Splenic rupture and fungal endocarditis in a pediatric patient with invasive fusariosis after allogeneic hematopoietic stem cell transplantation for aplastic anemia: A case report.

Background: Invasive mold infections are a well-known and life-threatening condition after allogeneic hematopoietic stem cell transplantation (HSCT). While Aspergillus species are recognized as predominant pathogens, Fusarium species should also be considered due to their broad environmental distribution and the expected poor outcome of invasive fusariosis. Particularly, splenic rupture as a complication of disseminated disease has not been reported yet. Case presentation: Two weeks after allogeneic HSCT for severe aplastic anemia, a 16-year-old boy presented with painful, erythematous skin nodules affecting the entire integument. As disseminated mycosis was considered, treatment with liposomal amphotericin B and voriconazole (VCZ) was initiated. Invasive fusariosis was diagnosed after histological and previously unpublished polymerase chain reaction-based examination of skin biopsies. Microbiological tests revealed Fusarium solani species. Despite stable neutrophil engraftment and uninterrupted treatment with VCZ, he developed mold disease-associated splenic rupture with hypovolemic shock and fungal endocarditis. The latter induced a cardiac thrombus and subsequent embolic cerebral infarctions with unilateral hemiparesis. Following cardiac surgery, the patient did not regain consciousness because of diffuse cerebral ischemia, and he died on day +92 after HSCT. Conclusion: Invasive fusariosis in immunocompromised patients is a life-threatening condition. Despite antimycotic treatment adapted to antifungal susceptibility testing, the patient reported here developed uncommon manifestations such as splenic rupture and fungal endocarditis.

Exclusion of Mucorales Co-Infection in a Patient with Aspergillus flavus Sinusitis by Fluorescence In Situ Hybridization (FISH).

Invasive fungal infections are associated with increased mortality in hematological patients. Despite considerable advances in antifungal therapy, the evaluation of suspected treatment failure is a common clinical challenge requiring extensive diagnostic testing to rule out potential causes, such as mixed infections. We present a 64-year-old patient with secondary AML, diabetes mellitus, febrile neutropenia, and sinusitis. While cultures from nasal tissue grew Aspergillus flavus, a microscopic examination of the tissue was suggestive of concomitant mucormycosis. However, fluorescence in situ hybridization (FISH) using specific probes targeting Aspergillus and Mucorales species ruled out mixed infection. This was confirmed by specific qPCR assays amplifying the DNA of Aspergillus, but not of Mucorales. These results provided a rational basis for step-down targeted therapy, i.e., the patient received posaconazole after seven days of calculated dual therapy with liposomal amphotericin B and posaconazole. Despite clinical response to the antifungal therapy, he died due to the progression of the underlying disease within two weeks after diagnosis of fungal infection. Molecular diagnostics applied to tissue blocks may reveal useful information on the etiology of invasive fungal infections, including challenging situations, such as with mixed infections. A thorough understanding of fungal etiology facilitates targeted therapy that may improve therapeutic success while limiting side effects.

A Clinical Case of COVID-19-Associated Pulmonary Aspergillosis (CAPA), Illustrating the Challenges in Diagnosis (Despite Overwhelming Mycological Evidence).

The COVID-19 pandemic has resulted in large numbers of patients requiring critical care management. With the established association between severe respiratory virus infection and invasive pulmonary aspergillosis (7.6% for COVID-19-associated pulmonary aspergillosis (CAPA)), the pandemic places a significant number of patients at potential risk from secondary invasive fungal disease. We described a case of CAPA with substantial supporting mycological evidence, highlighting the need to employ strategic diagnostic algorithms and weighted definitions to improve the accuracy in diagnosing CAPA.

Diagnostic Performance of (1→3)-ß-D-Glucan Alone and in Combination with Aspergillus PCR and Galactomannan in Serum of Pediatric Patients after Allogeneic Hematopoietic Stem Cell Transplantation.

Data on biomarker-assisted diagnosis of invasive aspergillosis (IA) in pediatric patients is scarce. Therefore, we conducted a cohort study over two years including 404 serum specimens of 26 pediatric patients after allogeneic hematopoietic stem cell transplantation (alloSCT). Sera were tested prospectively twice weekly for Aspergillus-specific DNA, galactomannan (GM), and retrospectively for (1→3)-ß-D-glucan (BDG). Three probable IA and two possible invasive fungal disease (IFD) cases were identified using the European Organization for Research and Treatment of Cancer and the Mycoses Study Group (EORTC/MSGERC) 2019 consensus definitions. Sensitivity and specificity for diagnosis of probable IA and possible IFD was 80% (95% confidential interval (CI): 28-99%) and 55% (95% CI: 32-77%) for BDG, 40% (95% CI: 5-85%) and 100% (95% CI: 83-100%) for GM, and 60% (95% CI: 15-95%) and 95% (95% CI: 75-100%) for Aspergillus-specific real-time PCR. However, sensitivities have to be interpreted with great caution due to the limited number of IA cases. Interestingly, the low specificity of BDG was largely caused by false-positive BDG results that clustered around the date of alloSCT. The following strategies were able to increase BDG specificity: two consecutive positive BDG tests for diagnosis (specificity 80% (95% CI: 56-94%)); using an optimized cutoff value of 306 pg/mL (specificity 90% (95% CI: 68-99%)) and testing BDG only after the acute posttransplant phase. In summary, BDG can help to diagnose IA in pediatric alloSCT recipients. However, due to the poor specificity either an increased cutoff value should be utilized or BDG results should be confirmed by an alternative Aspergillus assay.

Genome-scale metabolic modeling underscores the potential of Cutaneotrichosporon oleaginosus ATCC 20509 as a cell factory for biofuel production.

BACKGROUND: Cutaneotrichosporon oleaginosus ATCC 20509 is a fast-growing oleaginous basidiomycete yeast that is able to grow in a wide range of low-cost carbon sources including crude glycerol, a byproduct of biodiesel production. When glycerol is used as a carbon source, this yeast can accumulate more than 50% lipids (w/w) with high concentrations of mono-unsaturated fatty acids. RESULTS: To increase our understanding of this yeast and to provide a knowledge base for further industrial use, a FAIR re-annotated genome was used to build a genome-scale, constraint-based metabolic model containing 1553 reactions involving 1373 metabolites in 11 compartments. A new description of the biomass synthesis reaction was introduced to account for massive lipid accumulation in conditions with high carbon-to-nitrogen (C/N) ratio in the media. This condition-specific biomass objective function is shown to better predict conditions with high lipid accumulation using glucose, fructose, sucrose, xylose, and glycerol as sole carbon source. CONCLUSION: Contributing to the economic viability of biodiesel as renewable fuel, C. oleaginosus ATCC 20509 can effectively convert crude glycerol waste streams in lipids as a potential bioenergy source. Performance simulations are essential to identify optimal production conditions and to develop and fine tune a cost-effective production process. Our model suggests ATP-citrate lyase as a possible target to further improve lipid production.

Polymorphisms within the TNFSF4 and MAPKAPK2 Loci Influence the Risk of Developing Invasive Aspergillosis: A Two-Stage Case Control Study in the Context of the aspBIOmics Consortium.

Here, we assessed whether 36 single nucleotide polymorphisms (SNPs) within the TNFSF4 and MAPKAPK2 loci influence the risk of developing invasive aspergillosis (IA). We conducted a two-stage case control study including 911 high-risk patients diagnosed with hematological malignancies that were ascertained through the aspBIOmics consortium. The meta-analysis of the discovery and replication populations revealed that carriers of the TNFSF4 rs7526628T/T genotype had a significantly increased risk of developing IA (p = 0.00022). We also found that carriers of the TNFSF4 rs7526628T allele showed decreased serum levels of TNFSF14 protein (p = 0.0027), and that their macrophages had a decreased fungicidal activity (p = 0.048). In addition, we observed that each copy of the MAPKAPK2 rs12137965G allele increased the risk of IA by 60% (p = 0.0017), whereas each copy of the MAPKAPK2 rs17013271T allele was estimated to decrease the risk of developing the disease (p = 0.0029). Mechanistically, we found that carriers of the risk MAPKAPK2 rs12137965G allele showed increased numbers of CD38+IgM-IgD- plasmablasts in blood (p = 0.00086), whereas those harboring two copies of the allele had decreased serum concentrations of thymic stromal lymphopoietin (p = 0.00097). Finally, we also found that carriers of the protective MAPKAPK2 rs17013271T allele had decreased numbers of CD27-IgM-IgD- B cells (p = 0.00087) and significantly lower numbers of CD14+ and CD14+CD16- cells (p = 0.00018 and 0.00023). Altogether, these results suggest a role of the TNFSF4 and MAPKAPK2 genes in determining IA risk.

Multiplex PCR Based Strategy for Detection of Fungal Pathogen DNA in Patients with Suspected Invasive Fungal Infections.

A new and easy polymerase chain reaction (PCR) multiplex strategy, for the identification of the most common fungal species involved in invasive fungal infections (IFI) was developed in this work. Two panels with species-specific markers were designed, the Candida Panel for the identification of Candida species, and the Filamentous Fungi Panel for the identification of Aspergillus species and Rhizopusarrhizus. The method allowed the correct identification of all targeted pathogens using extracted DNA or by colony PCR, showed no cross-reactivity with nontargeted species and allowed identification of different species in mixed infections. Sensitivity reached 10 to 1 pg of DNA and was suitable for clinical samples from sterile sites, with a sensitivity of 89% and specificity of 100%. Overall, the study showed that the new method is suitable for the identification of the ten most important fungal species involved in IFI, not only from positive blood cultures but also from clinical samples from sterile sites. The method provides a unique characteristic, of seeing the peak in the specific region of the panel with the correct fluorescence dye, that aids the ruling out of unspecific amplifications. Furthermore, the panels can be further customized, selecting markers for different species and/or resistance genes.

The domestic pig as human-relevant large animal model to study adaptive antifungal immune responses against airborne Aspergillus fumigatus.

Pulmonary mucosal immune response is critical for preventing opportunistic Aspergillus fumigatus infections. Although fungus-specific CD4+ T cells in blood are described to reflect the actual host-pathogen interaction status, little is known about Aspergillus-specific pulmonary T-cell responses. Here, we exploit the domestic pig as human-relevant large animal model and introduce antigen-specific T-cell enrichment in pigs to address Aspergillus-specific T cells in the lung compared to peripheral blood. In healthy, environmentally Aspergillus-exposed pigs, the fungus-specific T cells are detectable in blood in similar frequencies as observed in healthy humans and exhibit a Th1 phenotype. Exposing pigs to 106 cfu/m3 conidia induces a long-lasting accumulation of Aspergillus-specific Th1 cells locally in the lung and also systemically. Temporary immunosuppression during Aspergillus-exposure showed a drastic reduction in the lung-infiltrating antifungal T-cell responses more than 2 weeks after abrogation of the suppressive treatment. This was reflected in blood, but to a much lesser extent. In conclusion, by using the human-relevant large animal model the pig, this study highlights that the blood clearly reflects the mucosal fungal-specific T-cell reactivity in environmentally exposed as well as experimentally exposed healthy pigs. But, immunosuppression significantly impacts the mucosal site in contrast to the initial systemic immune response.

The screening of blood by Aspergillus PCR and galactomannan ELISA precedes BAL detection in patients with proven and probable IA.

Early detection of Aspergillus infection has the potential to facilitate a more effective management of invasive disease. Data from probable/proven cases of invasive aspergillosis (IA) with a positive galactomannan enzyme-linked immunosorbent assay (GM) bronchoalveolar lavage fluid (BALF) was analyzed in respect to serum GM and/or polymerase chain reaction (PCR) screening of blood samples prior to, or concurrent with bronchoscopy. Concurrent serum GM testing is less sensitive than BALF itself. Nevertheless screening of blood using GM or PCR testing detected IA cases earlier (GM: 42% or PCR: 56%), particularly when combined (GM/PCR: 73%). Therefore, regular screening facilitates and improves early detection of IA in patients suffering from acute leukemia.

Detection of Fusarium Species in Clinical Specimens by Probe-Based Real-Time PCR.

The mold Fusarium is a ubiquitous fungus causing plant, animal and human infections. In humans, Fusarium spp. are the major cause of eye infections in patients wearing contact lenses or after local trauma. Systemic infections by Fusarium spp. mainly occur in immunosuppressed patients and can disseminate throughout the human body. Due to high levels of resistance to antifungals a fast identification of the causative agent is an urgent need. By using a probe-based real-time PCR assay specific for the genus Fusarium we analysed several different clinical specimens detecting Fusarium spp. commonly found in clinical samples in Germany. Also, a large collection of lung fluid samples of haematological patients was analysed (n = 243). In these, two samples (0.8%) were reproducibly positive, but only one could be confirmed by sequencing. For this case of probable invasive fungal disease (IFD) culture was positive for Fusarium species. Here we describe a rapid, probe-based real-time PCR assay to specifically detect DNA from a broad range of Fusarium species and its application to clinically relevant specimens.

Treatment with etanercept and low monocyte concentration contribute to the risk of invasive aspergillosis in patients post allogeneic stem cell transplantation.

Invasive aspergillosis (IA) is a life-threatening complication among allogeneic hematopoietic stem cell transplant (alloSCT) recipients. Despite well known risk factors and different available assays, diagnosis of invasive aspergillosis remains challenging. 103 clinical variables from patients with hematological malignancies and subsequent alloSCT were collected. Associations between collected variables and patients with (n = 36) and without IA (n = 36) were investigated by applying univariate and multivariable logistic regression. The predictive power of the final model was tested in an independent patient cohort (23 IA cases and 25 control patients). Findings were investigated further by in vitro studies, which analysed the effect of etanercept on A. fumigatus-stimulated macrophages at the gene expression and cytokine secretion. Additionally, the release of C-X-C motif chemokine ligand 10 (CXCL10) in patient sera was studied. Low monocyte concentration (p = 4.8 × 10-06), severe GvHD of the gut (grade 2-4) (p = 1.08 × 10-02) and etanercept treatment of GvHD (p = 3.5 × 10-03) were significantly associated with IA. Our studies showed that etanercept lowers CXCL10 concentrations in vitro and ex vivo and down-regulates genes involved in immune responses and TNF-alpha signaling. Our study offers clinicians new information regarding risk factors for IA including low monocyte counts and administration of etanercept. After necessary validation, such information may be used for decision making regarding antifungal prophylaxis or closely monitoring patients at risk.

Antifungal-resistant Mucorales in different indoor environments.

This paper sought to address the prevalence of Mucorales in different indoor environments in Portugal. Environmental samples (183 in total) were collected at dwellings (n = 79) and workplaces (bakeries, swine farms, taxis, waste-sorting plants) (n = 93) by passive sampling using electrostatic dust collector (EDC), air-conditioning filters, litter, and/or raw materials. Samples were inoculated onto non-selective MEA and DG18 media and were screened for antifungal drug-resistance in azole-supplemented agar Sabouraud media. A probe-based Mucorales-specific real-time PCR assay (Muc18S) was used to detect Mucorales in complement to conventional culture-based methods. Mucorales order was found as more prevalent in air-conditioning filters from waste-sorting fork lifters (35.7%). Amongst Mucorales isolates able to grow in azole-supplemented media, 16 isolates of Mucor sp., Rhizopus sp. or Rhizomucor sp. were not susceptible to 1 mg/L voriconazole, and four isolates of Mucor sp. or Rhizopus sp. were not susceptible to 4 mg/L itraconazole. In conclusion, combination of the culture-based and molecular methods proved to be reliable for Mucorales order identification in complex environmental samples.

Triacetylfusarinine C: A urine biomarker for diagnosis of invasive aspergillosis.

OBJECTIVES: Early diagnosis of invasive aspergillosis (IA) remains challenging, with available diagnostics being limited by inadequate sensitivities and specificities. Triacetylfusarinine C, a fungal siderophore that has been shown to accumulate in urine in animal models, is a potential new biomarker for diagnosis of IA. METHODS: We developed a method allowing absolute and matrix-independent mass spectrometric quantification of TAFC. Urine TAFC, normalized to creatinine, was determined in 44 samples from 24 patients with underlying hematologic malignancies and probable, possible or no IA according to current EORTC/MSG criteria and compared to other established biomarkers measured in urine and same-day blood samples. RESULTS: TAFC/creatinine sensitivity, specificity, positive and negative likelihood ratio for probable versus no IA (cut-off ≥ 3) were 0.86, 0.88, 6.86, 0.16 per patient. CONCLUSION: For the first time, we provide proof for the occurrence of TAFC in human urine. TAFC/creatinine index determination in urine showed promising results for diagnosis of IA offering the advantages of non-invasive sampling. Sensitivity and specificity were similar as reported for GM determination in serum and bronchoalveolar lavage, the gold standard mycological criterion for IA diagnosis.

Validation of a simplified in vitro Transwell® model of the alveolar surface to assess host immunity induced by different morphotypes of Aspergillus fumigatus.

Interactions between fungal pathogens such as Aspergillus fumigatus with host alveolar epithelium and innate immune cells are crucial in the defense against opportunistic fungal infections. In this study a simplified Transwell® system with a confluent layer of A549 cells acted as a model for the alveolar surface. A. fumigatus and dendritic cells were added to simulate the spatial and cellular complexity in the alveolus. Fungal growth into the lower chamber was validated by galactomannan assays. Addition of moDCs to the upper chamber led to a reduced GM signal and fungal growth, indicating moDC antifungal activity. Minimal cell death was documented by analyses of lactate dehydrogenase concentrations and pro-apoptotic gene expression. Measurement of trans-epithelial dextran blue movement confirmed tightness of the epithelial barrier even in presence of A. fumigatus. Cytokine measurements in supernatants from both chambers of the Transwell® system documented distinct response patterns during early and late stages of epithelial invasion, with A549 cells appearing to make a minimal contribution to cytokine release. Concentrations of cytokines in the lower chamber varied distinctly from the upper chamber, depending on the molecular weight of the cytokines. Low inter-assay variability of fungal biomarkers and cytokines was confirmed, highlighting that in vitro models closely mimicking conditions in the human lung can facilitate reproducible measurement of the dynamics of cytokine release and fungal penetration of host epithelia.

Neochloris oleoabundans is worth its salt: Transcriptomic analysis under salt and nitrogen stress.

Neochloris oleoabundans is an oleaginous microalgal species that can be cultivated in fresh water as well as salt water. Using salt water gives the opportunity to reduce production costs and the fresh water footprint for large scale cultivation. Production of triacylglycerols (TAG) usually includes a biomass growth phase in nitrogen-replete conditions followed by a TAG accumulation phase under nitrogen-deplete conditions. This is the first report that provides insight in the saline resistance mechanism of a fresh water oleaginous microalgae. To better understand the osmoregulatory mechanism of N. oleoabundans during growth and TAG accumulating conditions, the transcriptome was sequenced under four different conditions: fresh water nitrogen-replete and -deplete conditions, and salt water (525 mM dissolved salts, 448mM extra NaCl) nitrogen-replete and -deplete conditions. In this study, several pathways are identified to be responsible for salt water adaptation of N. oleoabundans under both nitrogen-replete and -deplete conditions. Proline and the ascorbate-glutathione cycle seem to be of importance for successful osmoregulation in N. oleoabundans. Genes involved in Proline biosynthesis were found to be upregulated in salt water. This was supported by Nuclear magnetic resonance (NMR) spectroscopy, which indicated an increase in proline content in the salt water nitrogen-replete condition. Additionally, the lipid accumulation pathway was studied to gain insight in the gene regulation in the first 24 hours after nitrogen was depleted. Oil accumulation is increased under nitrogen-deplete conditions in a comparable way in both fresh and salt water. The mechanism behind the biosynthesis of compatible osmolytes can be used to improve N. oleoabundans and other industrially relevant microalgal strains to create a more robust and sustainable production platform for microalgae derived products in the future.

A Comparison of Aspergillus and Mucorales PCR Testing of Different Bronchoalveolar Lavage Fluid Fractions from Patients with Suspected Invasive Pulmonary Fungal Disease.

In patients with hematological malignancies, bronchoalveolar lavage fluid (BALF) specimens are commonly used for the diagnosis of mold infections. However, it is not clear whether the cell pellet (P) or the supernatant fraction (S) of the BALF specimen is optimal for molecular diagnostic testing. Thus, 99 BALF specimens were collected from 96 hematology patients with or without allogeneic hematopoietic stem cell transplant. The cell pellets and supernatants were processed alone and in combination (S/P) for testing by two fungus-specific real-time PCR assays compliant with international recommendations. The results achieved with S/P were revealed to be superior in comparison to those achieved with S and P alone, with the use of each single fraction showing a reduced sensitivity for the detection of Aspergillus DNA (82% and 43% for S and P, respectively). In 57% of the samples, testing of the combination of S and P generated a lower quantification cycle value than testing of S or P alone. Molds would have been missed in 5 and 16 out of 28 samples if only S or P, respectively, was analyzed. No sample was positive by testing of S or P only. Similar results were obtained for the detection of Mucorales DNA in BALF specimens (reduced sensitivity of 67% and 50% for S and P, respectively). Study patients were categorized according to the current European Organization for the Research and Treatment of Cancer/Mycoses Study Group classification for invasive fungal disease (IFD), revealing that 35 patients had proven/probable IFD (36%), 47 patients had possible IFD (49%), and 14 patients had undetermined IFD (15%).

Comparative Analysis of Inflammatory Cytokine Release and Alveolar Epithelial Barrier Invasion in a Transwell® Bilayer Model of Mucormycosis.

Understanding the mechanisms of early invasion and epithelial defense in opportunistic mold infections is crucial for the evaluation of diagnostic biomarkers and novel treatment strategies. Recent studies revealed unique characteristics of the immunopathology of mucormycoses. We therefore adapted an alveolar Transwell® A549/HPAEC bilayer model for the assessment of epithelial barrier integrity and cytokine response to Rhizopus arrhizus, Rhizomucor pusillus, and Cunninghamella bertholletiae. Hyphal penetration of the alveolar barrier was validated by 18S ribosomal DNA detection in the endothelial compartment. Addition of dendritic cells (moDCs) to the alveolar compartment led to reduced fungal invasion and strongly enhanced pro-inflammatory cytokine response, whereas epithelial CCL2 and CCL5 release was reduced. Despite their phenotypic heterogeneity, the studied Mucorales species elicited the release of similar cytokine patterns by epithelial and dendritic cells. There were significantly elevated lactate dehydrogenase concentrations in the alveolar compartment and epithelial barrier permeability for dextran blue of different molecular weights in Mucorales-infected samples compared to Aspergillus fumigatus infection. Addition of monocyte-derived dendritic cells further aggravated LDH release and epithelial barrier permeability, highlighting the influence of the inflammatory response in mucormycosis-associated tissue damage. An important focus of this study was the evaluation of the reproducibility of readout parameters in independent experimental runs. Our results revealed consistently low coefficients of variation for cytokine concentrations and transcriptional levels of cytokine genes and cell integrity markers. As additional means of model validation, we confirmed that our bilayer model captures key principles of Mucorales biology such as accelerated growth in a hyperglycemic or ketoacidotic environment or reduced epithelial barrier invasion upon epithelial growth factor receptor blockade by gefitinib. Our findings indicate that the Transwell® bilayer model provides a reliable and reproducible tool for assessing host response in mucormycosis.

Monascus ruber as cell factory for lactic acid production at low pH.

A Monascus ruber strain was isolated that was able to grow on mineral medium at high sugar concentrations and 175g/l lactic acid at pH 2.8. Its genome and transcriptomes were sequenced and annotated. Genes encoding lactate dehydrogenase (LDH) were introduced to accomplish lactic acid production and two genes encoding pyruvate decarboxylase (PDC) were knocked out to subdue ethanol formation. The strain preferred lactic acid to glucose as carbon source, which hampered glucose consumption and therefore also lactic acid production. Lactic acid consumption was stopped by knocking out 4 cytochrome-dependent LDH (CLDH) genes, and evolutionary engineering was used to increase the glucose consumption rate. Application of this strain in a fed-batch fermentation resulted in a maximum lactic acid titer of 190g/l at pH 3.8 and 129g/l at pH 2.8, respectively 1.7 and 2.2 times higher than reported in literature before. Yield and productivity were on par with the best strains described in literature for lactic acid production at low pH.

A prospective study of fungal biomarkers to improve management of invasive fungal diseases in a mixed specialty critical care unit.

PURPOSE: The diagnosis of invasive fungal diseases (IFD) in critical care patients (CrCP) is difficult. The study investigated the performance of a set of biomarkers for diagnosis of IFD in a mixed specialty critical care unit (CrCU). METHODS: A prospective observational study in patients receiving critical care for ≥7days was performed. Serum samples were tested for the presence of: (1-3) - ß-d-glucan (BDG), galactomannan (GM), and Aspergillus fumigatus DNA. GM antigen detection was also performed on bronchoalveolar lavage (BAL) samples. The patients were classified using published definitions for IFD and a diagnostic algorithm for invasive pulmonary aspergillosis. Performance parameters of the assays were determined. RESULTS: In patients with proven and probable IFD, the sensitivity, specificity, PPV and NPV of a single positive BDG were 63%, 83%, 65% and 83% respectively. Specificity increased to 86% with 2 consecutive positive results. The mean BDG value of patients with proven and probable IFD was significantly higher compared to those with fungal colonization and no IFD (p value<0.0001). CONCLUSION: New diagnostic criteria which incorporate these biomarkers, in particular BDG, and host factors unique to critical care patients should enhance diagnosis of IFD and positively impact antifungal stewardship programs.

Genus- and Species-Specific PCR Detection Methods.

PCR-based detection of fungal pathogens offers a sensitive and specific tool for the diagnosis of invasive fungal infections. A large variety of different clinical specimen types can be used as original material. However, certain precautions, in addition to the published MIQE guidelines [1], need to be taken to prevent contaminations from airborne fungal spores and PCR reagents. In addition, the European Aspergillus PCR Initiative (EAPCRI) recently defined standards for Aspergillus PCR [2, 3], following these recommendations leads to superior sensitivity. The combination of fungal PCR with the galactomannan ELISA assay increases the sensitivity for the detection of Aspergillus DNA from blood, compared to a single assay only [4, 5].