Determining the analytical specificity of PCR-based assays for the diagnosis of IA: What is Aspergillus ?

C. Oliver Morton (1) , P. Lewis White (2) , Rosemary Barnes (3) , Lena Klingspor (4) , Manuel Cuenca-Estrella (5) , Katrien Lagrou (6) , Stéphane Bretagne (7) , Willem Melchers (8) , Carlo Mengoli (9) , Angela Caliendo (10) , Massimo Cogliati (11) , Yvette Debets-Ossenkopp (12) , Rebecca Gorton (13) , Ferry Hagen (14) , Catriona Halliday (15) , Petr Hamal (16) , Kathleen Harvey-Wood (17) , Katia Jaton (18) , Gemma Johnson (19) , Sarah Kidd (20) , Martina Lengerova (21) , Cornelia Lass-Flörl (22) , Chris Linton (23) , Laurence Millon (24) , C. Orla Morrissey (25) , Melinda Paholcsek (26) , Alida Fe Talento (27) , Markus Ruhnke (28) , Birgit Willinger (29) , J. Peter Donnelly (8) , Juergen Loeffler (30)

1 Western Sydney University
2 Public Health Wales [Cardiff, Royaume uni]
3 School of Medicine [Cardiff]
4 Karolinska University Hospital [Stockholm]
5 ISC - Instituto de Salud Carlos III [Madrid]
6 UCL - Université Catholique de Louvain = Catholic University of Louvain
7 Université Paris Diderot, Sorbonne Paris Cité, Paris, France
8 Radboud University Medical Center [Nijmegen]
9 Unipd - Università degli Studi di Padova = University of Padua
10 Warren Alpert Medical School of Brown University
11 UNIMI - Università degli Studi di Milano = University of Milan
12 Neuroscience Amsterdam, VU University Medical Centre, 1081HV 1117, Amsterdam
13 Royal Free Hospital [London, UK]
14 Canisius-Wilhelmina Hospital [Nijmegen, The Netherlands]
15 Clinical Mycology Reference Laboratory, Pathology West, Westmead, Australia
16 Palacky University Olomouc
17 Southern General Hospital, Glasgow
18 UNIL - Université de Lausanne = University of Lausanne
19 School of Biological and Chemical Sciences, Queen Mary University of London
20 National Mycology Reference Centre, SA Pathology, Adelaide
21 University Hospital Brno
22 IMU - Innsbruck Medical University = Medizinische Universität Innsbruck
23 Public Health England [London]
24 LCE - Laboratoire Chrono-environnement (UMR 6249)
25 Alfred Health
26 Medical and Health Science Center, University of Debrecen, Debrecen, Hungary
27 Trinity College Dublin
28 Abt. Onkologie und Hämatologie, Med. Klinik u. Poliklinik II
29 Medizinische Universität Wien = Medical University of Vienna
30 JMU - Julius-Maximilians-Universität Würzburg

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.

Mots clés: Aspergillus PCR; analytical specificity; cross reactivity; detection range

Domaines: Mycologie

C. Oliver Morton, P. Lewis White, Rosemary Barnes, Lena Klingspor, Manuel Cuenca-Estrella, et al.. Determining the analytical specificity of PCR-based assays for the diagnosis of IA: What is Aspergillus ?. Medical Mycology, 2016, 55 (4), pp.402-413. ⟨10.1093/mmy/myw093⟩. ⟨hal-01852085⟩