BACKGROUND:Clostridioides difficile infection (CDI) is a major cause of healthcare-associated diarrhoea with significant morbidity and mortality worldwide. In Germany, CDI incidence has declined in recent years. However, prospective multi-centre data on all-cause in-hospital mortality among CDI patients and on risk factors for death remain scarce. METHODS:We conducted a prospective multi-centre surveillance study in six German university hospitals between 2016 and 2020 to assess all-cause in-hospital mortality among CDI patients. Additionally, multi-variable logistic regression was used to identify risk factors for death. RESULTS:A total of 3592 CDI cases were recorded, of whom 46.7% were female. Overall all-cause in-hospital mortality was 11.7%, whereas CDI-attributable mortality was 0.4% (N = 16). Independent risk factors for death in CDI patients included increasing age (3% per year, adjusted odds ratio [aOR]: 1.03, 95% confidence interval [CI]: 1.02-1.04), longer hospital stay (1% per day, aOR: 1.01, 95% CI: 1.00-1.01), admission to internal medicine (aOR: 1.60, 95% CI: 1.32-1.94) or haematology-oncology (aOR: 2.38, 95% CI: 1.75-3.24) as proxies for patient complexity, and elevated creatinine levels within ± 2 days of sampling (6% per mg/dL, aOR: 1.06, 95% CI: 0.99-1.13). In contrast, diagnosis on a general ward (vs intensive care unit or intermediate care, irrespective of speciality) was associated with a lower risk of death (aOR: 0.34, 95% CI: 0.28-0.42). CONCLUSIONS:Our study provides a comprehensive assessment of all-cause in-hospital mortality among CDI patients in Germany. By identifying robust and easily accessible risk factors for death, our findings support improved risk stratification and may inform targeted management strategies for high-risk patient populations.

During apoptosis, the BCL-2 family members BAX and BAK oligomerize and form a pore to mediate the decisive step of mitochondrial outer membrane permeabilization. However, the contribution of additional cellular components to apoptotic pore dynamics remains poorly understood. Here we map the protein environment of the apoptotic pore using in situ proximity labeling and identify the mitochondrial carrier homolog protein MTCH2 localizing nearby BAX and BAK assemblies specifically under apoptotic conditions. We show that cells lacking MTCH2 exhibit delayed BAX and BAK oligomerization at the single-particle level, which can be rescued by addition of lysophosphatidic acid. Accordingly, MTCH2 depletion decreases not only apoptosis sensitivity but also sublethal mitochondrial permeabilization during bacterial infection, mitochondrial DNA release into the cytosol and cGAS-STING activation under impaired caspases. Our findings uncover a key role of MTCH2 in promoting BAX and BAK high-order assembly with functional consequences for apoptotic pore growth and downstream responses.

Chlamydiae are obligate intracellular bacteria that inhibit mitochondrial apoptosis to maintain integrity of the host cell. We have previously reported that a chlamydial outer membrane β-barrel protein, OmpA, can during ectopic expression inhibit mitochondrial apoptosis through direct interaction with the BCL-2-family effectors BAX and BAK. We here show that OmpA from Chlamydia trachomatis (Ctr) uses membrane vesicles for its delivery to the outer mitochondrial membrane during Ctr infection. Using a number of imaging and fractionation techniques, we show that OmpA during infection reaches mitochondria and is inserted into mitochondrial membranes. Chlamydia derived vesicles (CDV) from Ctr-infected cells contained OmpA as well as other outer membrane proteins and LPS. When added to uninfected cells, CDVs fused with mitochondrial membranes, causing the interaction of OmpA with BAK and the cytosolic retro-translocation of BAX. CDV addition to uninfected cells also protected the cells against apoptosis. We previously showed that OmpA works in co-ordination with VDAC2 to block apoptosis and here propose a structural model of this BAK inhibition by OmpA that reenacts the inhibition of BAK by VDAC2. The results provide evidence that OmpA from Chlamydia, as well as the structurally similar ortholog from the related Simkania, specifically exploits its relationship to mitochondrial porins to protect the infected cell against apoptosis and to enable intracellular growth of the bacteria in human cells.

BACKGROUND:Proton pump inhibitor (PPI) treatment leads to significant changes in the gut microbiota composition. We hypothesized that these alterations may transfer into distinct changes in the causative agents of spontaneous bacterial peritonitis (SBP) in patients with cirrhosis. AIMS:To investigate the impact of PPI treatment on the pathogen spectrum of SBP. METHODS:A total of 227 cirrhosis patients with culture-positive SBP from two German tertiary care centers were included and the pathogen spectrum of SBP was analysed with respect to PPI treatment. RESULTS:In the PPI-group, gram-positive bacteria were significantly more common compared to the non-PPI-group (62.1% vs. 34.0%, p < 0.001). This involved increased abundances of Enterococcus spp. (22.0% vs. 8.0%), Streptococcus spp. (14.7% vs. 6.0%) and Staphylococcus aureus (15.8% vs. 8.0%). Importantly, more than half of the Enterococcus isolates were susceptible only to antibiotics with extended gram-positive activity. Conversely, gram-negative bacteria were significantly more frequent in the non-PPI-group compared to the PPI-group (64.0% vs. 40.7%, p = 0.003), primarily due to a higher prevalence of E. coli (40.0% vs. 20.3%). In uni- and multivariable logistic regression analyses, PPI use was an independent predictor of gram-positive SBP (OR 2.719, 95% CI 1.343-5.506, p = 0.005), adjusted for nosocomial infection (OR 2.609, 95% CI 1.464-4.650, p = 0.001) and Child-Pugh score (OR 0.684, 0.552-0.848, p < 0.001). CONCLUSIONS:PPI treatment is associated with increased abundances of gram-positive pathogens, including Enterococcus spp., in culture-positive SBP. This result can help guide empiric antibiotic therapy of SBP.

SMAC is a mitochondrial intermembrane space protein, which is released during apoptosis and whose known function is antagonism of inhibitor of apoptosis proteins in the cytosol, to facilitate caspase activation. Recent data suggest that SMAC can also be released by sub-lethal signals in the apoptosis pathway, in the absence of cell death. We here explored potential functions of SMAC in non-apoptotic cells. We found that a portion of SMAC is spontaneously released into the cytosol in the absence of apoptosis, regulated by the BCL-2-family proteins BAX and BAK and the fission GTPase DRP1. In cancer cell lines, SMAC was required for the activation of caspases in lethal and non-lethal conditions, while this contribution to caspase-activation was much smaller in non-malignant fibroblast lines. In cells with high levels of cytosolic SMAC, SMAC deficiency reduced in vitro migration, invasion and anchorage-independent growth as well as metastasis in a xenograft model in zebrafish. SMAC-deficient cells further showed a reduced activity in interferon signaling, associated with reduced cytosolic presence of mitochondrial DNA and activation of the stimulator of interferon genes (STING), and SMAC expression levels correlated with interferon-induced genes in cancer data sets. We further found that SMAC can regulate mitochondrial morphology and integrity. Finally, high gene-expression of SMAC was associated with poor prognosis in patients of several cancer types. These results identify SMAC as a regulator of inflammation and growth behavior of cancer cells. They further report a mitochondrial function of SMAC and demonstrate a role of SMAC in human cancer biology across several cancer entities.

Helicobacter pylori is known for colonizing the gastric mucosa and instigating severe upper gastrointestinal diseases such as gastritis, gastroduodenal ulcers, and gastric cancer. To date, there is no data available on the oral cavity as transmission site, whether H. pylori can survive in the oral cavity or in human saliva. The aim of the study was to investigate the influence of oral microorganisms and human saliva on the survival of H. pylori in human saliva. H. pylori strains KE, a motile derivate of type strain H. pylori 26695, and H. pylori SS1, a clinical isolate from a gastric biopsy, were grown in human pooled saliva (pooled from 4 healthy human donors, 0.22 μm filter-sterilized) or in BBF (Brucella browth formula; control) either as mono-cultures or in co-culture with Streptococcus mutans, Streptococcus oralis, Actinomyces naeslundii, Lacticaseibacillus casei and Candida dubliniensis. Bacterial survival of H. pylori and the oral microorganisms were investigated using colony forming units (CFU) assay and MALDI-TOF MS at baseline and after 24, 48 and 168 h. In saliva, H. pylori KE demonstrated enhanced survival in co-culture with S. mutans, A. naeslundii, and C. dubliniensis, enduring for at least 48 h. In contrast, L. casei and S. oralis inhibited H. pylori KE in saliva. H. pylori KE could not be cultured after 168 h in saliva, neither in mono- nor co-culture. In contrast, H. pylori SS1 in saliva could be cultured after 168 h in co-culture with S. mutans and C. dubliniensis, but not in mono-culture. In BBF, H. pylori KE could be cultured after 168 h with S. mutans, L. casei and C. dubliniensis, and H. pylori SS1 with L. casei and C. dubliniensis, but not with S. mutans. Notably, the co-cultured microorganisms survived at high CFU numbers similar to those of the monocultures. The study suggests that H. pylori can transiently survive in human saliva and even with presence of certain oral microorganisms. However, it may not be a permanent resident of the oral microbiota. The co-survival with oral microorganisms emphasizes the necessity for studying the role of the oral microbiota in the infectious and transmission cycle of H. pylori.

IntroductionIn human neurocysticercosis (NCC), the cellular and molecular mechanisms of host-parasite interactions triggering brain inflammation and epileptic seizures in Sub-Saharan Africa are poorly understood. Emerging evidence indicates that the viability of the cyst of the pork tapeworm Taenia solium determines brain inflammation and, thus, symptom development and disease severity. We have previously shown that while viable cyst-released molecules promote immune regulation and often asymptomatic disease, the fluid from degenerating cysts causes inflammation in microglia and peripheral immune cells, potentially driving immune-mediated pathology. This study aims to elucidate the apoptotic signaling pathways underlying this process and their relevance for symptomatic disease in NCC patients.Materials and methodsHuman and porcine peripheral immune cells, as well as murine microglia, were exposed to T. solium cyst vesicular fluid (CVF). Apoptosis signaling pathways were analysed using flow cytometric FLICA (fluorochrome-labeled inhibitors of caspases) caspase 8 and 9 assays, while mitochondrial dysfunction was assessed via TMRE and MitoTracker Deep Red and Green fluorescent probes. Apoptosis-inducing CVF molecules were identified by differential mass spectrometry and functionally tested using specific inhibitors. Caspase activity and soluble mediators (FasL, ROS, TNFα) were measured in NCC asymptomatic and symptomatic patients’ sera, and inflammatory T cell infiltrates expressing caspases near viable and degenerating cysts in naturally infected pig brain slices were examined via immunohistology.ResultsWe found that vesicular fluid derived from cysts primarily induced apoptosis and caspase 3 and 9 activity, and only minimal necrosis, in a dose-dependent manner across central and peripheral immune cells. This effect was prominent in CD16+ monocytes, microglia, and in CD3+ T cell-expressing caspase 3 near degenerating brain cysts. Apoptotic signaling was predominantly mediated by a dynamic remodeling of caspase 9 pathway, accompanied by a significant loss of mitochondrial potential and a sharp decrease in Bid and Bcl2 transcription, favoring the intrinsic over the FasL-dependent extrinsic pathway and mechanisms. This process is primarily mediated by small molecules (< 30 kDa), and remained unaffected by heat and proteinase treatment. Notably, symptomatic NCC patients exhibited elevated FasL levels correlating with increased caspase activity, underscoring the potential contribution of apoptosis to disease pathogenesis.ConclusionsThis study identifies caspase 9-mediated apoptosis as a mechanism of helminth-induced brain inflammation and implicates FasL in symptomatic disease progression. These insights enhance our understanding of NCC immunopathogenesis and may inform future therapeutic strategies targeting apoptotic pathways.

A motile, rod-shaped and anaerobic strain WK13T was isolated from a secondary root canal infection of a human tooth. WK13T cells were Gram-stain-negative, catalase-positive and oxidase-negative. The major fatty acids (≥ 5.0%) were C16 : 0, C18 : 0, C16 : 1 ω7c, C18 : 1 ω9c and C18 : 2 ω6,9c. The DNA G+C content was 57.94 mol%. The major polar lipids were phosphatidylethanolamine, phosphatidylserine, diphosphatidylglycerol, phosphatidylcholine and lysophosphatidylcholine. There were no respiratory quinones detectable. Phylogenetic analysis based on 16S rRNA gene sequences indicated that strain WK13T belongs to the class Betaproteobacteria. WK13T showed a 93.6% and 93.5% 16S rRNA gene sequence similarity to the most closely related cultured species, Propionivibrio pelophilus strain DSM 12018T and Propionivibrio dicarboxylicus strain DSM 5885T, respectively. On the basis of physiological and biochemical data, the isolate is considered to represent a novel species of a new genus in the class Betaproteobacteria, for which we propose the name Dentiradicibacter hellwigii gen. nov., sp. nov. The type strain is WK13T (=DSM 112713T=NCTC 14938T).

Caspases-9, -3 and -7 are activated in the mitochondrial apoptosis pathway and lead to the apoptotic phenotype. Caspases also function to limit inflammation upon apoptotic mitochondrial permeabilization through degradation of the signalling proteins cGAS, MAVS and IRF3. Cells and mice lacking caspases have higher interferon levels and are resistant to viral infection. We report that in unstimulated, non-apoptotic cells caspase-3 functions to cleave specifically activated MAVS and very likely cGAS. In unstimulated HeLa cells, constitutive caspase-9- and -3- but not 7-dependent proteolytic events were observed. Inhibition of the mitochondrial apoptosis pathway in various healthy cells induced type I interferon (IFN I) through increased cGAS activity in the absence of changes to cGAS levels. We observed enhanced MAVS-dependent signals upon RIG-I-like helicase stimulation in the absence of BAX, caspase-9 or caspase-3 or upon caspase-inhibition. During activation, MAVS forms complexes, and blockade of mitochondrial apoptosis signalling increased complex abundance in unstimulated and stimulated cells. MAVS complexes were more sensitive to caspase-degradation than the monomer, and mutation of caspase-3-cleavage sites in MAVS spontaneously increased complex formation. Inhibition of voltage-dependent anion channel 1 (VDAC1) oligomerization blocked BAX/BAK- and caspase-regulated IFN induction, suggesting a stimulating role of leakage of mitochondrial DNA. We propose that low level, spontaneous activity of the mitochondrial apoptosis pathway, through specific caspase-3-mediated cleavage of only active signaling proteins, counteracts mitochondrial release of nucleic acids to reduce inflammation in the absence of infection. Caspase-3 therefore has a novel function in conformation- and activation-specific cleavage of substrates. ### Competing Interest Statement The authors have declared no competing interest.

Viral respiratory infections can be complicated by bacterial superinfections. SARS-CoV-2 patients may suffer from superinfections, and negative effects of additional infections have been identified. When analysing hospital data, patients typically leave the facility of observation, due to discharge or death, which leads to changes in the study cohort over time. This may distort the estimate of the impact of superinfection. Therefore, it is essential for the statistical analysis of hospital data to acknowledge this change of the risk set over time. We analysed superinfections in a retrospective cohort study with 268 critically ill patients, taking into account discharge and death as competing risks in the statistical analysis. We evaluated bacterial respiratory infections and bloodstream infections and used multi-state statistical modelling to account for the different patient states. We calculated risks of superinfection, probability of discharge or death over time and analysed subgroups according to age and sex. The observed pathogen spectrum was mainly composed of Enterobacterales, Nonfermenters but also Staphylococcus aureus. We identified an elevated mortality due to bacterial infection of the respiratory tract or bloodstream infection (adj. cause-specific HR 1.7, CI 1.15–2.52) as well as a reduced discharge rate (adj. cause-specific HR 0.51, CI 0.36–0.73). Female patients showed a tendency to have a reduced risk of acquiring a superinfection (adj. subdistribution HR 0.71, CI 0.48–1.04), and in case of infection a higher mortality compared to male patients (interaction effect HR 1.49, CI 0.67–3.30). The study accounts for competing risks and quantifies the risk of death associated with bacterial superinfection in critically ill COVID-19 patients. We observed an increased risk of death for patients who developed a superinfection, with Enterobacterales being the predominant agent. The results emphasize the need for microbiological sampling in SARS-CoV-2-infected patients. German Clinical Trials Register number: DRKS00031367, registration date: 01.03.2023 ( https://drks.de/search/de/trial/DRKS00031367 ).

The large family of BCL-2 proteins plays a well-established role in the regulation of mitochondrial apoptosis pathway, and the crosstalk between death receptor signaling and mitochondrial apoptosis. Accumulating evidence suggests, however, that various BCL-2 family members are also involved in the regulation of apoptosis-unrelated necrotic forms of cell death, and even non-cell death processes. In this review, we discuss the emerging role of BCL-2 family members, and in particular BIM, in the regulation of mitochondrial dynamics, morphology and energy metabolism, and associated consequences for drug-inuced necrotic cell death.

Chlamydiae are obligate intracellular bacteria that inhibit mitochondrial apoptosis to maintain integrity of the host cell. We have previously reported that a chlamydial outer membrane β-barrel protein, the porin OmpA, can during ectopic expression inhibit mitochondrial apoptosis through direct interaction with the BCL-2-family effectors BAX and BAK. We here show that OmpA from Chlamydia trachomatis ( Ctr ) uses membrane vesicles for its delivery to the outer mitochondrial membrane during Ctr infection. Using a number of imaging and fractionation techniques, we show that OmpA during infection reaches mitochondria and is inserted into mitochondrial membranes. Purified membrane vesicles (MV) from Ctr -infected cells contained OmpA. When added to uninfected cells, MV fused with mitochondrial membranes, causing the interaction of OmpA with BAK and the cytosolic retro-translocation of BAX. MV addition to uninfected cells protected the cells against apoptosis. We propose a structural model of this BAK inhibition by OmpA that reenacts the inhibition of BAK by the mitochondrial porin VDAC2. The results provide evidence that the porin from Chlamydia , as well as the structurally similar porin from the related Simkania , specifically exploits its relationship to mitochondrial porins to protect the infected cell against apoptosis and to enable intracellular growth of the bacteria in human cells. ### Competing Interest Statement The authors have declared no competing interest.

SMAC is a mitochondrial intermembrane space protein, which is released during apoptosis and whose known function is antagonism of inhibitor of apoptosis proteins in the cytosol, to facilitate caspase activation. Recent data suggest that SMAC can also be released by sub-lethal signals in the apoptosis pathway, in the absence of cell death. We here explored potential functions of SMAC in non-apoptotic cells. We found that a portion of SMAC is spontaneously released into the cytosol in the absence of apoptosis, regulated by the BCL-2-family proteins BAX and BAK and the fission GTPase DRP1. In cancer cell lines, SMAC was required for the activation of caspases in lethal and non-lethal conditions, while this contribution to caspase-activation was much smaller in non-malignant fibroblast lines. In cells with high levels of cytosolic SMAC, SMAC deficiency reduced in vitro migration, invasion and anchorage-independent growth. SMAC-deficient cells further showed a reduced activity in interferon signalling, associated with reduced cytosolic presence of mitochondrial DNA and activation of the stimulator of interferon genes (STING), and SMAC expression levels correlated with interferon-induced genes in cancer data sets. We further found that SMAC can regulate mitochondrial morphology and integrity. Finally, high gene-expression of SMAC was associated with poor prognosis in patients of several cancer types. These results identify SMAC as a regulator of inflammation and growth behaviour of cancer cells. They further report a mitochondrial function of SMAC and demonstrate a role of SMAC in human cancer biology across several cancer entities. ### Competing Interest Statement The authors have declared no competing interest.

Juvenile myelomonocytic leukemia (JMML) is caused by constitutively activated RAS signaling and characterized by increased proliferation and predominant myelomonocytic differentiation of hematopoietic cells. Using MxCre;Ptpn11D61Y/+ mice, which model human JMML, we show that RAS pathway activation affects apoptosis signaling through cell type-dependent regulation of BCL-2 family members. Apoptosis resistance observed in monocytes and granulocytes was mediated by overexpression of the anti-apoptotic and down-regulation of the pro-apoptotic members of the BCL-2 family. Two anti-apoptotic proteins, BCL-XL and MCL-1, were directly regulated by the oncogenic RAS signaling but, in addition, were influenced by microenvironmental signals. While BCL-XL and BCL-2 were required for the survival of monocytes, MCL-1 was essential for neutrophils. Interestingly, stem and progenitor cells expressing the oncogenic PTPN11 mutant showed no increased apoptosis resistance. BCL-XL inhibition was the most effective in killing myeloid cells in vitro but was insufficient to completely resolve myeloproliferation in vivo.

Acute graft-versus-host disease (aGVHD) is a life-threatening complication of allogeneic hematopoietic cell transplantation (allo-HCT), for which therapeutic options are limited. Strategies to promote intestinal tissue tolerance during aGVHD may improve patient outcomes. Using single-cell RNA sequencing, we identified a lipocalin-2 (LCN2)–expressing neutrophil population in mice with intestinal aGVHD. Transfer of LCN2-overexpressing neutrophils or treatment with recombinant LCN2 reduced aGVHD severity, whereas the lack of epithelial or hematopoietic LCN2 enhanced aGVHD severity and caused microbiome alterations. Mechanistically, LCN2 induced insulin-like growth factor 1 receptor (IGF-1R) signaling in macrophages through the LCN2 receptor SLC22A17, which increased interleukin-10 (IL-10) production and reduced major histocompatibility complex class II (MHCII) expression. Transfer of LCN2-pretreated macrophages reduced aGVHD severity but did not reduce graft-versus-leukemia effects. Furthermore, LCN2 expression correlated with IL-10 expression in intestinal biopsies in multiple cohorts of patients with aGVHD, and LCN2 induced IGF-1R signaling in human macrophages. Collectively, we identified a LCN2-expressing intestinal neutrophil population that reduced aGVHD severity by decreasing MHCII expression and increasing IL-10 production in macrophages. This work provides the foundation for administration of LCN2 as a therapeutic approach for aGVHD.

The gut microbiome is a dense and diverse community of different microorganisms that deeply influence human physiology and that have important interactions with pathogens. For the correct antibiotic treatment of infections, with its twin goals of effective inhibition of the pathogen and limitation of collateral damage to the microbiome, the identification of infectious organisms is key. Microbiological culturing is still the mainstay of pathogen identification, and anaerobic species are among the most demanding bacterial communities to culture. This study aimed to evaluate the impact of growth media on the culture of an-aerobic bacteria from human stool samples. Stool samples from eight human subjects were cultured each on a yeast extract cysteine blood agar (HCB) and a modified peptone-yeast extract-glucose (MPYG) plate and subjected to Illumina NGS analysis after DNA extraction and amplification. The results showed tight clustering of sequencing samples belonging to the same human subject. Various differences in bacterial richness and evenness could be observed between the two media, with HCB plates supporting the growth of a more diverse microbial community, and MPYG plates improving the growth rates of certain taxa. No statistical significance was observed between the groups. This study highlights the importance of choosing the appropriate growth media for anaerobic bacterial culture and adjusting culture conditions to target specific pathological conditions. HCB plates are suitable for standard microbiological diagnostics, while MPYG plates may be more appropriate for targeting specific conditions. This work emphasizes the role of next-generation sequencing in supporting future research in clinical microbiology.

Cellular senescence is a response to many stressful insults. DNA damage is a consistent feature of senescent cells, but in many cases its source remains unknown. Here, we identify the cellular endonuclease caspase-activated DNase (CAD) as a critical factor in the initiation of senescence. During apoptosis, CAD is activated by caspases and cleaves the genomic DNA of the dying cell. The CAD DNase is also activated by sub-lethal signals in the apoptotic pathway, causing DNA damage in the absence of cell death. We show that sub-lethal signals in the mitochondrial apoptotic pathway induce CAD-dependent senescence. Inducers of cellular senescence, such as oncogenic RAS, type-I interferon, and doxorubicin treatment, also depend on CAD presence for senescence induction. By directly activating CAD experimentally, we demonstrate that its activity is sufficient to induce senescence in human cells. We further investigate the contribution of CAD to senescence in vivo and find substantially reduced signs of senescence in organs of ageing CAD-deficient mice. Our results show that CAD-induced DNA damage in response to various stimuli is an essential contributor to cellular senescence.

ObjectivesAscites, often associated with critical pathologies such as liver cirrhosis or bowel perforation, can be complicated by fungal infection, increasing mortality especially in intensive care settings and demanding rapid diagnosis and adequate treatment. Traditional microbiological diagnostic methods have limited sensitivity in accurately identifying fungal pathogens in ascitic fluid. Alternative diagnostic methods may offer important insights to enable guiding of antifungal therapy and refining empirical treatment strategies. The objective of this study was to evaluate the potential of next-generation sequencing methods to identify specific fungal pathogens responsible for ascitic fluid infections.MethodsWe prospectively collected 50 ascitic fluid samples from ICU patients with suspected ascites infection. In addition to standard culture-based microbiological testing, an ascitic fluid aliquot underwent fungal DNA isolation and was analyzed by next-generation sequencing (NGS) methods for identification of fungal species.ResultsOf 50 ascitic samples collected, five samples showed growth of Candida spp. in culture. After DNA isolation and ITS2 PCR, detectable amplification was achieved in 10 samples. Sequencing of the 50 patients’ samples identified facultative pathogenic fungi in 19 patients. In 15 cases, culture alone would not have permitted the identification of all facultative pathogenic fungi. The identification of fungal DNA by sequencing was significantly associated with poor patient outcome and a number of clinical parameters.ConclusionsOur results show a higher sensitivity for NGS-based diagnostic methods in the identification of ascitic fluid fungal infections compared to culture-based diagnostics. This may be beneficial especially for patients in a critical care setting, who have an increased prevalence of comorbidities and high mortality. The implementation of such methods in standard diagnosis will require increased standardization of the workflows and interpretation of the sequencing results with respect to patients’ clinical picture.