Interlaboratory comparison of PCR-based identification of Candida and Aspergillus DNA in spiked blood samples Utz Reichard, 1 Dieter Buchheidt, 2 Cornelia Lass-Flo ¨ rl, 3 Juergen Loeffler, 4 Raimond Lugert, 1 Markus Ruhnke, 5 Kathrin Tintelnot, 6 Michael Weig 1 and Uwe Groß 1 1 Department of Medical Microbiology and National Reference Center for Systemic Mycoses, University Hospital of Goettingen, Germany, 2 Department of Internal Medicine, University Hospital of Mannheim, Germany, 3 Department of Hygiene and Clinical Microbiology, University of Innsbruck, Innsbruck, Austria, 4 Department of Internal Medicine, University Hospital of Wuerzburg, Germany, 5 Department of Internal Medicine, Charite ´ University Hospital of Berlin, Germany and 6 Section Mycology, Robert-Koch Institute, Berlin, Germany Summary Despite PCR per se being a powerful and sensitive technique, regarding the detection of fungi in patientsÕ blood, no consensus for a standardised PCR protocol yet exists. To complement other ongoing or accomplished studies which tackle this problem, the German Reference Center for Systemic Mycoses conducted an interlaboratory comparison starting with blood samples spiked with fungal cell elements. Altogether, six laboratories using in-house PCR-protocols from Germany and Austria participated in the trial. Blood samples were spiked with vital cells of Candida albicans or Aspergillus fumigatus. Candida was used in the yeast form, whereas Aspergillus cells were either spiked as conidia or as very young germlings, also known as smoo cells. Spiked blood samples contained between 10 and 10 000 cells ml )1 . Depending on the techniques used for fungal cell disruption and DNA-amplification, detection quality was variable between laboratories, but also differed within single laboratories in different trials particularly for samples spiked with less than 100 cells ml )1 . Altogether, at least regarding the detection of A. fumigatus, two of six laboratories showed constant reliable test results also with low fungal cell number spiked samples. Protocols used by these labs do not differ substantially from others. However, as particularities, one protocol included a conventional phenol chloroform extraction during the DNA preparation process and the other included a real time PCR-protocol based on FRET probes. Other laboratory comparisons on the basis of clinical samples should follow to further evaluate the procedures. The difficulties and problems of such trials in general are discussed. Key words: PCR, interlaboratory comparison, fungus, blood, Aspergillus, diagnosis. Introduction Diagnosis of systemic fungal infections such as deep candidosis and invasive aspergillosis still mainly rely on clinical findings and the cultural or microscopic verifi- cation of Candida or Aspergillus spp. in clinical samples (for current reviews see [1] and [2]). This verification, however, is often dependent on more or less invasive procedures, such as biopsies or bronchoalveolar lavages, which may be a major problem, particularly if patients are in poor general condition. Thus, there is a clear need to improve diagnosis of systemic fungal infections on the basis of antibody or antigen detection from blood samples. In this context, tests based on antibody detection are of minor value. Antibodies do not give a reliable answer whether an infection is systemic or not. The immune system might have been exposed to Correspondence: Utz Reichard, Department of Medical Microbiology, National Reference Center for Systemic Mycoses, University Hospital of Goettingen, Kreuzbergring 57, 37075 Goettingen, Germany. Tel.: +49 551 39 5856. Fax: +49 551 39 5860. E-mail: ureicha@gwdg.de Accepted for publication 16 December 2011 Original article Ó 2012 Blackwell Verlag GmbH doi:10.1111/j.1439-0507.2011.02167.x mycoses Diagnosis,Therapy and Prophylaxis of Fungal Diseases