ABSTRACT
Increasing evidence within the literature has identified the presence of biofilms in chronic wounds and proposed that they contribute to delayed wound healing. This research aimed to ...investigate the presence of biofilm in diabetic foot ulcers (DFUs) using microscopy and molecular approaches and define if these are predominantly mono‐ or multi‐species. Secondary objectives were to correlate wound observations against microscopy results in ascertaining if clinical cues are useful in detecting wound biofilm. DFU tissue specimens were obtained from 65 subjects. Scanning electron microscopy (SEM) and peptide nucleic acid fluorescent in situ hybridisation (PNA‐FISH) techniques with confocal laser scanning microscopy (CLSM) were used to visualise biofilm structures. Next‐generation DNA sequencing was performed to explore the microbial diversity. Clinical cues that included the presence of slough, excessive exudate, a gel material on the wound bed that reforms quickly following debridement, poor granulation and pyocyanin were correlated to microscopy results. Of the 65 DFU specimens evaluated by microscopy, all were characterised as containing biofilm (100%, P < 0·001). The presence of both mono‐species and multi‐species biofilms within the same tissue sections were detected, even when DNA sequencing analysis of DFU specimens revealed diverse polymicrobial communities. No clinical correlations were identified to aid clinicians in identifying wound biofilm. Microscopy visualisation, when combined with molecular approaches, confirms biofilms are ubiquitous in DFUs and form either mono‐ or multi‐species biofilms. Clinical cues to aid clinicians in detecting wound biofilm are not accurate for use in DFUs. A paradigm shift of managing DFUs needs to consider anti‐biofilm strategies.
Turner syndrome (TS) is the most common chromosomal abnormality in females. The diagnosis of TS is based on karyotyping of 30 blood lymphocytes. This technique does not rule out tissue mosaicism or ...low-grade mosaicism in the blood. Because of the associated risk of gonadoblastoma, mosaicism is especially important in case this involves a Y chromosome.
This study was set to determine the value of additional genetic studies such as fluorescent
hybridisation and the inclusion of buccal cells in search for mosaicism in TS patients.
This cross-sectional, descriptive study was performed in Human Genetics Department, Medical Research Institute, Alexandria University.
Fluorescence
hybridisation technique was applied to lymphocyte cultures as well as buccal smears using centromeric probes for X and Y chromosomes. Genotype phenotype correlation was also evaluated.
Descriptive study where categorical variables were described using number and percentage and continuous variables were described using mean and standard deviation.
Fluorescence
hybridisation technique study detected hidden mosaicism in 60% of studied patients; 20% of patients had a cell line containing Y material, while 40% had variable degrees of X, XX mosaicism, and in the remaining 40% no second cell line was detected. Fluorescence
hybridisation study helped identify the origin of the marker to be Y in all patients. The introduction of an additional cell line helped in identifying mosaicism in patients with monosomy X. Virilisation signs were only observed among TS patients with Y cell line mosaicism. The clinical manifestations were more severe in patients with monosomy X than other mosaic cases.
Molecular cytogenetic investigation for all suspected cases of TS should be considered for appropriate treatment plan and genetic counselling.
•Bladder malakoplakia is a rare and poorly recognized entity in veterinary medicine.•This granulomatous cystitis present in cases of bladder malakoplakia is characterized macroscopically by ...tumor-like formations and histologically by a histiocytic inflammatory response to a recurrent urinary bacterial infection. Malakoplakia could be the result of defective macrophage phagocytic activity.•Malakoplakia should be suspected in the presence of one or more proliferative bladder lesions in a young animal and in cases of persistent urinary tract infections.•Medical management alone can be considered with targeted and prolonged antibiotic therapy (antibiotic treatment for more than 2 months has been reported). An antibiotic with intracellular penetration should be used. Further studies are needed to refine the therapeutic options for this condition.
A 3-month-old female French Bulldog presented with hematuria, severe pollakiuria, and urinary incontinence lasting for 1.5 months. Broad-spectrum empirical antibiotic therapy and nonsteroidal anti-inflammatory drugs were initiated by the referring veterinarian. Due to a lack of improvement, the dog was referred. At referral examination, urinary clinical signs persisted (hematuria, severe pollakiuria) and a firm bladder was noted. Abdominal ultrasonography revealed severe, diffuse bladder wall thickening with a significant reduction in the bladder lumen. Urinary tract endoscopy showed whitish exophytic proliferations throughout the entire bladder wall. Histological bladder wall analysis led to a diagnosis of bladder malakoplakia. Prolonged antibiotic therapy with fluoroquinolones was prescribed and resulted in clinical remission despite persistent bacteria in the bladder wall. This report describes a case of successfully medically managed bladder malakoplakia, a very rare condition in veterinary medicine, well documented in humans.
Background and aim: A role for the intestinal microbial community (microbiota) in the onset and chronicity of Crohn’s disease (CD) is strongly suspected. However, investigation of such a complex ...ecosystem is difficult, even with culture independent molecular approaches. Methods: We used, for the first time, a comprehensive metagenomic approach to investigate the full range of intestinal microbial diversity. We used a fosmid vector to construct two libraries of genomic DNA isolated directly from faecal samples of six healthy donors and six patients with CD. Bacterial diversity was analysed by screening the two DNA libraries, each composed of 25 000 clones, for the 16S rRNA gene by DNA hybridisation. Results: Among 1190 selected clones, we identified 125 non-redundant ribotypes mainly represented by the phyla Bacteroidetes and Firmicutes. Among the Firmicutes, 43 distinct ribotypes were identified in the healthy microbiota, compared with only 13 in CD (p<0.025). Fluorescent in situ hybridisation directly targeting 16S rRNA in faecal samples analysed individually (n = 12) confirmed the significant reduction in the proportion of bacteria belonging to this phylum in CD patients (p<0.02). Conclusion: The metagenomic approach allowed us to detect a reduced complexity of the bacterial phylum Firmicutes as a signature of the faecal microbiota in patients with CD. It also indicated the presence of new bacterial species.
Human epidermal growth factor receptor 2 (HER2) is a prognostic biomarker and therapeutic target in carcinomas of the breast, stomach and colon. In 2018, clinical trial data confirmed the prognostic ...and predictive role of HER2 in uterine serous carcinoma, with a demonstrated survival benefit from combined chemotherapy and anti-HER2 targeted therapy in patients with advanced or recurrent disease. Approximately one-third of uterine serous carcinomas demonstrate HER2 protein overexpression and/or gene amplification and HER2 immunohistochemistry, supplemented by in situ hybridisation in equivocal cases, is fast becoming a reflex ancillary test at time of diagnosis. The potential role of HER2 in gynaecological tumours other than uterine serous carcinoma is yet to be firmly established. With the advent of personalised medicine, routine tumour sequencing and pursuit of targeted therapies, this is a field currently under active investigation. Emerging data suggest triaging endometrial carcinomas for HER2 analysis based on molecular classification may be superior to histotype-based testing, with copy-number high/p53 mutant tumours enriched for HER2 overexpression or amplification. Accordingly, many carcinosarcomas and a subset of clear cell and high-grade endometrioid carcinomas may be eligible for HER2 targeted therapy, although any clinical benefit in this context is currently undefined. For ovarian carcinomas, combined data support the role of HER2 as a prognostic biomarker, however its use as a therapeutic target is yet to be elucidated through clinical trials. In the cervix, reported rates of HER2 overexpression vary and are generally low, and currently there is insufficient evidence to justify routine HER2 testing in this context. Limited data suggest HER2 holds promise as a prognostic and predictive biomarker in vulvar Paget disease. Future clinical trials, with pathologist input to develop and refine site-specific scoring criteria, are required to establish what role HER2 might play more broadly in gynaecological cancer care.
The present study describes a new species of myxosporean, Auerbachia ignobili n. sp., infecting the hepatic bile ducts of Caranx ignobilis (Forsskål, 1775). Myxospores are club-shaped with a broad ...anterior region and a narrow, slightly curved and blunt caudal extension, measuring 17.4 ± 1.5 μm in length and 7.5 ± 7.4 μm in width. Shell valves asymmetrical, with a faint suture line, and enclosed a single, elongate-elliptical polar capsule with a ribbon-like polar filament, arranged in 5–6 coils. Developmental stages included early and late presporogonic stages, pansporoblast, and sporogonic stages with monosporic and disporic plasmodia. A. ignobili n. sp. differs from the other described species of Auerbachia in the shape and dimensions of the myxospores and polar capsules. The molecular analysis generated ∼1400 bp long SSU rDNA sequences and the present species exhibited a maximum similarity 94.04–94.91% with A. chakravartyi. Genetic distance analysis indicated the lowest interspecies divergence of 4.4% with A. chakravartyi. In phylogenetic analysis, A. ignobili n. sp. was positioned independently with a high bootstrap value (1/100) and appeared as sister to A. maamouni and A. chakravartyi. Fluorescent in situ hybridization and histology indicates that the parasite develops within the hepatic bile ducts. Histological studies did not reveal any pathological changes. Considering the morphological, morphometric, molecular, and phylogenetic differences coupled with the differences in host and geographic locations, the present myxosporean is treated as a new species and named A. ignobili n. sp.
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•Describes a novel myxosporean, Auerbachia ignobili n. sp. infecting Caranx ignobilis.•Parasite develops within the hepatic bile ducts and mature myxospores are stored in the gallbladder.•Provides the first detailed description of the developmental stages of an Auerbachia species.•The coelozoic nature of the parasite is confirmed using histology and Fluorescent in situ hybridization.•Forms the first report of a myxosporean from C. ignobilis in Indian waters.
Abstract
There has been considerable research into the understanding of the healthy skin microbiome. Similarly, there is also a considerable body of research into whether specific microbes contribute ...to skin disorders, with atopic dermatitis (AD) routinely linked to increased
Staphylococcus aureus (S. aureus)
colonisation. In this study, the epidermal surface of participants was sampled using swabs, while serial tape-stripping (35 tapes) was performed to sample through the stratum corneum. Samples were taken from AD patients and healthy controls, and the bacterial communities were profiled by metabarcoding the universal V3-V4 16S rRNA region. Results show that the majority of bacterial richness is located within the outermost layers of the stratum corneum, however there were many taxa that were found almost exclusively at the very outermost layer of the epidermis. We therefore hypothesise that tape-stripping can be performed to investigate the ‘core microbiome’ of participants by removing environmental contaminants. Interestingly, significant community variation between AD patients and healthy controls was only observable at the epidermal surface, yet a number of individual taxa were found to consistently differ with AD status across the entire epidermis (i.e. both the epidermal surface and within the epidermis). Sampling strategy could therefore be tailored dependent on the hypothesis, with sampling for forensic applications best performed using surface swabs and outer tapes, while profiling sub-surface communities may better reflect host genome and immunological status.
Periodontitis is an infection driven inflammatory disease caused by dental plaque accumulation that in turn causes microbial alterations which may lead to drastic consequences in the periodontium in ...susceptible individuals. Hence, the rationale of periodontal therapy is predominantly focused on the elimination or reduction of these periodontal pathogens. Despite following a wide range of preventive measures, controlling periodontal disease is challenging and treatment is usually initiated mostly after lesions become clinically detectable and tissues undergo irreversible damage. Microbiological diagnostic tests aid in the early detection of these lesions when they are still reversible giving an opportunity for non-invasive treatment. Microscopy, bacterial culture, immunological assays like Evalusite, Fluorescent In-Situ Hybridisation (FISH), Oraquick, enzymatic assays like Perioscan, Periogard, Pocketwatch, Periocheck, Matrix Metalloproteinase (MMP) dipstick test, Biolise, and molecular biology techniques like Polymerase Chain Reactions (PCR), Terminal Restriction Fragment Length Polymorphism (T-RFLP), 454 Pyrosequencing, Supported Oligonucleode Ligation and Detection (SOLiD) have been among the techniques employed. Some of these diagnostic aids were solely for scientific purposes, while others were adapted and updated for therapeutic use. The current paper focuses on the practical utility of the rapidly expanding plethora of microbiological diagnostic aids highlighting the concerns surrounding their applications in periodontal diagnosis.
To evaluate whether the epidermal growth factor receptor (EGFR), K-Ras and PTEN, all members of the EGFR signalling pathway, may affect the clinical response in cetuximab-treated metastatic ...colorectal cancer (mCRC) patients. Twenty-seven cetuximab-treated mCRC patients were evaluated for drug response and investigated for EGFR protein expression and gene status, K-Ras mutational status and PTEN protein expression. Ten patients achieved a partial response (PR) to cetuximab-based therapy. All 27 patients showed EGFR protein overexpression. Epidermal growth factor receptor gene amplification was observed in eight out of 27 (30%) and chromosome 7 marked polysomy in 16 (59%) patients. Partial response was observed in six out of eight patients with EGFR gene amplification, four out of 16 with marked polysomy and none out of three with eusomy (P<0.05). The K-Ras wild-type sequence was observed in 17 patients, and nine of them experienced a PR. Conversely, K-Ras was mutated in 10 cases, of which one patient experienced a PR (P<0.05). The PTEN protein was normally expressed in 16 patients, and 10 of them achieved a PR. In contrast, no benefit was documented in 11 patients with loss of PTEN activity (P<0.001). Patients with EGFR gene amplification or chromosome 7 marked polysomy respond to cetuximab. In addition to K-Ras mutations, we demonstrate for the first time that the loss of PTEN protein expression is associated with nonresponsiveness to cetuximab.
Aim
This study compared the faecal microbial composition of formula‐fed infants who did and did not have colic.
Methods
Faecal samples from formula‐fed infants under 16 weeks of age with (n = 38) and ...without (n = 39) colic were collected at Department of Pediatrics in Turin, Italy, between February 2014 and October 2015. The pH and faecal ammonia were determined and total bacteria, bifidobacteria, lactic acid bacteria and coliforms were quantified by fluorescent in situ hybridisation (FISH).
Results
Faecal ammonia was significantly higher in the colicky infants than in the controls (483 vs. 216 μg/g, p < 0.05). The FISH counts of total bacteria were lower in colicky infants (1.8E10 ± 1.5E10) than in the controls (3.4E10 ± 3.0E10) (p < 0.05). The relative abundance of coliform bacteria was significantly higher in colicky infants (p < 0.05). No differences were observed for the bifidobacteria and lactic acid bacteria counts between the two groups.
Conclusion
Our comparison of formula‐fed infants with and without colic revealed significant differences in total bacteria, Enterobacteriaceae and faecal ammonia. This study provides the stimulus for further studies of the gut microbiome, using new methods of analysis such as 16S metagenomics sequencing in order to lead to more tailored dietary approaches.
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