title: Aspergillus PCR formidable challenges and progress.
abstract: Invasive aspergillosis (IA) is an important cause of morbidity and mortality in immuno-compromised patients, with an incidence of 4-10%, and with a mortality rate of 80-90%, such as in allogeneic stem cell transplant recipients. Conventional diagnostic tests, like blood culture, are not useful in the diagnosis of Aspergillus spp. fungemia. Furthermore, non-culture based techniques that have been used in the past have lacked sensitivity and specificity in immunocompromised patients. New rapid methods which can detect IA early in the course of disease with high sensitivity and specificity are needed since treating these infections at an early stage is often essential for favourable outcomes. In this regard, the polymerase chain reaction (PCR) offers great promise for the rapid diagnosis of fungal infections, including detection of fungi that do not grow in blood cultures such as Aspergillus spp. At Karolinska University Hospital we have established a diagnostic assay, using a combination of a manual extraction and a robot for automated extraction of Candida and Aspergillus DNA, together with real-time PCR. To asses its clinical applicability, a large number of samples from patients with suspected invasive fungal infection have been analyzed with real time PCR. Data will be presented with focus on Aspergillus R-T PCR results in immunocompromised patients. A range of different PCR assays have been developed, targeting different gene regions and including a variety of amplicon detection methods. These molecular assays provide high potential in terms of sensitivity and specificity, but vary widely in their feasibility and have not, until now, been standardized. Despite this progress, there are certain questions to be addressed using these assays, such as the frequency of prospective sampling, as well as the number of positive results of a PCR assay required to initiate antifungal therapy. Furthermore, there are only a few standardized assays that are commercially available. This particular challenge will be addressed by the Working Group 'EAPCRI' (European Aspergillus PCR Initiative) under the auspices of ISHAM. Twenty-four centres have started to establish a European standard for Aspergillus-PCR. The principal goal of this initiative is to achieve a standard for PCR that can be incorporated into the next revision of the EORTC/MSG definitions for IA. Future prospective studies evaluating the potential benefits of early therapy based on R-T PCR in patients at high risk for IA infections are needed.
authors.affiliation: Karolinska Institute, Department of Laboratory Medicine, Division of Clinical Microbiology, Karolinska University Hospital, Stockholm, Sweden. lena.klingspor@ki.se
authors.forename: Lena
authors.initials: L
authors.lastname: Klingspor
authors.forename: Juergen
authors.initials: J
authors.lastname: Loeffler
mesh_terms: D001230:Aspergillus / Q000235:genetics / Q000302:isolation & purification*; D006801:Humans; D055744:Invasive Pulmonary Aspergillosis / Q000175:diagnosis*; D025202:Molecular Diagnostic Techniques / Q000379:methods; D020953:Neuroaspergillosis / Q000175:diagnosis*; D016133:Polymerase Chain Reaction / Q000379:methods*; D012680:Sensitivity and Specificity