Explanted Skull Flaps after Decompressive Hemicraniectomy Demonstrate Relevant Bone Avitality-Is Their Reimplantation Worth the Risk?

BACKGROUND: Reimplantations of autologous skull flaps after decompressive hemicraniectomies (DHs) are associated with high rates of postoperative bone flap resorption (BFR). We histologically assessed the cell viability of explanted bone flaps in certain periods of time after DH, in order to conclude whether precursors of BRF may be developed during their storage. METHODS: Skull bone flaps explanted during a DH between 2019 and 2020 were stored in a freezer at either -23 °C or -80 °C. After their thawing process, the skulls were collected. Parameters of bone metabolism, namely PTH1 and OPG, were analyzed via immunohistochemistry. H&E stain was used to assess the degree of avital bone tissue, whereas the repeated assays were performed after 6 months. RESULTS: A total of 17 stored skull flaps (8 at -23 °C; 9 at -80 °C) were analyzed. The duration of cryopreservation varied between 2 and 17 months. A relevant degree of bone avitality was observed in all skull flaps, which significantly increased at the repeated evaluation after 6 months (p < 0.001). Preservation at -23 °C (p = 0.006) as well as longer storage times (p < 0.001) were identified as prognostic factors for higher rates of bone avitality in a linear mixed regression model. CONCLUSIONS: Our novel finding shows a clear benefit from storage at -80° C, which should be carefully considered for the future management and storage of explanted skull flaps. Our analysis also further revealed a significant degree of bone avitality, a potential precursor of BFR, in skull flaps stored for several weeks. To this end, we should reconsider whether the reimplantation of autologous skull flaps instead of synthetic skull flaps is still justified.

Identification of micro-organism from positive blood cultures: comparison of three different short culturing methods to the Rapid Sepsityper workflow.

Sepsis is one of the leading causes of death worldwide. The rapid identification (ID) of the causative micro-organisms is crucial for the patients' clinical outcome. MALDI-TOF MS has been widely investigated to speed up the time-to-report for ID from positive blood cultures, and many different procedures and protocols were developed, all of them attributable either to the direct separation of microbial cells from the blood cells, or to a short subculture approach. In this study, the Rapid Sepsityper workflow (MBT Sepsityper IVD Kit, Bruker Daltonics GmbH and Co. KG, Bremen, Germany) was compared to three different short subculturing methods, established into the routine practice of three different clinical microbiology laboratories. A total of N=503 routine samples were included in this study and tested in parallel with the two approaches. Results of the rapid procedures were finally compared to routine proceedings with Gram-staining and overnight subculture. Among monomicrobial samples, the Rapid Sepsityper workflow enabled overall the correct identification of 388/443 (87.6 %) micro-organisms, while the short subculturing methods of 267/435 (61.8 %). Except for the performance with Streptococcus pneumoniae, in each one of the three sites the Rapid Sepsityper workflow proved to be superior to the short subculture method, regardless of the protocol applied, and it delivered a result from 1 to 5 h earlier.

Effective high-throughput RT-qPCR screening for SARS-CoV-2 infections in children.

Systematic SARS-CoV-2 testing is a valuable tool for infection control and surveillance. However, broad application of high sensitive RT-qPCR testing in children is often hampered due to unpleasant sample collection, limited RT-qPCR capacities and high costs. Here, we developed a high-throughput approach ('Lolli-Method') for SARS-CoV-2 detection in children, combining non-invasive sample collection with an RT-qPCR-pool testing strategy. SARS-CoV-2 infections were diagnosed with sensitivities of 100% and 93.9% when viral loads were >106 copies/ml and >103 copies/ml in corresponding Naso-/Oropharyngeal-swabs, respectively. For effective application of the Lolli-Method in schools and daycare facilities, SEIR-modeling indicated a preferred frequency of two tests per week. The developed test strategy was implemented in 3,700 schools and 698 daycare facilities in Germany, screening over 800,000 individuals twice per week. In a period of 3 months, 6,364 pool-RT-qPCRs tested positive (0.64%), ranging from 0.05% to 2.61% per week. Notably, infections correlated with local SARS-CoV-2 incidences and with a school social deprivation index. Moreover, in comparison with the alpha variant, statistical modeling revealed a 36.8% increase for multiple (≥2 children) infections per class following infections with the delta variant. We conclude that the Lolli-Method is a powerful tool for SARS-CoV-2 surveillance and can support infection control in schools and daycare facilities.

Multicentre study on the reproducibility of MALDI-TOF MS for nontuberculous mycobacteria identification.

The ability of MALDI-TOF for the identification of nontuberculous mycobacteria (NTM) has improved recently thanks to updated databases and optimized protein extraction procedures. Few multicentre studies on the reproducibility of MALDI-TOF have been performed so far, none on mycobacteria. The aim of this study was to evaluate the reproducibility of MALDI-TOF for the identification of NTM in 15 laboratories in 9 European countries. A total of 98 NTM clinical isolates were grown on Löwenstein-Jensen. Biomass was collected in tubes with water and ethanol, anonymized and sent out to the 15 participating laboratories. Isolates were identified using MALDI Biotyper (Bruker Daltonics). Up to 1330 MALDI-TOF identifications were collected in the study. A score ≥ 1.6 was obtained for 100% of isolates in 5 laboratories (68.2-98.6% in the other). Species-level identification provided by MALDI-TOF was 100% correct in 8 centres and 100% correct to complex-level in 12 laboratories. In most cases, the misidentifications obtained were associated with closely related species. The variability observed for a few isolates could be due to variations in the protein extraction procedure or to MALDI-TOF system status in each centre. In conclusion, MALDI-TOF showed to be a highly reproducible method and suitable for its implementation for NTM identification.

Bloodstream Infections Caused by Magnusiomyces capitatus and Magnusiomyces clavatus: Epidemiological, Clinical, and Microbiological Features of Two Emerging Yeast Species.

Magnusiomyces clavatus and Magnusiomyces capitatus are emerging yeasts with intrinsic resistance to many commonly used antifungal agents. Identification is difficult, and determination of susceptibility patterns with commercial and reference methods is equally challenging. For this reason, few data on invasive infections by Magnusiomyces spp. are available. Our objectives were to determine the epidemiology and susceptibility of Magnusiomyces isolates from bloodstream infections (BSI) isolated in Germany and Austria from 2001 to 2020. In seven institutions, a total of 34 Magnusiomyces BSI were identified. Identification was done by internal transcribed spacer (ITS) sequencing and matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS). Antifungal susceptibility was determined by EUCAST broth microdilution and gradient tests. Of the 34 isolates, M. clavatus was more common (n = 24) than M. capitatus (n = 10). BSI by Magnusiomyces spp. were more common in men (62%) and mostly occurred in patients with hemato-oncological malignancies (79%). The highest in vitro antifungal activity against M. clavatus/M. capitatus was observed for voriconazole (MIC50, 0.03/0.125 mg/L), followed by posaconazole (MIC50, 0.125/0.25 mg/L). M. clavatus isolates showed overall lower MICs than M. capitatus. With the exception of amphotericin B, low essential agreement between gradient test and microdilution was recorded for all antifungals (0 to 70%). Both species showed distinct morphologic traits on ChromAgar Orientation medium and Columbia blood agar, which can be used for differentiation if no MALDI-TOF MS or molecular identification is available. In conclusion, most BSI were caused by M. clavatus. The lowest MICs were recorded for voriconazole. Gradient tests demonstrated unacceptably low agreement and should preferably not be used for susceptibility testing of Magnusiomyces spp.

How MALDI-TOF mass spectrometry can aid the diagnosis of hard-to-identify pathogenic bacteria - the rare and the unknown.

Introduction: Ten years after its introduction into clinical microbiology, MALDI-TOF mass spectrometry has become the standard routine identification tool for bacteria in most laboratories. The technology has accelerated analyses and improved the quality of results. The greatest significance has been observed for bacteria that were challenging to be identified by traditional methods. Areas covered: We searched in existing literature (Pubmed) for reports how MALDI-TOF MS has contributed to identification of rare and unknown bacteria from different groups. We describe how this has improved the diagnostics in different groups of bacteria. Reference patterns for strains which yet cannot be assigned to a known species even enable the search for related bacteria in studies as well as in routine diagnostics. MALDI-TOF MS can help to discover and investigate new species and their clinical relevance. It is a powerful tool in the elucidation of the bacterial composition of complex microbiota in culturomics studies. Expert opinion: MALDI-TOF MS has improved the diagnosis of bacterial infections. It also enables knowledge generation for prospective diagnostics. The term 'hard-to-identify' might only be rarely attributed to bacteria in the future. Novel applications are being developed, e.g. subspecies differentiation, typing, and antibiotic resistance testing which may further contribute to improved microbial diagnostics.

MALDI-TOF bacterial subtyping to detect antibiotic resistance.

The spread of bacterial resistance has been continuously increasing in the recent decade. Multi-drug resistant (MDR) bacteria now represent one of the most worrisome public health issues, as they seriously complicate the treatment of infections, often leaving few therapeutic options. Enterobacteria and Staphylococcus aureus are among the most common bacterial pathogens, while Bacteroides fragilis is the most frequent anaerobic pathogen. All of these species can cause severe and life-threatening infections, and represent the most frequent causes of antibiotic-resistant healthcare-associated infections worldwide, as they frequently exhibit resistance to various classes of antibiotics. Resistance to carbapenems, the last resort beta-lactam agent, is a particularly threatening problem. Achieved by different mechanisms, leads to total inefficacy of any beta-lactam agent. During the recent years, MALDI-TOF mass spectrometry has become established as the reference method for bacterial identification in routine practice. It has proven to be a reliable and robust method to detect specific peaks in bacterial mass spectra, corresponding to specific resistance markers, enabling the instant detection of resistant isolates in real time during the standard routine identification process. Here, we investigated the performance of the subtyping module of the MALDI Biotyper system (Bruker Daltonik, GmbH) for the instant identification of KPC-producing Klebsiella pneumoniae, methicillin-resistant Staphylococcus aureus, and carbapenemase-producing Bacteroides fragilis during the identification workflow. We evaluated accuracy and potential impact on turnaround time. Furthermore, we investigated the possibility to extend the subtyping for detection of the KPC-specific marker to bacterial species other than K. pneumoniae.

A Full MALDI-Based Approach to Detect Plasmid-Encoded KPC-Producing Klebsiella pneumoniae.

KPC-producing Klebsiella pneumoniae represents a severe public health concern worldwide. The rapid detection of these isolates is of fundamental importance for the adoption of proper antibiotic treatment and infection control measures, and new applications of MALDI-TOF MS technology fit this purpose. In this study, we present a full MALDI-based approach to detect plasmid-encoded KPC-producing strains, accomplished by the automated detection of a KPC-specific peak (at 11,109 m/z) by a specific algorithm integrated into the MALDI Biotyper system (Bruker Daltonik), and the confirmation of carbapenemase activity by STAR-Carba imipenem hydrolysis assay. A total of 6209 K. pneumoniae isolates from Italy and Germany were investigated for the presence of the KPC-related peak, and a subset of them (n = 243) underwent confirmation of carbapenemase activity by STAR-Carba assay. The novel approach was further applied directly to positive blood culture bottles (n = 204), using the bacterial pellet obtained with Sepsityper kit (Bruker Daltonik). The novel approach enabled a reliable and very fast detection of KPC-producing K. pneumoniae strains, from colonies as well as directly from positive blood cultures. The automated peak detection enabled the instant detection of KPC-producing K. pneumoniae during the routine identification process, with excellent specificity (100%) and a good sensitivity (85.1%). The sensitivity is likely mainly related to the prevalence of the specific plasmid harboring clones among all the KPC-producing circulating strains. STAR-Carba carbapenemase confirmation showed 100% sensitivity and specificity, both from colonies and from positive blood cultures.

Accurate differentiation of Mycobacterium chimaera from Mycobacterium intracellulare by MALDI-TOF MS analysis.

PURPOSE: The increasing number of infections caused by nontuberculous mycobacteria (NTM) has prompted the need for rapid and precise identification methods of these pathogens. Several studies report the applicability of MALDI-TOF mass spectrometry (MS) for identification of NTM. However, some closely related species have very similar spectral mass fingerprints, and until recently, Mycobacterium chimaera and M. intracellulare could not be separated from each other by MALDI-TOF MS. METHODOLOGY: The conventional identification methods used in routine diagnostics have similar limitations. Recently, the differentiation of these two species within the Mycobacterium avium complex has become increasingly important due to reports of M. chimaera infections related to open heart surgery in Europe and in the USA. In this report, a method for the distinct differentiation of M. chimaera and M. intracellulare using a more detailed analysis of MALDI-TOF mass spectra is presented. KEY FINDINGS: Species-specific peaks could be identified and it was possible to assign all isolates (100 %) from reference strain collections as well as clinical isolates to the correct species. CONCLUSIONS: We have developed a model for the accurate identification of M. chimaera and M. intracellulare by MALDI-TOF MS. This approach has the potential for routine use in microbiology laboratories, as the model itself can be easily implemented into the software of the currently available systems by MALDI-TOF MS manufacturers.

Identification of microorganisms grown on chromogenic media by MALDI-TOF MS.

Matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS) and chromogenic media are widely used in clinical microbiology laboratories to facilitate the rapid selection and identification of pathogens. The aim of this study was to evaluate whether usage of chromogenic media limits the diagnostic performance of MALDI-TOF MS for microbial identification. A total of 386 microorganisms collected and analyzed at five laboratories were included. Isolates were cultured on relevant chromogenic media and non-selective agar plates in parallel and identified using the Bruker MALDI-TOF MS. Among the tested isolates, no misidentification was recorded and there was no medium-related difference in the identification level. However, score values were overall slightly but significantly lower for isolates grown on chromogenic media. In conclusion, the use of chromogenic culture media tested here had no relevant impact on MALDI-TOF MS performance for diagnostic purposes.

[Rhinoorbitocerebral zygomycosis caused by Rhizopus microsporus in a roe deer (Capreolus capreolus)]. / Rhinoorbitozerebrale Zygomykose durch Rhizopus microsporus bei einem Reh (Capreolus capreolus).

An one-year-old male roe deer (Capreolus capreolus) with abnormal behaviour was shot in order to exclude rabies virus infection. The 12.8 kg weighing animal was emaciated and revealed an asymmetric head with protruding left eye and expositional keratitis. There was a grey whitish soft mass within the caudal nasal cavity, which had infiltrated the frontal cerebrum through the cribriform plate and the retrobulbar tissue through the orbita. Histologically, the mass consisted of a chronic granulomatous inflammation with plentiful fungal hyphae. Fungal culture revealed mold fungi of the zygomycotic genus Rhizomucor, which were differentiated as Rhizopus microsporus by MALDI-TOF mass spectrometry and DNA-sequencing. Rhinoorbitocerebral zygomycosis has to be considered as a differential diagnosis for nasal and orbital tumour-like lesions and as a cause of abnormal behaviour of roe deer.

Description of the metallo-ß-lactamase GIM-1 in Acinetobacter pittii.

OBJECTIVES: To characterize the mechanisms involved in the reduced carbapenem susceptibility of five Acinetobacter pittii strains isolated from different regions of Germany. METHODS: The strains were analysed by susceptibility testing, phenotypic tests for metallo-ß-lactamase production, sequencing of the integron structure and strain typing by PFGE, as well as multilocus sequence typing (MLST) and plasmid analysis by S1 restriction and hybridization. RESULTS: Despite GIM-1 production, the MICs of imipenem were only 4 mg/L for four strains and some methods of phenotypic MBL detection failed. According to PFGE and MLST, the strains belonged to four different clones, but blaGIM-1 was present in identical integron structures in all strains and carried on plasmids of ∼60 kb. CONCLUSIONS: For the first time, GIM-1 has been demonstrated in A. pittii. This resistance mechanism has previously been reported only in Enterobacteriaceae and Pseudomonas aeruginosa. As GIM-1 was found in strains with diverse clonal backgrounds, but encoded on plasmids of a similar size, further spread among Acinetobacter spp. seems possible. The detection of GIM-1 production might be challenging in some strains due to the low MICs of carbapenems.