
Melchers
University of Padua, Padua, Italy.
Carlo
C
Mengoli
Department of Medicine, Alpert Medical School of Brown University, Providence, Rhode Island and Aspergillus Technology Consortium, USA.
Angela M
AM
Caliendo
Dip. Scienze Biomediche per la Salute, Università degli Studi di Milano, Milan, Italy.
Massimo
M
Cogliati
VU University Medical Centre, Amsterdam. The Netherlands.
Yvette
Y
Debets-Ossenkopp
Royal Free Hospital, London, United Kingdom.
Rebecca
R
Gorton
Canisius-Wilhelmina Hospital, Nijmegen. The Netherlands.
Ferry
F
Hagen
Clinical Mycology Reference Laboratory, Pathology West, Westmead, Australia.
Catriona
C
Halliday
Department of Microbiology, Faculty of Medicine and Dentistry, Palacky University Olomouc, Czech Republic.
Petr
P
Hamal
Southern General Hospital, Glasgow, Scotland. UK.
Kathleen
K
Harvey-Wood
Institute of Microbiology, University Hospital of Lausanne, Switzerland.
Katia
K
Jaton
Blizard Institute of Cell and Molecular Science, Queen Mary University of London, London, United Kingdom.
Gemma
G
Johnson
National Mycology Reference Centre, SA Pathology, Adelaide. Australia.
Sarah
S
Kidd
Department of Internal Medicine - Hematology and Oncology, University Hospital Brno, Brno, Czech Republic.
Martina
M
Lengerova
Innsbruck Medical University, Innsbruck, Austria.
Cornelia
C
Lass-Florl
UK Mycology Reference Lab, Public Health England, Bristol, United Kingdom.
Chris
C
Linton
Laboratoire de Parasitologie-Mycologie Centre Hospitalier Universitaire, Besançon, France.
Laurence
L
Millon
Alfred Health and Monash University, Melbourne, Australia.
C Orla
CO
Morrissey
University of Debrecen Medical and Health Science Center, Debrecen. Hungary.
Melinda
M
Paholcsek
Department of Clinical Microbiology, Trinity College, Dublin, Ireland.
Alida Fe
AF
Talento
Charité Medical School, University of Berlin, Berlin. Germany.
Markus
M
Ruhnke
Medical University of Vienna, Vienna, Austria.
Birgit
B
Willinger
Radboud University Medical Centre, Nijmegen, Netherlands.
J Peter
JP
Donnelly
Wuerzburg University, Wuerzburg, Germany.
Juergen
J
Loeffler
D001230:Aspergillus / Q000145:classification / Q000235:genetics / Q000302:isolation & purification*; D006801:Humans; D055744:Invasive Pulmonary Aspergillosis / Q000175:diagnosis*; D025202:Molecular Diagnostic Techniques / Q000379:methods*; D016133:Polymerase Chain Reaction / Q000379:methods*; D012680:Sensitivity and Specificity
Aspergillus PCR; analytical specificity; cross reactivity; detection range