ABSTRACT PCR has been used as an aid in the diagnosis of invasive aspergillosis for almost 2 decades. A lack of standardization has limited both its acceptance as a diagnostic tool and multicenter clinical evaluations, preventing its inclusion in disease-defining criteria. In 2006, the European Aspergillus PCR Initiative was formed. The aim of the initiative was to provide optimal standardized protocols for the widespread clinical evaluation of the Aspergillus PCR to determine its diagnostic role and allow inclusion in disease diagnosis criteria. Quality control panels were developed and circulated to centers for evaluation of the existing methodology before recommendations based on the initial results were proposed for further panels. The centers were anonymously classified as “compliant” or “noncompliant,” according to whether they had followed the proposed recommendations before the performance parameters were determined and meta-regression analysis was performed. Most PCR amplification systems provided similar detection thresholds, although positivity was a function of the fungal burden. When PCR amplification was combined with DNA extraction, 50% of the centers failed to achieve the same level of detection. Meta-regression analysis showed positive correlations between sensitivity and extraction protocols incorporating the proposed recommendations and the use of bead beating, white cell lysis buffer, and an internal control PCR. The use of elution volumes above 100 μl showed a negative correlation with sensitivity. The efficiency of the Aspergillus PCR is limited by the extraction procedure and not by PCR amplification. For PCR testing of whole blood, it is essential that large blood volumes (≥3 ml) be efficiently lysed before bead beating to disrupt the fungal cell and performance of an internal control PCR to exclude false negativity. DNA should be eluted in volumes of <100 μl.
Macrophages and neutrophils kill the airborne fungal pathogen Aspergillus fumigatus. The dependency of this killing process on reactive oxygen intermediates (ROI) has been strongly suggested. Therefore, we investigated the enzymatic ROI detoxifying system by proteome analysis of A. fumigatus challenged by H(2)O(2). Since many of the identified proteins and genes are apparently regulated by a putative Saccharomyces cerevisiae Yap1 homolog, the corresponding gene of A. fumigatus was identified and designated Afyap1. Nuclear localization of a functional AfYap1-eGFP fusion was stress dependent. Deletion of the Afyap1 gene led to drastically increased sensitivity of the deletion mutant against H(2)O(2) and menadione, but not against diamide and NO radicals. Proteome analysis of the DeltaAfyap1 mutant strain challenged with 2 mM H(2)O(2) indicated that 29 proteins are controlled directly or indirectly by AfYap1, including catalase 2. Despite its importance for defense against reactive agents, the Afyap1 deletion mutant did not show attenuated virulence in a murine model of Aspergillus infection. These data challenge the hypothesis that ROI such as superoxide anions and peroxides play a direct role in killing of A. fumigatus in an immunocompromised host. This conclusion was further supported by the finding that killing of A. fumigatus wild-type and DeltaAfyap1 mutant germlings by human neutrophilic granulocytes worked equally well irrespective of whether the ROI scavenger glutathione or an NADPH-oxidase inhibitor was added to the cells.
To determine the sensitivity of polymerase chain reaction (PCR) assays for the diagnosis of invasive aspergillosis, results of quantitative culture, PCR-ELISA, and a quantitative LightCycler assay (Roche Diagnostics) of blood and organ specimens of experimentally infected mice and rabbits were compared. By PCR-ELISA, 297 of 379 murine lung specimens were positive, but only 235 of 379 were culture positive. Whereas 64 culture-negative lungs were positive by PCR, Aspergillus was grown from only 2 PCR-negative samples. The PCR assay was 19.4 times more sensitive than culture. None of the 68 blood cultures from mice and rabbits were positive for Aspergillus fumigatus, whereas PCR detected Aspergillus DNA in 17 of 68 blood samples. Quantitative PCR analysis of blood samples showed a fungus load of 101-102 cfu/mL of blood. The data confirm the superior sensitivity of PCR for the diagnosis of experimental Aspergillus infections.
This is a protocol for a Cochrane Review (Diagnostic test accuracy). The objectives are as follows: To provide an overall summary of the diagnostic accuracy of PCR‐based tests on blood specimens for the diagnosis of IA in the immunocompromised host.
Invasive aspergillosis is a significant threat to health and is a major cause of mortality in immunocompromised individuals. Understanding the interaction between the fungus and the immune system is important in determining how the immunocompetent host remains disease free. Several studies examining the direct interaction between Aspergillus fumigatus and purified innate immune cells have been conducted to measure the responses of both the host cells and the pathogen. It has been revealed that innate immune cells have different modes of action ranging from effective fungal killing by neutrophils to the less aggressive response of dendritic cells. Natural killer cells do not phagocytose the fungus unlike the other innate immune cells mentioned but appear to mediate their antifungal effect through the release of gamma interferon. Transcriptional analysis of A. fumigatus interacting with these cells has indicated that it can adapt to the harsh microenvironment of the phagosome and produces toxins, ribotoxin and gliotoxin, that can induce cell death in the majority of innate immune cells. These data point toward potential novel antifungal treatments including the use of innate immune cells as antifungal vaccines.
ABSTRACT Samples from patients at high risk for invasive aspergillosis (IA) were prospectively collected and analyzed for the presence of molecular markers of fungal infection. Serum specimens were screened for galactomannan and Aspergillus DNA, and whole-blood specimens were screened only for Aspergillus DNA. Fungal infections were categorized according to the European Organization for Research and Treatment of Cancer/Invasive Fungal Infections Cooperative Group, National Institute of Allergy and Infectious Diseases Mycoses Study Group (EORTC/MSG) criteria. Forty-seven cases (proven and probable IA) and 31 controls (no evidence of IA) were selected retrospectively for this case-control study, comprising 803 samples, in order to determine the performance of whole-blood PCR, serum PCR, and serum galactomannan testing. Although no single assay was able to detect every case of IA, a combination of different assays provided the best performance. There was no significant difference between the use of whole-blood and serum specimens for PCR-based diagnosis of IA, but there was a trend for whole blood to be more sensitive (85% versus 79%) and to yield an earlier positive result (36 days versus 15 days) than for serum. However, DNA extraction from serum specimens is easier and faster than that from whole-blood specimens, and it allows the same specimen to be used for both galactomannan and PCR assays. In conclusion, the appropriate sample type for DNA extraction should be determined by the local requirements and the technical platforms available at each individual center. A combination of biomarker tests offered the best diagnostic utility for detecting IA.
This overview of reviews (i.e., an umbrella review) is designed to reappraise the validity of systematic reviews (SRs) and meta-analyses related to the performance of Aspergillus PCR tests for the diagnosis of invasive aspergillosis in immunocompromised patients. The methodological quality of the SRs was assessed using the AMSTAR-2 checklist; the quality of the evidence (QOE) within each SR was appraised following the GRADE approach. Eight out of 12 SRs were evaluated for qualitative and quantitative assessment. Five SRs evaluated Aspergillus PCR on bronchoalveolar lavage fluid (BAL) and three on blood specimens. The eight SRs included 167 overlapping reports (59 evaluating PCR in blood specimens, and 108 in BAL), based on 107 individual primary studies (98 trials with a cohort design, and 19 with a case−control design). In BAL specimens, the mean sensitivity and specificity ranged from 0.57 to 0.91, and from 0.92 to 0.97, respectively (QOE: very low to low). In blood specimens (whole blood or serum), the mean sensitivity ranged from 0.57 to 0.84, and the mean specificity from 0.58 to 0.95 (QOE: low to moderate). Across studies, only a low proportion of AMSTAR-2 critical domains were unmet (1.8%), demonstrating a high quality of methodological assessment. Conclusions. Based on the overall methodological assessment of the reviews included, on average we can have high confidence in the quality of results generated by the SRs.
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