Effect of Antibiotic Eye Drops on the Nasal Microbiome in Healthy Subjects-A Pilot Study.

BACKGROUND: Antibiotic eye drops are frequently used in clinical practice. Due to the anatomical connection via the nasolacrimal duct, it seems possible that they have an influence on the nasal/pharyngeal microbiome. This was investigated by using two different commonly used antibiotic eye drops. METHODS: 20 subjects were randomized to four groups of five subjects receiving eye drops containing gentamicin, ciprofloxacin, or, as controls, unpreserved povidone or benzalkonium chloride-preserved povidone. Nasal and pharyngeal swabs were performed before and after the instillation period. Swabs were analyzed by Illumina next-generation sequencing (NGS)-based 16S rRNA analysis. Bacterial culture was performed on solid media, and bacterial isolates were identified to the species level by MALDI-TOF MS. Species-dependent antimicrobial susceptibility testing was performed using single isolates and pools of isolates. RESULTS: Bacterial richness in the nose increased numerically from 163 ± 30 to 243 ± 100 OTUs (gentamicin) and from 114 ± 17 to 144 ± 45 OTUs (ciprofloxacin). Phylogenetic diversity index (pd) of different bacterial strains in the nasal microbiome increased from 12.4 ± 1.0 to 16.9 ± 5.6 pd (gentamicin) and from 10.2 ± 1.4 to 11.8 ± 3.1 pd (ciprofloxacin). Unpreserved povidone eye drops resulted in minimal changes in bacterial counts. Preservative-containing povidone eye drops resulted in no change. A minor increase (1-2-fold) in the minimal inhibitory concentration (MIC) was observed in single streptococcal isolates. CONCLUSIONS: Antibiotic eye drops could affect the nasal microbiome. After an instillation period of seven days, an increase in the diversity and richness of bacterial strains in the nasal microbiome was observed.

Candida auris in Austria-What Is New and What Is Different.

Candida auris is a novel and emerging pathogenic yeast which represents a serious global health threat. Since its first description in Japan 2009, it has been associated with large hospital outbreaks all over the world and is often resistant to more than one antifungal drug class. To date, five C. auris isolates have been detected in Austria. Morphological characterization and antifungal susceptibility profiles against echinocandins, azoles, polyenes and pyrimidines, as well as the new antifungals ibrexafungerp and manogepix, were determined. In order to assess pathogenicity of these isolates, an infection model in Galleria mellonella was performed and whole genome sequencing (WGS) analysis was conducted to determine the phylogeographic origin. We could characterize four isolates as South Asian clade I and one isolate as African clade III. All of them had elevated minimal inhibitory concentrations to at least two different antifungal classes. The new antifungal manogepix showed high in vitro efficacy against all five C. auris isolates. One isolate, belonging to the African clade III, showed an aggregating phenotype, while the other isolates belonging to South Asian clade I were non-aggregating. In the Galleria mellonella infection model, the isolate belonging to African clade III exhibited the lowest in vivo pathogenicity. As the occurrence of C. auris increases globally, it is important to raise awareness to prevent transmission and hospital outbreaks.

Atypical enteropathogenic E. coli are associated with disease activity in ulcerative colitis.

With increasing urbanization and industrialization, the prevalence of inflammatory bowel diseases (IBDs) has steadily been rising over the past two decades. IBD involves flares of gastrointestinal (GI) inflammation accompanied by microbiota perturbations. However, microbial mechanisms that trigger such flares remain elusive. Here, we analyzed the association of the emerging pathogen atypical enteropathogenic E. coli (aEPEC) with IBD disease activity. The presence of diarrheagenic E. coli was assessed in stool samples from 630 IBD patients and 234 age- and sex-matched controls without GI symptoms. Microbiota was analyzed with 16S ribosomal RNA gene amplicon sequencing, and 57 clinical aEPEC isolates were subjected to whole-genome sequencing and in vitro pathogenicity experiments including biofilm formation, epithelial barrier function and the ability to induce pro-inflammatory signaling. The presence of aEPEC correlated with laboratory, clinical and endoscopic disease activity in ulcerative colitis (UC), as well as microbiota dysbiosis. In vitro, aEPEC strains induce epithelial p21-activated kinases, disrupt the epithelial barrier and display potent biofilm formation. The effector proteins espV and espG2 distinguish aEPEC cultured from UC and Crohn's disease patients, respectively. EspV-positive aEPEC harbor more virulence factors and have a higher pro-inflammatory potential, which is counteracted by 5-ASA. aEPEC may tip a fragile immune-microbiota homeostasis and thereby contribute to flares in UC. aEPEC isolates from UC patients display properties to disrupt the epithelial barrier and to induce pro-inflammatory signaling in vitro.

Disturbances in microbial skin recolonization and cutaneous immune response following allogeneic stem cell transfer.

The composition of the gut microbiome influences the clinical course after allogeneic hematopoietic stem cell transplantation (HSCT), but little is known about the relevance of skin microorganisms. In a single-center, observational study, we recruited a cohort of 50 patients before undergoing conditioning treatment and took both stool and skin samples up to one year after HSCT. We could confirm intestinal dysbiosis following HSCT and report that the skin microbiome is likewise perturbed in HSCT-recipients. Overall bacterial colonization of the skin was decreased after conditioning. Particularly patients that developed acute skin graft-versus-host disease (aGVHD) presented with an overabundance of Staphylococcus spp. In addition, a loss in alpha diversity was indicative of aGVHD development already before disease onset and correlated with disease severity. Further, co-localization of CD45⁺ leukocytes and staphylococci was observed in the skin of aGVHD patients even before disease development and paralleled with upregulated genes required for antigen-presentation in mononuclear phagocytes. Overall, our data reveal disturbances of the skin microbiome as well as cutaneous immune response in HSCT recipients with changes associated with cutaneous aGVHD.

Mucosal Biofilms Are an Endoscopic Feature of Irritable Bowel Syndrome and Ulcerative Colitis.

BACKGROUND & AIMS: Irritable bowel syndrome (IBS) and inflammatory bowel diseases result in a substantial reduction in quality of life and a considerable socioeconomic impact. In IBS, diagnosis and treatment options are limited, but evidence for involvement of the gut microbiome in disease pathophysiology is emerging. Here we analyzed the prevalence of endoscopically visible mucosal biofilms in gastrointestinal disease and associated changes in microbiome composition and metabolism. METHODS: The presence of mucosal biofilms was assessed in 1426 patients at 2 European university-based endoscopy centers. One-hundred and seventeen patients were selected for in-depth molecular and microscopic analysis using 16S ribosomal RNA gene amplicon-sequencing of colonic biopsies and fecal samples, confocal microscopy with deep learning-based image analysis, scanning electron microscopy, metabolomics, and in vitro biofilm formation assays. RESULTS: Biofilms were present in 57% of patients with IBS and 34% of patients with ulcerative colitis compared with 6% of controls (P < .001). These yellow-green adherent layers of the ileum and right-sided colon were microscopically confirmed to be dense bacterial biofilms. 16S-sequencing links the presence of biofilms to a dysbiotic gut microbiome, including overgrowth of Escherichia coli and Ruminococcus gnavus. R. gnavus isolates cultivated from patient biofilms also formed biofilms in vitro. Metabolomic analysis found an accumulation of bile acids within biofilms that correlated with fecal bile acid excretion, linking this phenotype with a mechanism of diarrhea. CONCLUSIONS: The presence of mucosal biofilms is an endoscopic feature in a subgroup of IBS and ulcerative colitis with disrupted bile acid metabolism and bacterial dysbiosis. They provide novel insight into the pathophysiology of IBS and ulcerative colitis, illustrating that biofilm can be seen as a tipping point in the development of dysbiosis and disease.

Lessons from low seroprevalence of SARS-CoV-2 antibodies in schoolchildren: A cross-sectional study.

BACKGROUND: Children are discussed as hidden SARS-CoV-2 virus reservoir because of predominantly mild or even asymptomatic course of disease. The objective of this cross-sectional study in May-July 2020 was to assess the prevalence of SARS-CoV-2 antibodies and virus RNA in schoolchildren, consistent with previous infection by contact tracing. METHODS: School authorities approached parents for voluntary participation. Interested families were contacted by the study team. A nasal and oropharyngeal swab, a blood sample, and a questionnaire were employed. Primary endpoint was the frequency of SARS-CoV-2 real-time PCR (RT-PCR) and antibody-positive children. Antibody positivity was assessed by a highly sensitive first-line ELISA, and a neutralization assay and two other immunoassays as confirmatory assays. RESULTS: Of 2069 children (median age 13 years, IQR 10-15), 2 cases (0.1%) tested positive for SARS-CoV-2 RNA and 26 cases (1.3%) tested positive for specific antibodies. SARS-CoV-2-specific antibodies exhibited detectable virus-neutralizing activity in 92% (24 of 26 samples). Seropositivity was associated with a history of mild clinical symptoms in 14 children (53.8%), while 12 children (46.2%) remained asymptomatic. Among 13 seropositive children being tested concomitantly with their siblings, only one pair of siblings was seropositive. Contact tracing revealed adult family members and school teachers as potential index cases. CONCLUSION: In schoolchildren, the infection rate with SARS-CoV-2 is low and associated with a mild or asymptomatic course of disease. Virus spreading seemed to occur more likely in intergenerational contacts than among siblings in the same household. The presence of neutralizing SARS-CoV-2 antibodies in children may reflect protective adaptive immunity.

Apparent Intracellular Helicobacter pylori Detected by Immunohistochemistry: The Missing Link in Eradication Failure.

BACKGROUND: Helicobacter pylori is primarily an extracellularly living bacterium. However, seemingly intracellular occurrence can often be detected by immunohistochemical stains. Considering antimicrobial resistance, we investigated the impact of the apparent intracellular H. pylori (aiHp) on treatment failure of first-line triple therapies. METHODS: Gastric biopsies of 814 patients infected with H. pylori naive to treatment were analyzed before and after eradication therapy by immunohistochemistry. Of these, 373 received treatment consisting of amoxicillin, clarithromycin, and proton pump inhibitor (AC/PPI). Availability of polymerase chain reaction-based clarithromycin susceptibility test results from pretreatment gastric biopsies was a precondition for matching 52 aiHp to 52 non-aiHp cases within the AC/PPI group. RESULTS: AiHp were detected mostly in low counts predominantly in corpus biopsies, rarely in antrum biopsies (95.2% vs 24.6%); they were found in 497 (61%) of all patients and in 192 of 373 patients (51.5%) in the AC/PPI group. The eradication rate in aiHp versus non-aiHp cases was 44.4% versus 72.9% in the entire sample and 45.3% versus 66.8% in the AC/PPI group. Among the 104 paired patients, respective values were 46.2% versus 78.8%; in clarithromycin-susceptible cases, 60.6% versus 91.9%. Both aiHp and resistance to clarithromycin proved to be highly significant (P ≤ .001) and independent predictors of eradication failure. Twelve of 13 aiHp cases with a clarithromycin-sensitive strain who failed eradication developed resistance to the antibiotic. CONCLUSIONS: AiHp found by immunohistochemical staining especially in corpus biopsies proved to be a risk factor for failure of first-line triple therapies; occurrence of aiHp should be considered with regard to therapy options.

Oral probiotics to reduce vaginal group B streptococcal colonization in late pregnancy.

This study aimed to evaluate the potential of oral probiotics to eradicate vaginal GBS colonization during the third trimester of pregnancy. We screened 1058 women for GBS colonization at 33-37 gestational weeks using a combination of vaginal-to-rectal swab and culture-based methods. Women who tested GBS positive were randomized to either the verum group, receiving a dietary probiotic supplement of four viable strains of Lactobacillus twice-daily for 14 days, or to the placebo group. Women underwent follow-up smears, whereat GBS colonization upon follow-up was considered the primary endpoint. We found that 215 women (20.3%) were positive for GBS upon screening, of which 82 (38.1%) were eligible for study inclusion; 41 (50%) of these were randomized to the verum and placebo groups each. After treatment, 21/33 (63.6%) members of the verum group, and 21/27 (77.8%) of the placebo group were still GBS positive (p = 0.24). Four (9.8%) women in the verum group and one (2.4%) in the placebo group experienced preterm birth (p = 0.20); smokers showed significantly higher rates of preterm birth (p = 0.03). Hence, the findings did not support the hypothesis that oral probiotics can eradicate GBS during pregnancy, although we observed a trend toward reduced GBS persistence after probiotic intake.

Review: Diagnosis of Helicobacter pylori infection.

Endoscopic imaging of the stomach is improving. In addition to narrow band imaging, other methods, for example, blue light imaging and linked color imaging, are now available and can be combined with artificial intelligence systems to obtain information on the gastric mucosa and detect early gastric cancer. Immunohistochemistry is only recommended as an ancillary stain in case of chronic active gastritis without Helicobacter pylori detection by standard staining, and recommendations to exclude false negative H. pylori results have been made. Molecular methods using real-time PCR, droplet digital PCR, or amplification refractory mutation system PCR have shown a high accuracy, both for detecting H. pylori and for clarithromycin susceptibility testing, and can now be used in clinical practice for targeted therapy. The most reliable non-invasive test remains the 13 C-urea breath test. Large data sets show that DOB values are higher in women and that the cut-off for positivity could be decreased to 2.74 DOB. Stool antigen tests using monoclonal antibodies are widely used and may be a good alternative to UBT, particularly in countries with a high prevalence of H. pylori infection. Attempts to improve serology by looking at specific immunodominant antigens to distinguish current and past infection have been made. The interest of Gastropanel® which also tests pepsinogen levels was confirmed.

Prevalence and Outcome of Secondary Hemophagocytic Lymphohistiocytosis Among SIRS Patients: Results from a Prospective Cohort Study.

Secondary hemophagocytic lymphohistiocytosis (sHLH) is a life-threatening condition clinically presenting as SIRS (Systemic Inflammatory Response Syndrome). However, there is no comprehensive data concerning diagnostic algorithms, prevalence, outcome and biomarker performance in SIRS patients. We conducted a prospective observational cohort study on 451 consecutive patients fulfilling ≥2 SIRS criteria. The Hscore and the HLH-2004 criteria were used to determine the presence of sHLH, and the correlation of the screening-biomarkers ferritin, sCD25, and sCD163 with both scores was assessed. Out of 451 standard-care SIRS patients, five patients had high Hscores (≥169), suggesting incipient or HLH-like disease, and these patients were in urgent need for intensified therapy. However, none of these patients fulfilled five HLH-2004 criteria required for formal diagnosis. From the studied biomarkers, ferritin correlated strongest to both the HLH-2004 criteria and the Hscore (rs = 0.72, 0.41, respectively), and was the best predictor of 30-day survival (HR:1.012 per 100 µg/L, 95% CI: 1.004-1.021), when adjusted for patient's age, sex, bacteremia and malignant underlying-disease. Also, the HLH-2004 (HR per point increase: 1.435, 95% CI: 1.1012-2.086) and the Hscore (HR per point increase:1.011, 95% CI: 1.002-1.020) were independent predictors of 30-day-survival. The Hscore detected patients in hyperinflammatory states requiring urgent therapy escalation. Degrees of hyperinflammation, as assessed by ferritin and both HLH scores, are associated with worse outcomes.

Combination of Nasal and Expanded Skin Swabs Enhances the Detection Rate of Staphylococcus aureus Colonization in Premature Infants.

Nasal cultures are commonly used to detect carriers of Staphylococcus aureus (SA) in infants. Combination of nasal and skin swabs has been shown to enhance the detection rate of SA colonization in adult hospitalized patients. Combining nasal swabs with expanded body skin swabs enhanced detection of SA colonization in premature infants in a tertiary care neonatal department.

Analysis of antifungal resistance genes in Candida albicans and Candida glabrata using next generation sequencing.

INTRODUCTION/OBJECTIVES: An increase in antifungal resistant Candida strains has been reported in recent years. The aim of this study was to detect mutations in resistance genes of azole-resistant, echinocandin-resistant or multi-resistant strains using next generation sequencing technology, which allows the analysis of multiple resistance mechanisms in a high throughput setting. METHODS: Forty clinical Candida isolates (16 C. albicans and 24 C. glabrata strains) with MICs for azoles and echinocandins above the clinical EUCAST breakpoint were examined. The genes ERG11, ERG3, TAC1 and GSC1 (FKS1) in C. albicans, as well as ERG11, CgPDR1, FKS1 and FKS2 in C. glabrata were sequenced. RESULTS: Fifty-four different missense mutations were identified, 13 of which have not been reported before. All nine echinocandin-resistant Candida isolates showed mutations in the hot spot (HS) regions of FKS1, FKS2 or GSC1. In ERG3 two homozygous premature stop codons were identified in two highly azole-resistant and moderately echinocandin-resistant C. albicans strains. Seven point mutations in ERG11 were determined in azole-resistant C. albicans whereas in azole-resistant C. glabrata, no ERG11 mutations were detected. In 10 out of 13 azole-resistant C. glabrata, 12 different potential gain-of-function mutations in the transcription factor CgPDR1 were verified, which are associated with an overexpression of the efflux pumps CDR1/2. CONCLUSION: This study showed that next generation sequencing allows the thorough investigation of a large number of isolates more cost efficient and faster than conventional Sanger sequencing. Targeting different resistance genes and a large sample size of highly resistant strains allows a better determination of the relevance of the different mutations, and to differentiate between causal mutations and polymorphisms.

Emergence of a dalbavancin induced glycopeptide/lipoglycopeptide non-susceptible Staphylococcus aureus during treatment of a cardiac device-related endocarditis.

In the present study, we demonstrated the emergence of dalbavancin non-susceptible and teicoplanin-resistant Staphylococcus aureus small colony variants which were selected in vivo through long-term treatment with dalbavancin. A 36-year-old man presented with a cardiac device-related S. aureus endocarditis and received long-term therapy with dalbavancin. Consecutively, two glycopeptide/lipoglycopeptide susceptible and two non-susceptible S. aureus isolates were obtained from blood cultures and the explanted pacemaker wire. The isolates were characterized by: standard typing methods, antimicrobial susceptibility testing, auxotrophic profiling, proliferation assays, scanning and transmission electron microscopy, as well as whole genome sequencing. The isolated SCVs demonstrated a vancomycin-susceptible but dalbavancin non-susceptible and teicoplanin-resistant phenotype whereof the respective MICs of the last isolate were 16- and 84-fold higher than the susceptible strains. All four strains were indistinguishable or at least closely related by standard typing methods (spa, MLST, and PFGE), and whole genome sequencing revealed only eight sequence variants. A consecutive increase in cell wall thickness (up to 2.1-fold), an impaired cell separation with incomplete or multiple cross walls and significantly reduced growth rates were observed in the present study. Therefore, the mutations in pbp2 and the DHH domain of GdpP were identified as the most probable candidates due to their implication in the biosynthesis and metabolism of the staphylococcal cell wall. For the first time, we demonstrated in vivo induced dalbavancin non-susceptible/teicoplanin resistant, but vancomycin and daptomycin susceptible S. aureus SCVs without lipopeptide or glycopeptide pretreatment, thus, indicating the emergence of a novel lipoglycopeptide resistance mechanism.

Substantial diagnostic impact of blood culture independent molecular methods in bloodstream infections: Superior performance of PCR/ESI-MS.

This study analyzed the performance of different molecular technologies along with blood culture (BC) in the diagnosis of bloodstream infections (BSI) in patients from internal medicine wards - including intensive care units (ICUs) - and the emergency room. Patients with systemic inflammatory response syndrome were prospectively included. BCs and EDTA whole blood were obtained simultaneously. The latter was analyzed by PCR combined with electrospray ionization mass spectrometry (PCR/ESI-MS; IRIDICA BAC BSI assay, Abbott) and by SeptiFast (Roche). Cases were classified as BSI according to adapted European Centre for Disease Prevention and Control criteria. Out of 462 analyzed episodes, 193 with valid test results fulfilled the inclusion criteria and were further evaluated. Sixty-nine (35.8%) were classified as BSI. PCR/ESI-MS showed a significantly better overall performance than BC (p = 0.004) or SeptiFast (p = 0.034). Only in patients from the ICU the performance of SeptiFast was comparable to that of PCR/ESI-MS. Mainly due to the negative effect of antimicrobial pre-treatment on BC results, the cumulative performance of each of the molecular tests with BC was significantly higher than that of BC alone (p < 0.001). SeptiFast and in particular the broad-range pathogen detection system PCR/ESI-MS proved to be an essential addition to BC-based diagnostics in BSI.

Machine learning for fast identification of bacteraemia in SIRS patients treated on standard care wards: a cohort study.

Bacteraemia is a life-threating condition requiring immediate diagnostic and therapeutic actions. Blood culture (BC) analyses often result in a low true positive result rate, indicating its improper usage. A predictive model might assist clinicians in deciding for whom to conduct or to avoid BC analysis in patients having a relevant bacteraemia risk. Predictive models were established by using linear and non-linear machine learning methods. To obtain proper data, a unique data set was collected prior to model estimation in a prospective cohort study, screening 3,370 standard care patients with suspected bacteraemia. Data from 466 patients fulfilling two or more systemic inflammatory response syndrome criteria (bacteraemia rate: 28.8%) were finally used. A 29 parameter panel of clinical data, cytokine expression levels and standard laboratory markers was used for model training. Model tuning was performed in a ten-fold cross validation and tuned models were validated in a test set (80:20 random split). The random forest strategy presented the best result in the test set validation (ROC-AUC: 0.729, 95%CI: 0.679-0.779). However, procalcitonin (PCT), as the best individual variable, yielded a similar ROC-AUC (0.729, 95%CI: 0.679-0.779). Thus, machine learning methods failed to improve the moderate diagnostic accuracy of PCT.

Incidence of risk factors for bloodstream infections in patients with major burns receiving intensive care: A retrospective single-center cohort study.

OBJECTIVES: The objective was primarily to identify risk factors for bloodstream infections (BSI) caused by different pathogens. METHODS: A retrospective single-center cohort study was performed on 472 burn patients with an abbreviated burn severity index (ABSI)≥3, a total burn surface area (TBSA)≥10%, and an ICU stay of at least 24h. Risk factors for different BSI pathogens were analyzed by competing risks regression model of Fine and Gray. RESULTS: A total of 114 burn patients developed 171 episodes of BSIs caused by gram-negative bacteria (n=78;46%), gram-positive bacteria (n=69;40%), and fungi (n=24;14%) median after 14days (range, 1-164), 16days (range, 1-170), and 16days (range, 0-89), respectively. A total of 24/114 patients (21%) had fatal outcomes. Isolation of the most common bloodstream isolates Enterococcus sp. (n=26), followed by Candida sp. and Pseudomonas sp. (n=22 for both) was significantly associated with increased TBSA (p≤0.006) and ABSI (p<0.0001) and need for fasciotomy (p<0.01). The death risk of patients with MDR gram-negative bacteremia was significantly increased by a hazard ratio of 12.6 (95% CI:4.8-32.8; p<0.0001). CONCLUSIONS: A greater TBSA and ABSI were associated with a significantly higher incidence of BSIs caused by Pseudomonas sp., Enterococcus sp. and Candida sp.

Clinical characteristics of a large cohort of patients with positive culture of Fusobacterium necrophorum.

Background: Fusobacterium necrophorum is a rare pathogen, mostly affecting young adults, causing infections of the head and neck, typically described as the Lemierre's syndrome. Today this symptom complex has become increasingly rare and has almost turned to a 'forgotten disease'. Methods: We performed a retrospective, descriptive study to identify the clinical features of patients with positive culture of F. necrophorum. Additionally, the antibiotic susceptibility profile of the pathogens was analysed. Results: During a period of 22 years 36 patients with at least one isolate of F. necrophorum were identified. Mostly tonsillar and peritonsillar abscesses were found, 10 patients were identified with bacteraemia, but only 4 patients presented with symptoms like sore throat, fever and swollen cervical lymph nodes, which may suggest Lemierre's. Most of the isolates (33/35) showed sensitivity to all tested antibiotics. Conclusion: Appropriate techniques are needed to detect F. necropho rum, especially from throat swabs, in the microbiological laboratory. Current clinical and microbiological practice may lead to under-diagnosis of infections caused by F. necrophorum. Further research is needed to define the colonization rate and to optimize methods for detection as well as identification of virulence.

Detection of fungal pathogens by a new broad range real-time PCR assay targeting the fungal ITS2 region.

PURPOSE: The rise in the incidence of fungal infections and the expanding spectrum of fungal pathogens make early and broad detection of fungal pathogens essential. In the present study, a panfungal real-time PCR assay for the broad-range detection of fungal DNA (Fungi assay) in a wide variety of clinical specimens was developed. METHODOLOGY: Our in-house, HybProbe real-time PCR assay targets the ITS2 region of fungal DNA. The applicability was evaluated by testing 105 clinical samples from 98 patients with suspected fungal infection. Samples included tissue biopsies, paraffin embedded tissues, aspirates, EDTA-anticoagulated blood, cerebrospinal fluids and bronchoalveolar lavages. RESULTS: Fungal pathogens were identified by the Fungi assay in 47 samples. In all of these cases, conventional methods and clinical data were also indicative for a fungal infection. Five samples were interpreted false negative. blast analyses of the amplicons derived from 11 samples revealed the presence of environmental fungal species while other tests and clinical data did not suggest a fungal infection. This fact might indicate contaminated samples. The remaining 42 samples were negative by the Fungi assay as well as the conventional methods and were therefore regarded as true negatives. Thus, sensitivity was 90.4 % and specificity 79.2 %. CONCLUSION: The Fungi assay improved the targeted diagnosis of fungal infections allowing pathogen identification in samples that were histologically positive but culture negative. For reliable diagnosis, results have to be interpreted in context with conventional methods and clinical data.