ASPERGILLUS PCR - FORMIDABLE CHALLENGES AND PROGRESS

Lena Klingspor, MD PhD

Author address: 

Karolinska Institute, Department of Laboratory Medicine

Abstract: 

Invasive aspergillosis (IA,) are important causes of morbidity and mortality in immunocompromised patients, with an incidence of 4-10%, and with a mortality rate of 80-90%, in allogeneic stem cell transplant recipients. Conventional diagnostic tests, such as blood culture, show poor sensitivity for the detection of Aspergillus spp. Non-culture based techniques that has been used in the past, has lacked sensitivity and specificity in immuno-compromised 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 a favourable outcome. Especially, the polymerase chain reaction (PCR) offers great promise for the rapid diagnosis of fungal infections, including detection of fungi that does not grow in blood cultures such as Aspergillus spp. At Karolinska University Hospital we have established an assay, using a combination of a manual extraction and a robot for automated extraction of Candida and Aspergillus DNA, in combination with real-time PCR. To asses its clinical applicability, a large number of patient samples, from patients with suspected invasive fungal infection have been analysed with real time PCR. Data will be presented with focus on Aspergillus R-T PCR results in immunocompromised patients However, a range of different PCR assays (conventional-, nested-, real-time- based) have been developed, targeting different gene regions (cytochrome p450, heat shock proteins, 18S, 5.8S, 28S, ITS) and including a variety of amplicon detection methods, such as gel electrophoresis, hybridization with specific probes, ELISA and restriction fragment length polymorphism (RFLP). These molecular assays provide high potential in terms of sensitivity and specificity, but vary widely in their feasibility and are up until now not standardized. Despite of this progress, there are certain questions to be addresses using those assays, such as the risk of contamination with spores and amplicons (which can be minimized by the use of cabinets and real-time PCR assays), the frequency of prospective sampling as well as the number of positive results of a PCR assay required to initiate antifungal therapy. Furthermore, only few commercially available, standardized assays are available. This particular challenge will be addressed by the Working Group "œEAPCRI"œ(European Aspergillus PCR Initiative) under the auspices of ISHAM. Twenty-four centres have started to establish an 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. Besides the use of PCR assays for the diagnosis of IFI in symptomatic patients, this highly sensitive technology can be also performed to preemptively monitor patients at risk to develop IFI Thus, they might help to reduce empirical antifungal therapy and might be valuable tools for early initiation and monitoring of preemptive antifungal therapy. 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.
2008

abstract No: 

204

Full conference title: 

3rd Advances Against Aspergillosis
    • AAA 3rd (2008)