Fluorescence in Situ Hybridization (FISH) for the Diagnosis of Periprosthetic Joint Infection in Formalin-Fixed Paraffin-Embedded Surgical Tissues.

BACKGROUND: As the number of arthroplasties performed increases, periprosthetic joint infection (PJI) represents a common and challenging problem. The Musculoskeletal Infection Society (MSIS) recommends diagnosing PJI according to its guidelines. The aim of the current study was to assess whether fluorescence in situ hybridization (FISH) analysis of formalin-fixed paraffin-embedded periprosthetic membranes can successfully improve the diagnosis of infection in patients with orthopaedic implants. METHODS: We retrospectively analyzed 88 periprosthetic membranes of joint prostheses using FISH analysis according to a standard protocol, with a probe targeting a sequence found in most bacteria. We compared the results with routine clinical classification according to the guidelines of the MSIS, microbiological culture, and histopathological classification according to Morawietz and Krenn. We additionally performed FISH analysis using 2 species-specific probes for several culture-positive cases. RESULTS: FISH successfully detected bacteria in 38 (95%) of 40 periprosthetic membranes that were rated positive by clinical classification. FISH results compared with clinical classification demonstrated a sensitivity of 95% (95% confidence interval [CI], 83.08% to 99.39%), a specificity of 85.42% (95% CI, 72.24% to 93.93%), a positive predictive value of 84.44% (95% CI, 70.55% to 93.50%), and a negative predictive value of 95.35% (95% CI, 84.19% to 99.43%). FISH results compared with histopathological classification demonstrated a sensitivity of 95.12% (95% CI, 83.47% to 99.40%), a specificity of 87.23% (95% CI, 74.26% to 95.17%), a positive predictive value of 86.67% (95% CI, 73.21% to 94.95%), and a negative predictive value of 95.35% (95% CI, 84.19% to 99.43%). We successfully detected Pseudomonas aeruginosa and Staphylococcus aureus with species-specific FISH probes in all cases that were positive for these respective bacteria by microbiological culture. CONCLUSIONS: FISH-based diagnosis of PJI is feasible and can be used as an additional diagnostic criterion. FISH not only can detect bacteria in periprosthetic membranes but can also differentiate pathogens at the species level. FISH represents a fast and reliable tool for detecting PJI in periprosthetic membranes, especially in combination with clinical and histopathological classification. LEVEL OF EVIDENCE: Diagnostic Level III. See Instructions for Authors for a complete description of levels of evidence.

Fluorescence In Situ Hybridization for Diagnosis of Whipple's Disease in Formalin-Fixed Paraffin-Embedded Tissue.

Whipple's disease (WD) is a rare chronic systemic infection with a wide range of clinical symptoms, routinely diagnosed in biopsies from the small intestine and other tissues by periodic acid-Schiff (PAS) diastase staining and immunohistological analysis with specific antibodies. The aim of our study was to improve the pathological diagnosis of WD. Therefore, we analyzed the potential of fluorescence in situ hybridization (FISH) for diagnosing WD, using a Tropheryma (T.) whipplei-specific probe. 19 formalin-fixed paraffin-embedded (FFPE) duodenal biopsy specimens of 12 patients with treated (6/12) and untreated (6/12) WD were retrospectively examined using PAS diastase staining, immunohistochemistry, and FISH. 20 biopsy specimens with normal intestinal mucosa, Helicobacter pylori, or mycobacterial infection, respectively, served as controls. We successfully detected T. whipplei in tissue biopsies with a sensitivity of 83% in untreated (5/6) and 40% in treated (4/10) cases of WD. In our study, we show that FISH-based diagnosis of individual vital T. whipplei in FFPE specimens is feasible and can be considered as ancillary diagnostic tool for the diagnosis of WD in FFPE material. We show that FISH not only detect active WD but also be helpful as an indicator for the efficiency of antibiotic treatment and for detection of recurrence of disease when the signal of PAS diastase and immunohistochemistry lags behind the recurrence of disease, especially if the clinical course of the patient and antimicrobial treatment is considered.

Assessment of long-term cultivated human precision-cut lung slices as an ex vivo system for evaluation of chronic cytotoxicity and functionality.

BACKGROUND: Investigation of basic chronic inflammatory mechanisms and development of new therapeutics targeting the respiratory tract requires appropriate testing systems, including those to monitor long- persistence. Human precision-cut lung slices (PCLS) have been demonstrated to mimic the human respiratory tract and have potential of an alternative, ex-vivo system to replace or augment in-vitro testing and animal models. So far, most research on PCLS has been conducted for short cultivation periods (≤72 h), while analyses of slowly metabolized therapeutics require long-term survival of PCLS in culture. In the present study, we evaluated viability, physiology and structural integrity of PCLS cultured for up to 15 days. METHODS: PCLS were cultured for 15 days and various parameters were assessed at different time points. RESULTS: Structural integrity and viability of cultured PCLS remained constant for 15 days. Moreover, bronchoconstriction was inducible over the whole period of cultivation, though with decreased sensitivity (EC501d = 4 × 10-8 M vs. EC5015d = 4 × 10-6 M) and reduced maximum of initial airway area (1d = 0.5% vs. 15d = 18.7%). In contrast, even though still clearly inducible compared to medium control, LPS-induced TNF-α secretion decreased significantly from day 1 to day 15 of culture. CONCLUSIONS: Overall, though long-term cultivation of PCLS need further investigation for cytokine secretion, possibly on a cellular level, PCLS are feasible for bronchoconstriction studies and toxicity assays.

Nasopharyngeal colonization with Streptococcus pneumoniae triggers dendritic cell dependent antibody responses against invasive disease in mice.

Nasopharyngeal colonization with Streptococcus pneumoniae (Spn) is an important precondition for the development of pneumococcal pneumonia. At the same time, nasopharyngeal colonization with Spn has been shown to mount adaptive immune responses against Spn in mice and humans. Cellular responses of the nasopharyngeal compartment, including the nasal-associated lymphoid tissue, to pneumococcal colonization and their importance for developing adaptive immune responses are poorly defined. We show that nasopharyngeal colonization with S. pneumoniae led to substantial expansion of dendritic cells (DCs) both in nasopharyngeal tissue and nasal-associated lymphoid tissue of mice. Depletion of DCs achieved by either diphtheria toxin (DT) treatment of chimeric zDC+/DTR mice, or by use of FMS-like tyrosine kinase 3 ligand (Flt3L) KO mice exhibiting congenitally reduced DC pool sizes, significantly diminished antibody responses after colonization with Spn, along with impaired protective immunity against invasive pneumococcal disease. Collectively, the data show that classical DCs contribute to pneumococcal colonization induced adaptive immune responses against invasive pneumococcal disease in two different mouse models. These data may be useful for future nasopharyngeal vaccination strategies against pneumococcal diseases in humans.

Biosynthetic Nanostructured Cellulose Patch for Chest Wall Reconstruction: Five-Month Follow-up in a Porcine Model.

PURPOSE: Ideal approaches and materials for reconstruction of large chest wall defects remain a topic of debate. We sought to explore the suitability of a reinforced nanostructured cellulose (NC) patch for chest wall reconstruction in an animal model. MATERIALS AND METHODS: In four domestic pigs, a standardized 10 × 10 cm chest wall defect was created by resecting three rib segments. Subsequently the defect was reconstructed via a biosynthetic NC patch (16 × 12 cm) reinforced by polytetrafluoroethylene mesh. After 1, 2, 4, and 5 months respectively, gross examination of NC patches was performed following sacrifice of the animals. Specimens of NC patches and surrounding connective tissue underwent histological examinations after staining with Hematoxylin-eosin and Elastica van Gieson. RESULTS: All animals survived their observation period without encountering major adverse events. On gross examination all NC patches were intact and well integrated into the surrounding tissue. Histological examination showed clearly demarked zones of foreign body reaction at the patch/host-tissue interface. After 5 months a slight increase in foreign body reaction, fibrous capsule formation and cellular infiltration were observed. No signs of fibroblast proliferation or neovascularization were seen within NC patches at any point. CONCLUSIONS: Our findings suggest a quick healing process and good overall biocompatibility following NC patch implantation.NC might prove an efficient and suitable biomaterial for complex chest wall reconstruction.

Adhesion Prevention Efficacy of Composite Meshes Parietex®, Proceed® and 4DryField® PH Covered Polypropylene Meshes in an IPOM Rat Model.

Background: Adhesions to intraperitoneally implanted meshes (IPOM) are a common problem following hernia surgery and may cause severe complications. Recently, we showed that missing peritoneal coverage of the intestine is a decisive factor for adhesion formation and 4DryField® PH (4DF) gel significantly prevents intestine-to-mesh adhesions even with use of uncoated Ultrapro® polypropylene mesh (UPM). The present study investigates adhesion prevention capability of coated Parietex® mesh (PTM) and Proceed® mesh (PCM) in comparison to 4DF treated UPM. Methods: 20 rats were randomized into two groups. A 1.5 x 2 cm patch of PTM or PCM was attached to the abdominal wall and the cecum was depleted from peritoneum by abrasion. After seven days incidence of intestine-to-mesh adhesions was evaluated using Lauder and Hoffmann adhesion scores. Histological specimens were evaluated; statistics were performed using student's t-test. The data were compared with recently published data of 4DF treated uncoated UPM. Results: Use of PTM or PCM did not significantly diminish development of intestine-to-mesh adhesions (adhesion reduction rate PTM: 29%, p = 0.069 and PCM: 25%, p = 0.078). Histological results confirmed macroscopic finding of agglutination of intestine and abdominal wall with the mesh in between. Compared to these data, the use of UPM combined with 4DF gel reveals significantly better adhesion prevention capability (p < 0.0001) as shown in earlier studies. However, in clinical situation interindividual differences in adhesion induction mechanisms cannot be excluded by this experimental approach as healing responses towards the different materials might vary. Conclusion: This study shows that in case of impaired intestinal peritoneum coated PTM and PCM do not provide significant adhesion prevention. In contrast, use of UPM combined with 4DF gel achieved a significant reduction of adhesions. Hence, in case of injury of the visceral peritoneum, application of a polysaccharide barrier device such as 4DF gel might be considered more effective in reducing intestine-to-mesh adhesions than coated mesh devices.

Molecular Profiling in Lung Biopsies of Human Pulmonary Allografts to Predict Chronic Lung Allograft Dysfunction.

Chronic lung allograft dysfunction (CLAD) is the main reason for poor long-term outcome of lung transplantation, with bronchiolitis obliterans (BO) representing the predominant pathological feature. BO is defined as a progressive fibrous obliteration of the small airways, thought to be triggered by a combination of nonimmune bronchial injury and alloimmune and autoimmune mechanisms. Because biopsy samples are too insensitive to reliably detect BO and a decline in lung function test results, which is clinically used to define CLAD, does not detect early stages, there is need for alternative biomarkers for early diagnosis. Herein, we analyzed the cellular composition and differential expression of 45 tissue remodeling-associated genes in transbronchial lung biopsy specimens from two cohorts with 18 patients each: patients who did not develop CLAD within 3 years after transplantation (48 biopsy specimens) and patients rapidly developing CLAD within the first 3 postoperative years (57 biopsy specimens). Integrating the mRNA expression levels of the five most significantly dysregulated genes from the transforming growth factor-ß axis (BMP4, IL6, MMP1, SMAD1, and THBS1) into a score, patient groups could be confidently separated and the outcome predicted (P < 0.001). We conclude that overexpression of fibrosis-associated genes may be valuable as a tissue-based molecular biomarker to more accurately diagnose or predict the development of CLAD.