Aspergillus fumigatus induces microRNA-132 in human monocytes and dendritic cells.

Aspergillus fumigatus is responsible for severe and often fatal infections in immunocompromised patients. The human immune response against this pathogenic mould is still not fully understood. Recently, microRNAs (miRNAs) have been characterized as regulators of inflammation and immune response in various diseases. MiRNAs specifically bind to mRNA target sequences, thereby leading to gene silencing by target degradation and/or translational repression. To investigate the possible role of miRNAs during A. fumigatus infection, we studied the expression of two major immune relevant miRNAs, miR-132 and miR-155, in human monocytes and dendritic cells (DCs). Both cell types are crucial for the immune response against A. fumigatus. Here, we demonstrate for the first time that miR-132 and miR-155 are differentially expressed in monocytes and DCs upon stimulation with A. fumigatus or bacterial lipopolysaccharide (LPS). Interestingly, miR-132 was induced by A. fumigatus but not by LPS in both cell types. Our data suggest that miR-132 may be a relevant regulator of the immune response directed against A. fumigatus.

A novel extraction method combining plasma with a whole-blood fraction shows excellent sensitivity and reproducibility for patients at high risk for invasive aspergillosis.

Diagnosis of invasive aspergillosis (IA) is still a major problem in routine clinical practice. Early diagnosis is essential for a good patient prognosis. PCR is a highly sensitive method for the detection of nucleic acids and could play an important role in improving the diagnosis of fungal infections. Therefore, a novel DNA extraction method, ultraclean production (UCP), was developed allowing purification of both cellular and cell-free circulating fungal DNA. In this prospective study we evaluated the commercially available UCP extraction system and compared it to an in-house system. Sixty-three patients at high risk for IA were screened twice weekly, and DNA extracted by both methods was cross-analyzed, in triplicate, by two different real-time PCR assays. The negative predictive values were high for all methods (94.3 to 100%), qualifying them as screening methods, but the sensitivity and diagnostic odds ratios were higher using the UCP extraction method. Sensitivity ranged from 33.3 to 66.7% using the in-house extracts to 100% using the UCP extraction method. Most of the unclassified patients showed no positive PCR results; however, single-positive PCR replicates were observed in some cases. These can bear clinical relevance but should be interpreted with additional clinical and laboratory data. The PCR assays from the UCP extracts showed greater reproducibility than the in-house method for probable IA patients. The standardized UCP extraction method yielded superior results, with regard to sensitivity and reproducibility, than the in-house method. This was independent of the PCR assay used to detect fungal DNA in the sample extracts.

Pathogen-specific DNA enrichment does not increase sensitivity of PCR for diagnosis of invasive aspergillosis in neutropenic patients.

PCR assays designed for the diagnosis of invasive aspergillosis (IA) in high-risk patients have to detect minute amounts of target DNA to reach sufficient analytical sensitivity to be of clinical use. This prospective study assessed the use of a novel strategy for selective pathogen DNA enrichment for enhancing the performance of diagnostic PCR in a direct comparison with a highly sensitive in-house quantitative PCR (qPCR) assay and the galactomannan enzyme-linked immunosorbent assay (ELISA). Surprisingly, and in contrast to experience with other patient groups, the novel protocol for selective pathogen DNA enrichment did not enhance but instead significantly impaired sensitivity. This could be explained by the small amounts of host DNA in the specimens, which were derived mostly from severely neutropenic patients. In the qPCR assay, positive samples required an average of 43.5 amplification cycles (range, 39.2 to 50) for detection in the in-house PCR. Repetitive testing of selected samples showed test positivity to be variable, most likely due to the small amounts of target DNA. Despite this, the in-house protocol proved helpful in the diagnosis of IA, detecting 2 out of 3 patients with probable IA and 10 out of 19 patients with possible IA. Our results underline the necessity for diagnostic PCR protocols that help diagnose IA to be highly sensitive and show that selective pathogen DNA enrichment using affinity purification may not be useful in severely neutropenic patients.