Early and adequate antimicrobial therapy has been shown to improve the clinical outcome in bloodstream infections (BSI). To provide rapid pathogen identification for targeted treatment, we applied matrix-assisted laser desorption-ionization time of flight (MALDI-TOF) mass spectrometry fingerprinting to bacteria directly recovered from blood culture bottles. A total of 304 aerobic and anaerobic blood cultures, reported positive by a Bactec 9240 system, were subjected in parallel to differential centrifugation with subsequent mass spectrometry fingerprinting and reference identification using established microbiological methods. A representative spectrum of bloodstream pathogens was recovered from 277 samples that grew a single bacterial isolate. Species identification by direct mass spectrometry fingerprinting matched reference identification in 95% of these samples and worked equally well for aerobic and anaerobic culture bottles. Application of commonly used score cutoffs to classify the fingerprinting results led to an identification rate of 87%. Mismatching mostly resulted from insufficient bacterial numbers and preferentially occurred with Gram-positive samples. The respective spectra showed low concordance to database references and were effectively rejected by score thresholds. Spiking experiments and examination of the respective study samples even suggested applicability of the method to mixed cultures. With turnaround times around 100 min, the approach allowed for reliable pathogen identification at the day of blood culture positivity, providing treatment-relevant information within the critical phase of septic illness.

In 2011 northern Germany experienced a large outbreak of Shiga-Toxigenic Escherichia coli O104:H4. The large amount of samples sent to microbiology laboratories for epidemiological assessment highlighted the importance of fast and inexpensive typing procedures. We have therefore evaluated the applicability of a MALDI-TOF mass spectrometry based strategy for outbreak strain identification. Specific peaks in the outbreak strain’s spectrum were identified by comparative analysis of archived pre-outbreak spectra that had been acquired for routine species-level identification. Proteins underlying these discriminatory peaks were identified by liquid chromatography tandem mass spectrometry and validated against publicly available databases. The resulting typing scheme was evaluated against PCR genotyping with 294 E. coli isolates from clinical samples collected during the outbreak. Comparative spectrum analysis revealed two characteristic peaks at m/z 6711 and m/z 10883. The underlying proteins were found to be of low prevalence among genome sequenced E. coli strains. Marker peak detection correctly classified 292 of 293 study isolates, including all 104 outbreak isolates. MALDI-TOF mass spectrometry allowed for reliable outbreak strain identification during a large outbreak of Shiga-Toxigenic E. coli. The applied typing strategy could probably be adapted to other typing tasks and might facilitate epidemiological surveys as part of the routine pathogen identification workflow.

Background: Infectious meningitis is a serious disease and patient outcome relies on fast and reliable diagnostics. A syndromic panel testing approach like the FilmArray ME can accelerate diagnosis and therefore decrease the time to pathogen specific therapy. Yet, its clinical utility is controversial, mainly because of a remaining uncertainty in correct interpretation of results, limited data on its performance on clinical specimens and its relatively high costs. The aim of this study was to analyze clinical performance of the assay in a real life setting at a tertiary university hospital using a pragmatic and simple sample selection strategy to reduce the overall cost burden. Methods: Over a period of 18 months we received 4623 CSF samples (2338 hospitalizations, 1601 individuals). FilmArray ME analysis was restricted to CSF-samples with a high pretest probability of infectious meningitis, e.g. positive Gram-stain, samples in which leukocytes and/or bacteria were evident or urgent suspicion of infection was communicated by clinicians. N = 171 samples matched to our risk criteria and were subjected to FilmArray ME analysis. Those samples were also analyzed by reference methods: culture only (n = 45), PCR only (n = 20) or both methods (n = 106). Results: 56/171 (32.75%) were FilmArray ME positive. Bacterial pathogens were detected in 30/56 (53.57%), viral pathogens were detected in 27/56 (48.21%) and yeast DNA was detected in 1/56 (1.79%) of positive samples. Double detection occurred in 2/56 samples. In 52/56 (92.86%) FilmArray ME positive samples, results could be confirmed by the reference assays (sensitivity = 96.30%, specificity =96.58%). Conclusion: The FilmArray ME assay is a fast and reliable diagnostic tool for the management of infectious meningitis and can easily be implemented in routine diagnostic workflows. However, correlation of test results and underlying clinical symptoms requires experienced users and the awareness of potentially false negative or false positive results. Moreover, considering the need for antimicrobial susceptibility testing, the use of molecular tests as a stand-alone diagnostic cannot be recommended.

In this study, we directly compared coronavirus disease 2019 (COVID-19) patients hospitalized during the first (27 February–28 July 2020) and second (29 July–31 December 2020) wave of the pandemic at a large tertiary center in northern Germany. Patients who presented during the first (n = 174) and second (n = 331) wave did not differ in age (median [IQR], 59 years [46, 71] vs. 58 years [42, 73]; p = 0.82) or age-adjusted Charlson Comorbidity Index (median [IQR], 2 [1, 4] vs. 2 [0, 4]; p = 0.50). During the second wave, a higher proportion of patients were treated as outpatients (11% [n = 20] vs. 20% [n = 67]), fewer patients were admitted to the intensive care unit (43% [n = 75] vs. 29% [n = 96]), and duration of hospitalization was significantly shorter (median days [IQR], 14 [8, 34] vs. 11 [5, 19]; p < 0.001). However, in-hospital mortality was high throughout the pandemic and did not differ between the two periods (16% [n = 27] vs. 16% [n = 54]; p = 0.89). While novel treatment strategies and increased knowledge about the clinical management of COVID-19 may have resulted in a less severe disease course in some patients, in-hospital mortality remained unaltered at a high level. These findings highlight the unabated need for efforts to hamper severe acute respiratory syndrome coronavirus type 2 (SARS-CoV-2) transmission, to increase vaccination coverage, and to develop novel treatment strategies to prevent mortality and decrease morbidity.

Purpose: Beta-D-Glucan (BDG) testing has been suggested to support the diagnosis of candidemia and invasive candidiasis. The actual benefit in critically ill high-risk patients in intensive care units (ICU) has not been verified so far. Methods: In ICU patients receiving empirical echinocandin treatment for suspected invasive candidiasis (IC), serial BDG testing using the Fujifilm Wako Beta-Glucan Test was performed, starting on the first day of echinocandin administration and every 24-48 h afterwards. Diagnostic accuracy was determined for single testing and serial testing strategies using a range of cut-off values. In addition, we compared the added value of these testing strategies when their results were introduced as additional predictors into a multivariable logistic regression model controlling for established risk factors of IC. Results: A total of 174 ICU patients, forty-six of which (25.7%) classified as cases of IC, were included in our study. Initial BDG testing showed moderate sensitivity (74%, 95%CI 59–86%) and poor specificity (45%, 95% CI 36–54%) for IC which could hardly be improved by follow-up testing. While raw BDG values or test results obtained with very high thresholds improved the predictive performance of our multivariable logistic regression model for IC, neither single nor serial testing with the manufacturer-proposed low-level cut-off showed substantial benefit. Conclusions: In our study of critically ill intensive care patients at high risk for candidemia or invasive candidiasis, diagnostic accuracy of BDG testing was insufficient to inform treatment decisions. Improved classification was only achieved for cases with very high BDG values.

Background: Mucormycosis is a rapidly progressive and often fatal invasive fungal infection in immunocompromised patients. Diagnosis remains challenging because microscopy and culture have limited sensitivity, while targeted molecular assays may fail to detect uncommon Mucorales species. Case Presentation: We report a fatal case of disseminated Mucor indicus infection with pulmonary and cerebral involvement in a heart transplant recipient receiving immunosuppressive therapy and posaconazole prophylaxis. Initial microscopy of bronchial secretions demonstrated broad pauciseptate hyphae consistent with Mucorales, whereas targeted PCR assays remained negative. Species identification was subsequently achieved by culture, MALDI-TOF mass spectrometry, and internal transcribed spacer (ITS) sequencing. Following neurological deterioration, brain biopsy confirmed cerebral involvement histopathologically, and ITS sequencing detected M. indicus. Comparative DNA- and RNA-based metagenomic sequencing identified Mucorales sequences, although read abundance remained unexpectedly low despite extensive histopathological fungal burden. Nevertheless, independent mapping of metagenomic reads against databases comprising all genomic viral and fungal reference sequences readily identified Mucor indicus as the sole pathogen receiving significant scores. Conclusions: This case illustrates that targeted Mucorales PCR may fail to detect an uncommon species despite evidence of invasive fungal infection. Microscopy, culture, histopathology, ITS sequencing, and comprehensive metagenomic analyses provided complementary evidence for disseminated Mucor indicus infection. Integrated diagnostics combining histopathology, culture, and complementary molecular methods remain essential for accurate diagnosis of invasive mucormycosis.