HighlightsHistory of seven fungal species used as models for studying development and pathogenicity.Outline of central stages of their life cycle and their infection processes.Molecular toolkits used to study different aspects of pathogenicity.Insight gained from genome sequencing projects.Current research trends and future challenges.

Different bacteria and fungi live as commensal organisms as part of the human microbiota, but shifts to a pathogenic state potentially leading to septic infections commonly occur in immunocompromised individuals. Several studies have reported synergistic or antagonistic interactions between individual bacteria and fungi which might be of clinical relevance. Here, we present first evidence for the interaction between Klebsiella pneumoniae and several Aspergillus species including A. fumigatus, A. terreus, A. niger and A. flavus which cohabit in the lungs and the intestines. Microbiological and molecular methods were employed to investigate the interaction in vitro, and the results indicate that Klebsiella pneumoniae is able to prevent Aspergillus spp. spore germination and hyphal development. The inhibitory effect is reversible, as demonstrated by growth recovery of Aspergillus spp. upon inhibition or elimination of the bacteria, and is apparently dependent on the physical interaction with metabolically active bacteria. Molecular analysis of Klebsiella-Aspergillus interaction has shown upregulation of Aspergillus cell wall-related genes and downregulation of hyphae-related genes, suggesting that Klebsiella induces cell wall stress response mechanisms and suppresses filamentous growth. Characterization of polymicrobial interactions may provide the basis for improved clinical management of mixed infections by setting the stage for appropriate diagnostics and ultimately for optimized treatment strategies.

Nucleic acid amplification techniques permitting sensitive and rapid screening in patients at risk for invasive fungal infections are an important addition to conventional fungal diagnostic methods. However, contamination with fungal DNA may be a serious threat to the validity of fungal amplification-based assays. Besides rigorous handling procedures to avoid false-positive test results from exogenous sources, we have implemented protocols for comprehensive assessment of fungal contamination in all materials involved in the analytical process. Traces of fungal DNA were found in different commercially available PCR reagents, including lyophilized primers, TaqMan probes, and master mix solutions. These contaminants resulted in a considerable rate of false-positive tests in panfungal real-time PCR analysis. To address this problem, we have established a decontamination protocol based on the activity of a double-strand specific DNase. Using this approach, we have significantly reduced the frequency of false-positive test results attributable to contaminated reagents. On the basis of our findings, we strongly recommend routine monitoring of all reagents used in fungal PCR assays for the presence of relevant contaminants. As long as fungal-grade reagents are not readily available, pretreatment methods facilitating elimination of fungal DNA are critical for reducing the risk of falsepositive results in highly sensitive molecular fungal detection assays. N ucleic acid amplification-based techniques can detect minute quantities of fungal DNA and can enable fast and sensitive screening in patients at risk for invasive fungal infections (IFI). They can overcome the limitations of conventional diagnostic methods that are slow or have inadequate sensitivities (1-3). However, a major drawback of highly sensitive PCR applications is that even low-level contaminations can lead to false-positive results (4). This is particularly evident when broad-range screening methods are applied because their ability to detect contaminating DNA from many different fungal species renders them more vulnerable to trace amounts of nucleic acid contamination than species-specific assays (1). This problem is already well described for universal bacterial assays, where PCR testing based on 16S rRNA gene amplification can be compromised by DNA contamination of reagents derived from bacterial sources such as Taq DNA polymerase or uracil-N-glycosylase (5, 6).Problems with fungal contamination during PCR-based assays have been previously reported. Airborne conidia or spores and trace amounts of fungal nucleic acids in commercially available enzymes used for fungal cell lysis and in components of DNA extraction kits were identified as possible sources of contamination (7-10). Here we provide the first evidence for the presence of fungal DNA contaminants in lyophilized primers and TaqMan probes as well as in master mix solutions for real-time PCR analysis purchased at renowned biotechnology companies. Using a TaqMan-based panfungal PCR assay (11) establi...