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.

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%).

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.

Prospective Biomarker Screening for Diagnosis of Invasive Aspergillosis in High-Risk Pediatric Patients.

Combined biomarker screening is increasingly used to diagnose invasive aspergillosis (IA) in high-risk patients. In adults, the combination of galactomannan (GM) and fungal DNA detection has proven to be beneficial in the diagnosis of IA. Data in purely pediatric cohorts are scarce. Here, we monitored 39 children shortly before and after allogeneic stem cell transplantation twice weekly by use of a commercial GM enzyme-linked immunosorbent assay (ELISA) and a PCR assay based on amplification of the pan-Aspergillus ITS1/5.8S ribosomal operon. In addition, clinical data were recorded and classification of IA was performed according to the European Organization for the Research and Treatment of Cancer/Mycoses Study Group (EORTC/MSG) criteria. Among the 39 high-risk children, we identified 4 patients (10.3%) with probable and 2 (5.1%) with possible IA. All patients with probable IA were repeatedly positive for both tests (means of 9.5 and 6.8 positive GM and PCR samples, respectively), whereas both possible IA cases were detected by PCR. The sensitivity and specificity were, respectively, 67% and 89% for GM and 100% and 63% for PCR. Positive and negative predictive values were, respectively, 50% and 100% for GM and 27% and 100% for PCR. For the combined testing approach, both values were 100%. The number of positive samples seemed to be lower in patients undergoing antifungal therapy. Sporadically positive tests occurred in 12% (GM) and 42% (PCR) of unclassified patients. In summary, our data show that combined monitoring for GM and fungal DNA also results in a high diagnostic accuracy in pediatric patients. Future studies have to determine whether combined testing is suitable for early detection of subclinical disease and how antifungal prophylaxis impacts assay performance.

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.

Proteomic Profiling of Serological Responses to Aspergillus fumigatus Antigens in Patients with Invasive Aspergillosis.

Aspergillus fumigatus is the species that most commonly causes the opportunistic infection invasive aspergillosis (IA) in patients being treated for hematological malignancies. Little is known about the A. fumigatus proteins that trigger the production of Aspergillus-specific IgG antibodies during the course of IA. To characterize the serological response to A. fumigatus protein antigens, mycelial proteins were separated by 2-D gel electrophoresis. The gels were immunoblotted with sera from patients with probable and proven IA and control patients without IA. We identified 49 different fungal proteins, which gave a positive IgG antibody signal. Most of these antigens play a role in primary metabolism and stress responses. Overall, our analysis identified 18 novel protein antigens from A. fumigatus. To determine whether these antigens can be used as diagnostic or prognostic markers or exhibit a protective activity, we employed supervised machine learning with decision trees. We identified two candidates for further analysis, the protein antigens CpcB and Shm2. Heterologously produced Shm2 induced a strongly proinflammatory response in human peripheral blood mononuclear cells after in vitro stimulation. In contrast, CpcB did not activate the immune response of PBMCs. These findings could serve as the basis for the development of an immunotherapy of IA.

Comparison of Performance Characteristics of Aspergillus PCR in Testing a Range of Blood-Based Samples in Accordance with International Methodological Recommendations.

Standardized methodologies for the molecular detection of invasive aspergillosis (IA) have been established by the European Aspergillus PCR Initiative for the testing of whole blood, serum, and plasma. While some comparison of the performance of Aspergillus PCR when testing these different sample types has been performed, no single study has evaluated all three using the recommended protocols. Standardized Aspergillus PCR was performed on 423 whole-blood pellets (WBP), 583 plasma samples, and 419 serum samples obtained from hematology patients according to the recommendations. This analysis formed a bicenter retrospective anonymous case-control study, with diagnosis according to the revised European Organization for Research and Treatment of Cancer/Invasive Fungal Infections Cooperative Group and National Institute of Allergy and Infectious Diseases Mycoses Study Group (EORTC/MSG) consensus definitions (11 probable cases and 36 controls). Values for clinical performance using individual and combined samples were calculated. For all samples, PCR positivity was significantly associated with cases of IA (for plasma, P = 0.0019; for serum, P = 0.0049; and for WBP, P = 0.0089). Plasma PCR generated the highest sensitivity (91%); the sensitivities for serum and WBP PCR were 80% and 55%, respectively. The highest specificity was achieved when testing WBP (96%), which was significantly superior to the specificities achieved when testing serum (69%, P = 0.0238) and plasma (53%, P = 0.0002). No cases were PCR negative in all specimen types, and no controls were PCR positive in all specimens. This study confirms that Aspergillus PCR testing of plasma provides robust performance while utilizing commercial automated DNA extraction processes. Combining PCR testing of different blood fractions allows IA to be both confidently diagnosed and excluded. A requirement for multiple PCR-positive plasma samples provides similar diagnostic utility and is technically less demanding. Time to diagnosis may be enhanced by testing multiple contemporaneously obtained sample types.

Precultivation of Bacillus coagulans DSM2314 in the presence of furfural decreases inhibitory effects of lignocellulosic by-products during L(+)-lactic acid fermentation.

By-products resulting from thermo-chemical pretreatment of lignocellulose can inhibit fermentation of lignocellulosic sugars to lactic acid. Furfural is such a by-product, which is formed during acid pretreatment of lignocellulose. pH-controlled fermentations with 1 L starting volume, containing YP medium and a mixture of lignocellulosic by-products, were inoculated with precultures of Bacillus coagulans DSM2314 to which 1 g/L furfural was added. The addition of furfural to precultures resulted in an increase in L(+)-lactic acid productivity by a factor 2 to 1.39 g/L/h, an increase in lactic acid production from 54 to 71 g and an increase in conversion yields of sugar to lactic acid from 68 to 88 % W/W in subsequent fermentations. The improved performance was not caused by furfural consumption or conversion, indicating that the cells acquired a higher tolerance towards this by-product. The improvement coincided with a significant elongation of B. coagulans cells. Via RNA-Seq analysis, an upregulation of pathways involved in the synthesis of cell wall components such as bacillosamine, peptidoglycan and spermidine was observed in elongated cells. Furthermore, the gene SigB and genes promoted by SigB, such as NhaX and YsnF, were upregulated in the presence of furfural. These genes are involved in stress responses in bacilli.

Analytical Comparison of In Vitro-Spiked Human Serum and Plasma for PCR-Based Detection of Aspergillus fumigatus DNA: a Study by the European Aspergillus PCR Initiative.

The use of serum or plasma for Aspergillus PCR testing facilitates automated and standardized technology. Recommendations for serum testing are available, and while serum and plasma are regularly considered interchangeable for use in fungal diagnostics, differences in galactomannan enzyme immunoassay (GM-EIA) performance have been reported and are attributed to clot formation. Therefore, it is important to assess plasma PCR testing to determine if previous recommendations for serum are applicable and also to compare analytical performance with that of serum PCR. Molecular methods testing serum and plasma were compared through multicenter distribution of quality control panels, with additional studies to investigate the effect of clot formation and blood fractionation on DNA availability. Analytical sensitivity and time to positivity (TTP) were compared, and a regression analysis was performed to identify variables that enhanced plasma PCR performance. When testing plasma, sample volume, preextraction-to-postextraction volume ratio, PCR volume, duplicate testing, and the use of an internal control for PCR were positively associated with performance. When whole-blood samples were spiked and then fractionated, the analytical sensitivity and TTP were superior when testing plasma. Centrifugation had no effect on DNA availability, whereas the presence of clot material significantly lowered the concentration (P = 0.028). Technically, there are no major differences in the molecular processing of serum and plasma, but the formation of clot material potentially reduces available DNA in serum. During disease, Aspergillus DNA burdens in blood are often at the limits of PCR performance. Using plasma might improve performance while maintaining the methodological simplicity of serum testing.

Clinical Performance of Aspergillus PCR for Testing Serum and Plasma: a Study by the European Aspergillus PCR Initiative.

Aspergillus PCR testing of serum provides technical simplicity but with potentially reduced sensitivity compared to whole-blood testing. With diseases for which screening to exclude disease represents an optimal strategy, sensitivity is paramount. The associated analytical study confirmed that DNA concentrations were greater in plasma than those in serum. The aim of the current investigation was to confirm analytical findings by comparing the performance of Aspergillus PCR testing of plasma and serum in the clinical setting. Standardized Aspergillus PCR was performed on plasma and serum samples concurrently obtained from hematology patients in a multicenter retrospective anonymous case-control study, with cases diagnosed according to European Organization for Research and Treatment of Cancer/Invasive Fungal Infections Cooperative Group and the National Institute of Allergy and Infectious Diseases Mycoses Study Group (EORTC/MSG) consensus definitions (19 proven/probable cases and 42 controls). Clinical performance and clinical utility (time to positivity) were calculated for both kinds of samples. The sensitivity and specificity for Aspergillus PCR when testing serum were 68.4% and 76.2%, respectively, and for plasma, they were 94.7% and 83.3%, respectively. Eighty-five percent of serum and plasma PCR results were concordant. On average, plasma PCR was positive 16.8 days before diagnosis and was the earliest indicator of infection in 13 cases, combined with other biomarkers in five cases. On average, serum PCR was positive 10.8 days before diagnosis and was the earliest indicator of infection in six cases, combined with other biomarkers in three cases. These results confirm the analytical finding that the sensitivity of Aspergillus PCR using plasma is superior to that using serum. PCR positivity occurs earlier when testing plasma and provides sufficient sensitivity for the screening of invasive aspergillosis while maintaining methodological simplicity.

High-yield lithiation of azobenzenes by tin-lithium exchange.

The lithiation of halogenated azobenzenes by halogen-lithium exchange commonly leads to substantial degradation of the azo group to give hydrazine derivatives besides the desired aryl lithium species. Yields of quenching reactions with electrophiles are therefore low. This work shows that a transmetalation reaction of easily accessible stannylated azobenzenes with methyllithium leads to a near-quantitative lithiation of azobenzenes in para, meta, and ortho positions. To investigate the scope of the reaction, various lithiated azobenzenes were quenched with a variety of electrophiles. Furthermore, mechanistic (119) Sn NMR spectroscopic studies on the formation of lithiated azobenzenes are presented. A tin ate complex of the azobenzene was detected and characterized at low temperature.

Metabolic engineering of itaconate production in Escherichia coli.

Interest in sustainable development has led to efforts to replace petrochemical-based monomers with biomass-based ones. Itaconic acid, a C5-dicarboxylic acid, is a potential monomer for the chemical industry with many prospective applications. cis-aconitate decarboxylase (CadA) is the key enzyme of itaconate production, converting the citric acid cycle intermediate cis-aconitate into itaconate. Heterologous expression of cadA from Aspergillus terreus in Escherichia coli resulted in low CadA activities and production of trace amounts of itaconate on Luria-Bertani (LB) medium (<10 mg/L). CadA was primarily present as inclusion bodies, explaining the low activity. The activity was significantly improved by using lower cultivation temperatures and mineral medium, and this resulted in enhanced itaconate titres (240 mg/L). The itaconate titre was further increased by introducing citrate synthase and aconitase from Corynebacterium glutamicum and by deleting the genes encoding phosphate acetyltransferase and lactate dehydrogenase. These deletions in E. coli's central metabolism resulted in the accumulation of pyruvate, which is a precursor for itaconate biosynthesis. As a result, itaconate production in aerobic bioreactor cultures was increased up to 690 mg/L. The maximum yield obtained was 0.09 mol itaconate/mol glucose. Strategies for a further improvement of itaconate production are discussed.

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.

Combined real-time PCR and galactomannan surveillance improves diagnosis of invasive aspergillosis in high risk patients with haematological malignancies.

Invasive aspergillosis (IA) is a leading complication of intensive treatment for haematological malignancies. Earlier diagnosis should facilitate effective antifungal therapy and prevent progression to invasive disease, which is often lethal. Polymerase chain reaction (PCR) assays, targeting the 28S and ITS ribosomal gene regions respectively, were evaluated for early detection of Aspergillus DNA and for diagnostic utility in patients receiving treatment in two busy haematopoietic stem cell transplant centres. Patients undergoing intensive chemotherapy, autologous or allogeneic transplant were eligible for inclusion in the study. EDTA blood and serum samples for circulating Aspergillus biomarkers, including galactomannan (GM), were collected twice-weekly on a prospective basis from all study patients who were categorized according to international consensus criteria for defining invasive fungal disease (IFD). Of 278 patients recruited there were 44 probable IA cases and only one proven case. Moderate sensitivity and specificity, poor positive predictive value (50-80%), but good negative predictive value (>80-90%) were common to both PCR assays. Overall biomarker performance could be improved by combining positive results of either PCR assay with GM taken within a 12-d period. The addition of PCR to GM monitoring in high-risk patients with haematological malignancies provides greater diagnostic accuracy in invasive aspergillosis.

Multicenter comparison of serum and whole-blood specimens for detection of Aspergillus DNA in high-risk hematological patients.

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.

Analysis of the polymerization initiation and activity of Pasteurella multocida heparosan synthase PmHS2, an enzyme with glycosyltransferase and UDP-sugar hydrolase activity.

Heparosan synthase catalyzes the polymerization of heparosan (-4GlcUAß1-4GlcNAcα1-)(n) by transferring alternatively the monosaccharide units from UDP-GlcUA and UDP-GlcNAc to an acceptor molecule. Details on the heparosan chain initiation by Pasteurella multocida heparosan synthase PmHS2 and its influence on the polymerization process have not been reported yet. By site-directed mutagenesis of PmHS2, the single action transferases PmHS2-GlcUA(+) and PmHS2-GlcNAc(+) were obtained. When incubated together in the standard polymerization conditions, the PmHS2-GlcUA(+)/PmHS2-GlcNAc(+) showed comparable polymerization properties as determined for PmHS2. We investigated the first step occurring in heparosan chain initiation by the use of the single action transferases and by studying the PmHS2 polymerization process in the presence of heparosan templates and various UDP-sugar concentrations. We observed that PmHS2 favored the initiation of the heparosan chains when incubated in the presence of an excess of UDP-GlcNAc. It resulted in a higher number of heparosan chains with a lower average molecular weight or in the synthesis of two distinct groups of heparosan chain length, in the absence or in the presence of heparosan templates, respectively. These data suggest that PmHS2 transfers GlcUA from UDP-GlcUA moiety to a UDP-GlcNAc acceptor molecule to initiate the heparosan polymerization; as a consequence, not only the UDP-sugar concentration but also the amount of each UDP-sugar is influencing the PmHS2 polymerization process. In addition, it was shown that PmHS2 hydrolyzes the UDP-sugars, UDP-GlcUA being more degraded than UDP-GlcNAc. However, PmHS2 incubated in the presence of both UDP-sugars favors the synthesis of heparosan polymers over the hydrolysis of UDP-sugars.

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.

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.

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.