Search

Objective: Diabetic patients suffer more frequently from biofilm-associated infections than normoglycemic patients. Well described in the literature is a relationship between elevated blood glucose levels in patients and the occurrence of biofilm-associated wound infections. Nevertheless, the underlying pathophysiological pathways leading to this increased infection vulnerability and its effects on biofilm development still need to be elucidated. We developed in our laboratory a model to allow the investigation of a biofilm-associated wound infection in diabetic mice under controlled insulin treatment. Methods: A dorsal skinfold chamber was used on 16 weeks old BKS.Cg-Dock7 +/+ Lepr/J mice and a wound within the observation field of the dorsal skinfold chamber was created. These wounds were infected with ATCC 49230 (10 cells/mL). Simultaneously, we implanted implants for sustained insulin release into the ventral subcutaneous tissue (N=5 mice). Mice of the control group (N=5) were treated with sham implants. Serum glucose levels were registered before intervention and daily after the operation. Densitometrical analysis of the wound size was performed at day 0, 3, and 6 after intervention. Mice were sacrificed on day 6 and wound tissue was submitted to fluorescence hybridization (FISH) and colony forming unit (CFU) analysis in addition to immunohistocheal staining to observe wound healing. Experiments were carried out in accordance with the National Institute of Health Guidelines for the Care and Use of Laboratory Animals (protocol number 05/19).Results: The insulin implants were able to reduce blood glucose levels in the mice. Hence, the diabetic mice in the intervention group were normoglycemic after the implantation. The combination with the dorsal skinfold chamber allowed for continuous, in vivo measurements of the infection development. Implantation of the insulin implant and the dorsal skinfold chamber was a tolerable condition for the diabetic mice. We succeeded to realize reproducible biofilm infections in the animals. Discussion: We developed a novel model to assess interactions between blood glucose level and -induced biofilm-associated wound infections. The combination of the dorsal skinfold chamber model with a sustained insulin treatment has not been described so far. It allows a broad field of glucose and insulin dependent studies of infection.

(1) Background: The successful treatment of infective endocarditis (IE) relies on detecting causative pathogens to administer targeted antibiotic therapy. In addition to standard microbiological cultivation of pathogens from tissue obtained during heart valve surgery, the potential of molecular biological methods was evaluated. (2) Methods: A retrospective study was performed on heart valve tissue from 207 patients who underwent heart valve surgery for IE. FISHseq (fluorescence in situ hybridization combined with 16S rRNA gene PCR and sequencing) was performed in addition to conventional culture-based microbiological diagnostics. The diagnostic performance of FISHseq was compared with the conventional methods and evaluated in the clinical context. (3) Results: Overall, FISHseq provided a significantly higher rate of specific pathogen detection than conventional valve culture (68.1% vs. 33.3%, p < 0.001). By complementing the findings from blood culture and valve culture, FISHseq was able to provide a new microbiological diagnosis in 10% of cases, confirm the cultural findings in 24.2% of cases and provide greater diagnostic accuracy in 27.5% of cases. FISHseq could identify a pathogen in blood-culture-negative IE in 46.2% of cases, while valve culture provided only 13.5% positive results (p < 0.001). (4) Conclusions: This study demonstrates that using FISHseq as an additional molecular biological technique for diagnostics in IE adds substantial diagnostic value, with potential implications for the treatment of IE. It provides pathogen detection, especially in cases where conventional microbiological cultivation is negative or inconclusive. [ABSTRACT FROM AUTHOR]

Infective endocarditis (IE) is an infection of the endocardium, and the heart valves associated with high morbidity and mortality. Fluorescence in situ Hybridization (FISH) is a molecular imaging technique used for diagnosis of IE based on histological heart valve tissue sections. FISH allows detection and identification of microorganisms and gives information about their quantity and spatial distribution. This information is important to guide appropriate antibiotic treatment. However, as manual FISH image analysis is time- and costexpensive, an automated image analysis pipeline (consisting of tissue segmentation, bacteria detection, and spot detection modules) is proposed to assist locating potential regions with microorganisms. The proposed approach was evaluated in a study, where five observers manually assessed a set of 171 fields-of-view (FoVs) captured in 400-fold magnification from 10 randomly chosen WSI for the presence of microorganisms, morphologically detected by the nucleic acid stain DAPI. The task of the observers was to mark the presented image using a 2-class score (‘positive/questionable’ or ‘negative’). The human assessment was compared to the results suggested by the algorithm. The proposed algorithm locates and ranks potential regions with microorganisms in heart valve sections so that experts can validate them in higher power FoVs for the presence of bacteria and identify their species. The automated system for preselecting and recommending adequate FoVs is thus a starting point to support experts and save human resources. It is now ready to be further developed for the detection of bacteria by FISH.

Hospital-acquired infections caused by multidrug-resistant (MDR) pathogens pose major challenges (1). Whereas the concept of transmission of pathogenic bacterial organisms through contact with medical staff is well established, other ways [...]

The polymicrobial nature of periodontal diseases is reflected by the diversity of phylotypes detected in subgingival plaque and the finding that consortia of suspected pathogens rather than single species are associated with disease development. A number of these microorganisms have been demonstrated in vitro to interact and enhance biofilm integration, survival or even pathogenic features. To examine the in vivo relevance of these proposed interactions, we extended the spatial arrangement analysis tool of the software daime (digital image analysis in microbial ecology). This modification enabled the quantitative analysis of microbial co-localization in images of subgingival biofilm species, where the biomass was confined to fractions of the whole-image area, a situation common for medical samples. Selected representatives of the disease-associated red and orange complexes that were previously suggested to interact with each other in vitro (Tannerella forsythia with Fusobacterium nucleatum and Porphyromonas gingivalis with Prevotella intermedia) were chosen for analysis and labeled with specific fluorescent probes via fluorescence in situ hybridization. Pair cross-correlation analysis of in vivo grown biofilms revealed tight clustering of F. nucleatum/periodonticum and T. forsythia at short distances (up to 6 µm) with a pronounced peak at 1.5 µm. While these results confirmed previous in vitro observations for F. nucleatum and T. forsythia, random spatial distribution was detected between P. gingivalis and P. intermedia in the in vivo samples. In conclusion, we successfully employed spatial arrangement analysis on the single cell level in clinically relevant medical samples and demonstrated the utility of this approach for the in vivo validation of in vitro observations by analyzing statistically relevant numbers of different patients. More importantly, the culture-independent nature of this approach enables similar quantitative analyses for "as-yet-uncultured" phylotypes which cannot be characterized in vitro.

Background and purpose: To assess the clinical spectrum of central nervous system (CNS) involvement as well as cerebrospinal fluid (CSF) and neuroimaging findings in patients with Whipple's disease (WD) and to analyze the association of neurological symptoms with CSF and imaging findings. Methods: Neurological involvement was retrospectively analyzed in a series of 36 patients diagnosed with WD at a single center between 1992 and 2019. Findings of 81 comprehensive CSF examinations from 36 patients, including polymerase chain reaction (PCR) tests for Tropheryma whipplei (TW) in CSF from 35 patients, were systematically evaluated. The prevalence of ischemic stroke in patients with WD was compared to a matched control cohort. Results: Neurological symptoms occurred in 23 of 36 (63.9%) patients, with cognitive, motor, and oculomotor dysfunction being most frequent. TW was detected by PCR in CSF of 13 of 22 (59.1%) patients with and four of 13 (30.8%, p = 0.0496) patients without neurological symptoms. Total CSF protein (p = 0.044) and lactate (p = 0.035) were moderately elevated in WD with neurologic symptoms compared with WD without. No intrathecal immunoglobulin synthesis was observed. Three of 36 (8.3%) patients had hydrocephalus due to aqueductal stenosis. Patients with WD had an unexpectedly high prevalence of ischemic stroke (10/36, 27.7%) compared to matched controls (10/360, 3.2%). Conclusions: Neurological involvement in patients with WD is common. Detection of TW DNA in CSF is only partly associated with neurological symptoms. Elevated CSF parameters suggest CNS parenchymal infection. Stroke is a hitherto underrecognized manifestation of WD. These findings suggest that mechanisms beyond CNS infection contribute to the spectrum of CNS involvement in WD. [ABSTRACT FROM AUTHOR]

Background The microbial etiology of prosthetic valve infective endocarditis (PVE) can be difficult to identify. Our aim was to investigate the benefit of molecular imaging technique fluorescence in situ hybridization (FISH) combined with 16S rRNA-gene polymerase chain reaction (PCR) and sequencing (FISHseq) for the analysis of infected prosthetic heart valves. Methods We retrospectively evaluated the diagnostic outcome of 113 prosthetic valves from 105 patients with suspected PVE, treated in 2003–2013 in the Department of Cardiac Surgery, Charité University Medicine Berlin. Each prosthetic valve underwent cultural diagnostics and was routinely examined by FISH combined with 16S rRNA gene PCR and sequencing. We compared classical microbiological culture outcomes (blood and valve cultures) with FISHseq results and evaluated the diagnostic impact of the molecular imaging technique. Results Conventional microbiological diagnostic alone turned out to be insufficient, as 67% of preoperative blood cultures were noninformative (negative, inconclusive, or not obtained) and 67% of valve cultures remained negative. FISHseq improved the conventional cultural diagnostic methods in PVE in 30% of the cases and increased diagnostic accuracy. Of the valve culture–negative PVE cases, FISHseq succeeded in identifying the causative pathogen in 35%. Conclusions FISHseq improves PVE diagnostics, complementing conventional cultural methods. In addition to species identification, FISH provides information about the severity of PVE and state of the pathogens (eg, stage of biofilm formation, activity, and localization on and within the prosthetic material). As a molecular imaging technique, FISHseq enables the unambiguous discrimination of skin flora as contaminant or infectious agent. [ABSTRACT FROM AUTHOR]

Aims In patients with infections of cardiac implantable electronic devices (CIEDs), the identification of causative pathogens is complicated by biofilm formations and previous antibiotic therapy. In this work, the impact of an additional fluorescence in situ hybridization (FISH), in combination with polymerase chain reaction and sequencing (FISHseq) was investigated. Methods and results In 36 patients with CIED infections, FISHseq of explanted devices was performed and compared with standard microbiological cultivation of preoperative and intraoperative samples. The mean age was 61.9 (±16.2) years; 25 (69.4%) were males. Most patients (62.9%) had heart failure with reduced ejection fraction. Infections occurred as endoplastits (n = 26), isolated local generator pocket infection (n = 8), or both (n = 2); CIED included cardiac resynchronization therapy defibrillator (n = 17), implantable cardioverter defibrillator (n = 11), and pacemaker (n = 8) devices. The overall positive FISHseq detection rate was 97%. Intraoperatively, pathogens were isolated in 42 vs. 53% in standard cultivation vs. FISHseq, respectively. In 16 of 17 FISHseq-negative patients, the nucleic acid strain DAPI (4′,6-diamidino-2-phenylindole) indicated inactive microorganisms, which were partially organized in biofilms (n = 4) or microcolonies (n = 2). In 13 patients in whom no pathogen was identified preoperatively, standard cultivation and FISHseq identified pathogens in 3 (23%) vs. 8 (62%), respectively. For the confirmation of preoperatively known bacteria, a combined approach was most efficient. Conclusion Fluorescence in situ hybridization sequencing is a valuable tool to detect causative microorganisms in CIED infections. The combination of FISHseq with preoperative cultivation showed the highest efficacy in detecting pathogens. Additional cultivation of intraoperative tissue samples or swabs yielded more confirmation of pathogens known from preoperative culture. [ABSTRACT FROM AUTHOR]

Due to suspicion of lymphoma or lung cancer 1 week later, an SP 18 sp FDG (B-D) plus a SP 68 sp Ga-labeled fibroblast activation protein inhibitor ( SP 68 sp Ga-FAPI) PET/CT scan (E-G) were performed. However, given an already known diagnosis of granulomatous disease like tularemia, SP 68 sp Ga-FAPI PET/CT might be suitable to assess extent and activity of chronic inflammation. [Extracted from the article]