Anticoagulation-Associated Delays in Acute Hip Fractures.

Introduction Delayed surgical management of hip fractures increases mortality. The Irish and UK guidelines recommend surgery within 48 hours of presentation.1-3 Our study aimed to evaluate the prevalence of hip fracture surgery delays secondary to anticoagulation, while also documenting anaesthesia preferences, rates of appropriate dosing as well as rates of venous thromboembolism, bleeding events and cerebrovascular accidents. Methods Single centre retrospective analysis of hip fracture patients admitted during a one-year period. Results In the year 2022, 48 out of 251 (19.1%) hip fracture surgeries were delayed beyond 48-hours. Notably, 26 (54.1%) of these patients were on a DOAC. In 23 (88.5%) cases, anticoagulation was the sole cause of delay. Only 9 (39.1%) of these delayed cases were documented by anaesthetics. Of patients who experienced delays on anticoagulation, patient weights were documented in 7/22 (31.8%) of cases, hindering dosing review. Of documented weights, 9/22 (41%) were receiving the wrong dose anticoagulant. Only 4/22 (18%) of those delayed due to anticoagulation had an acute kidney injury on admission. 7/22 (31.8%) of delayed patients due to anticoagulation received general anaesthetic while, 15/22 (68.1%) received spinal anaesthetic. No major bleeding, or clotting events were reported six months post-operatively. Patients aged over 80 years accounted for 15/22 (69.5%) of those delayed due to a DOAC. Our study revealed that 23 out of 48 (47.9%) of the hip fracture surgical delays in our centre in the year 2022 were associated with patients taking DOACs on admission. We advocate for the elimination of these delays in alignment with the latest best evidence.

The occurrence of the filarial nematode Dirofilaria repens in canine hosts from Maio Island, Cape Verde.

The prevalence of canine Dirofilaria infection in Maio Island (Cape Verde) was analysed by serology, morphological and molecular identification of the parasite species. Blood and sera were collected from 150 dogs and 80 cats aged over 6 months from various localities of the island. DNA was extracted from blood and samples were screened by polymerase chain reaction (PCR) using microfilaria-specific primers. No Dirofilaria immitis was found in dogs while D. repens microfilariae were found in 5.3% of dogs and 6% were positive by PCR. The species identity was confirmed by sequencing of PCR products, which showed almost 100% homology with D. repens European sequences published in GenBank. No difference in Dirofilaria infection was observed between males and females or in dogs with different weights. However, older dogs and those from the western part of Maio Island were more frequently infected. No Dirofilaria was found in cats. This study represents the first evidence of D. repens in Cape Verde (West Africa) and highlights the need for implementing control measures and for a better surveillance of dirofilariosis in Africa.

The lysis cassette of DLP12 defective prophage is regulated by RpoE.

Expression of the lysis cassette (essD, ybcT, rzpD/rzoD) from the defective lambdoid prophage at the 12th minute of Escherichia coli's genome (DLP12) is required in some strains for proper curli expression and biofilm formation. Regulating production of the lytic enzymes encoded by these genes is critical for maintaining cell wall integrity. In lambdoid phages, late-gene regulation is mediated by the vegetative sigma factor RpoD and the lambda antiterminator Qλ. We previously demonstrated that DLP12 contains a Q-like protein (QDLP12) that positively regulates transcription of the lysis cassette, but the sigma factor responsible for this transcription initiation remained to be elucidated. In silico analysis of essDp revealed the presence of a putative - 35 and - 10 sigma site recognized by the extracytoplasmic stress response sigma factor, RpoE. In this work, we report that RpoE overexpression promoted transcription from essDp in vivo, and in vitro using purified RNAP. We demonstrate that the - 35 region is important for RpoE binding in vitro and that this region is also important for QDLP12-mediated transcription of essDp in vivo. A bacterial two-hybrid assay indicated that QDLP12 and RpoE physically interact in vivo, consistent with what is seen for Qλ and RpoD. We propose that RpoE regulates transcription of the DLP12 lysis genes through interaction with QDLP12 and that proper expression is dependent on an intact - 35 sigma region in essDp. This work provides evidence that the unique Q-dependent regulatory mechanism of lambdoid phages has been co-opted by E. coli harbouring defective DLP12 and has been integrated into the tightly controlled RpoE regulon.

A Q-like transcription factor regulates biofilm development in Escherichia coli by controlling expression of the DLP12 lysis cassette.

The DLP12 lysis cassette (essD, ybcT, rzpD/rzoD) is required in certain Escherichia coli strains for normal curli expression and biofilm development. Tightly controlled regulation of the lysis cassette is of particular importance, since its overexpression causes host cell lysis. In silico analysis revealed a putative intrinsic transcriptional terminator 100 bp upstream of essD and within 2000 bp of ybcQ (Q(DLP12)), a putative lambda (λ) Q-like antiterminator. We hypothesized that Q(DLP12) may be required for effective expression of the lysis cassette. In this work we report on the role of Q(DLP12) as a positive regulator of DLP12 lysis cassette expression. Mutants lacking Q(DLP12) exhibited a biofilm-defective phenotype analogous to that of the lysis cassette knockouts. This defect occurred through the downregulation of curli transcription, which is also consistent with that seen in the lysis cassette mutants and was restored by complementation by ectopic expression of Q(DLP12). In addition, Q(DLP12) overexpression caused cell lysis, as demonstrated by leakage of ß-galactosidase activity from cells. This was accompanied by upregulation of the DLP12 lysis cassette as demonstrated by increased essD transcription, which was documented with gfp-reporter assays, RT-PCR and chromatin immunoprecipitation (ChIP). We provide evidence that this Q-mediated effect resulted from direct interaction of Q(DLP12) with the lysis cassette promoter (essDp), as demonstrated by electrophoretic gel mobility shift assay (EMSA). We propose that Q(DLP12) encodes a functional transcriptional regulator, which promotes expression of the DLP12 lysis cassette. This work provides evidence of a regulator from a defective prophage affecting host cell physiology.

Manchester triage in acute pulmonary embolism: can it unmask the grand impersonator?

BACKGROUND: Acute pulmonary embolism (PE) is associated with high mortality risk. Early diagnosis is difficult because of non-specific clinical presentation and delay in imaging confirmation. Manchester Triage (MT) prioritises patients on the basis of illness severity and potentially recognises those with higher mortality risk. No studies of the role and impact of MT on rapid PE diagnosis and in-hospital mortality (IHM) have been carried out. OBJECTIVE: To assess the appropriateness of MT in this set of patients presenting acutely to the emergency department (ED), and to determine whether it assists in a rapid diagnosis, acts as a protective triage tool and affects short-term mortality. METHODS: Single-centre retrospective study of 176 consecutive patients with PE, assessed by MT in the ED between January 2006 and October 2010 (mean age 70.5±15.7 years, 38.6% men). The primary outcome measure was all-cause IHM. RESULTS: IHM was seen in 30 (17%) patients. More than half of the patients with PE (54%) were classified as target time for first medical observation (MOb) ≤10 min. 73.3% of IHM occurred in this group (p=0.020) with several increased markers of illness severity. MOb ≤10 min was not associated with faster PE imaging confirmation. The average door-to-diagnosis time (PEDx) was 26.8±36.8 h and PEDx >17.0 h was associated with higher IHM (p=0.017). On multivariate analysis, thrombolysis and MOb ≤10 min were included in an IHM predictor model. CONCLUSION: MT has high sensitivity in identifying patients with PE at risk. Those patients assigned as MOb ≤10 min have increased markers of illness severity and higher IHM. MT acts as a protective system in this challenging set and should be used as a patient's first assessment, aiding the emergency medical team to recognise those in need of urgent assessment and treatment.

Draft genome sequences of two Pseudomonas aeruginosa clinical isolates with different antibiotic susceptibilities.

Pseudomonas aeruginosa is a primary cause of opportunistic infections. We have sequenced and annotated the genomes of two P. aeruginosa clinical isolates evidencing different antibiotic susceptibilities. Registered differences in the composition of their accessory genomes may provide clues on P. aeruginosa strategies to thrive in different environments like infection loci.

Role of DLP12 lysis genes in Escherichia coli biofilm formation.

Phages have recently been implicated as important in biofilm development, although the mechanisms whereby phages impact biofilms remain unclear. One defective lambdoid phage carried by Escherichia coli K-12 is DLP12. Among the genes found in DLP12 are essD, ybcS and rzpD/rzoD, which are homologues of the Lambda phage genes encoding cell-lysis proteins (S, R and Rz/Rz(1)). The role that these DLP12 lysis genes play in biofilm formation was examined in deletion mutants of E. coli PHL628, a curli-overproducing, biofilm-forming K-12 derivative. Strains lacking essD, ybcS and rzpD/rzoD were unable to form wild-type biofilms. While all mutants were compromised in attachment to abiotic surfaces and aggregated less well than the wild-type, the effect of the essD knockout on biofilm formation was less dramatic than that of deleting ybcS or rzpD/rzoD. These results were consistent with electron micrographs of the mutants, which showed a decreased number of curli fibres on cell surfaces. Also consistent with this finding, we observed that expression from the promoter of csgB, which encodes the curli subunits, was downregulated in the mutants. As curli production is transcriptionally downregulated in response to cell wall stress, we challenged the mutants with SDS and found them to be more sensitive to the detergent than the wild-type. We also examined the release of (14)C-labelled peptidoglycan from the mutants and found that they did not lose labelled peptidoglycan to the same extent as the wild-type. Given that curli production is known to be suppressed by N-acetylglucosamine 6-phosphate (NAG-6P), a metabolite produced during peptidoglycan recycling, we deleted nagK, the N-acetylglucosamine kinase gene, from the lysis mutants and found that this restored curli production. This suggested that deletion of the lysis genes affected cell wall status, which was transduced to the curli operon by NAG-6P via an as yet unknown mechanism. These observations provide evidence that the S, R and Rz/Rz(1) gene homologues encoded by DLP12 are not merely genetic junk, but rather play an important, though undefined, role in cell wall maintenance.

SdrF, a Staphylococcus epidermidis surface protein, contributes to the initiation of ventricular assist device driveline-related infections.

Staphylococcus epidermidis remains the predominant pathogen in prosthetic-device infections. Ventricular assist devices, a recently developed form of therapy for end-stage congestive heart failure, have had considerable success. However, infections, most often caused by Staphylococcus epidermidis, have limited their long-term use. The transcutaneous driveline entry site acts as a potential portal of entry for bacteria, allowing development of either localized or systemic infections. A novel in vitro binding assay using explanted drivelines obtained from patients undergoing transplantation and a heterologous lactococcal system of surface protein expression were used to identify S. epidermidis surface components involved in the pathogenesis of driveline infections. Of the four components tested, SdrF, SdrG, PIA, and GehD, SdrF was identified as the primary ligand. SdrF adherence was mediated via its B domain attaching to host collagen deposited on the surface of the driveline. Antibodies directed against SdrF reduced adherence of S. epidermidis to the drivelines. SdrF was also found to adhere with high affinity to Dacron, the hydrophobic polymeric outer surface of drivelines. Solid phase binding assays showed that SdrF was also able to adhere to other hydrophobic artificial materials such as polystyrene. A murine model of infection was developed and used to test the role of SdrF during in vivo driveline infection. SdrF alone was able to mediate bacterial adherence to implanted drivelines. Anti-SdrF antibodies reduced S. epidermidis colonization of implanted drivelines. SdrF appears to play a key role in the initiation of ventricular assist device driveline infections caused by S. epidermidis. This pluripotential adherence capacity provides a potential pathway to infection with SdrF-positive commensal staphylococci first adhering to the external Dacron-coated driveline at the transcutaneous entry site, then spreading along the collagen-coated internal portion of the driveline to establish a localized infection. This capacity may also have relevance for other prosthetic device-related infections.

Confirmatory assays for transient changes of omics in soil invertebrates - Copper materials in a multigenerational exposure.

Environmental risk assessment (ERA) based on effects caused by chronic and longer term exposure is highly relevant. Further, if mechanistic based approaches (e.g. omics) can be included, beyond apical endpoints (e.g. reproduction), the prediction of effects increases. For Cu NMs (and CuCl2) this has been studied in detail, covering multi-omics and apical effects using the soil standard species Enchytraeus crypticus. The intermediate level effects like cell/tissue and organ alterations represent a missing link. In the present study we aimed to: 1) perform long term exposure to Cu materials (full life cycle and multigeneration, 46 and 224 days) to collect samples; 2) perform histology and immunohistochemistry on collected samples at 12 time points and 17 treatments; 3) integrate all levels of biological organization onto an adverse outcome pathway (AOP) framework. CuO NMs and CuCl2 caused both similar and different stress response, either at molecular initiating events (MIE) or key events (KEs) of higher level of biological organization. Cell/Tissue and organ level, post-transcriptional and transcriptional mechanisms, through histone modifications and microRNA related protein, were similarly affected. While both Cu forms affected the Notch signalling pathway, CuCl2 also caused oxidative stress. Different mechanisms of DNA methylation (epigenetics) were activated by CuO NMs and CuCl2 at the MIE.

Histologic characterization of dilated cardiomyopathy in Estrela mountain dogs.

Dilated cardiomyopathy (DCM) is the second-most-important acquired cardiovascular disease in dogs (excluding heartworm disease in some geographic regions) and a major cause of morbidity and mortality in Estrela Mountain Dogs. The objective of this study is to describe the histologic features of DCM in Estrela Mountain Dogs, with special attention to the localization and quantification of attenuated wavy fibers (AWFs), fibrosis, and fatty infiltration. Myocardial samples from 10 areas were collected from the hearts of 10 dogs with DCM and 7 dogs without signs of cardiac disease-namely, the basal, middle, and apical portions of the free wall of both cardiac ventricles and the interventricular septum, as well as the left ventricular papillary muscle. In each sample, the presence or absence of AWFs was noted, and fatty infiltration and fibrosis were quantified. Fatty infiltration, fibrosis, and AWFs were observed in the myocardium of all dogs with DCM, in contrast to what has been described in other breeds. The left ventricular myocardium was the best tissue for diagnosis of DCM, based on these histologic features. The authors concluded that quantification of fibrosis and observation of AWFs in the left ventricular myocardium are useful in the histologic diagnosis of DCM in Estrela Mountain dogs.

Histological study of canine mammary gland during the oestrous cycle.

The development of the mammary gland is a puzzling phenomenon and the research on this field has been focussed mostly on the carcinogenesis, with a less goal-oriented concern in basic histology. In order to determine the histological features of normal mammary gland in the different oestrous phases we used 39 non-pregnant female dogs of various breeds and ages. The animals were grouped in: pre-pubertal, pro-oestrous, oestrous, early and late dioestrous, early and late anoestrous phases. Major changes of the canine mammary histology throughout the oestrous cycle were identified in this study. A rudimentary gland with few ducts in the base of the nipple was observed in pre-pubertal female individuals and pubertal pro-oestrous female ones. In the oestrus, small inactive lobules associated with ductal branching and inconspicuous regressive changes were observed, while in early dioestrus, a ductal arborization was present. In late dioestrus, a complete lobuloalveolar differentiation and secretory capacity was achieved. The regressive histological features were abundant on early anoestrus, and markedly generalized on late anoestrus. The regressive process was longer in the more caudal gland pairs. This work provides baseline knowledge of canine mammary gland that may be relevant for interspecies comparative purposes and for pathologists dealing with mammary gland tumours.

Comparative overview of the effects of aerobic and resistance exercise on anxiety-like behavior, cognitive flexibility, and hippocampal synaptic plasticity parameters in healthy rats.

Clinical studies show that physical exercise has anxiolytic and pro-cognitive properties for both healthy individuals and psychiatric patients. Most of these data refer to the effects of aerobic exercise. However, other modalities such as resistance exercise deserve more attention because they may also modulate brain function. This study aimed to compare the effects of an aerobic exercise protocol on a treadmill and a resistance exercise protocol on a ladder apparatus on anxiety-like behavior, cognitive flexibility, and neuroplasticity parameters in healthy animals. Adult male Wistar rats were divided into three groups: sedentary control, aerobic training, and resistance training. Subsequently, they were evaluated in the elevated plus-maze (EPM), light-dark box, and modified hole board (mHB) tests. The expressions of synaptophysin and postsynaptic plasticity protein 95 in the dorsal and ventral hippocampus were analyzed by immunofluorescence. The results demonstrated an anxiolytic effect promoted by exercise in the EPM, particularly in the animals submitted to aerobic training, and a mild pro-learning effect of both exercise modalities was observed in the mHB test. All groups showed similar outcomes in the other evaluations. Therefore, the exercise modalities investigated in the present study did not provide considerable modifications to such aspects of the emotional/cognitive functions and neuroplasticity under physiological contexts. Perhaps the two types of exercise acted in neurobiological pathways not analyzed in this study, or the effects may emerge under pathological contexts. These hypotheses should be tested in future studies.

Randomized, active-controlled study of once-weekly alendronate 280 mg high dose oral buffered solution for treatment of Paget's disease.

UNLABELLED: Daily oral tablet bisphosphonate therapy for Paget's disease of bone may cause serious upper gastrointestinal adverse events. A once-weekly alendronate 280 mg oral buffered solution was compared with an alendronate 40 mg/day tablet. While both were similarly effective, the tablet appeared to be better tolerated in this study. INTRODUCTION: Although daily doses of oral bisphosphonates are a generally safe and effective treatment for Paget's disease of bone (PDB), some patients may experience upper gastrointestinal adverse events (UGI AEs) or find the dosing requirements inconvenient and become noncompliant. A once-weekly (OW) oral dose of bisphosphonate in buffered solution (OBS) may be as effective, better tolerated, and more convenient. METHODS: Sixty-three patients were randomized to either alendronate (ALN) 280 mg OW OBS (n = 42) or an ALN 40 mg/day tablet (n = 21) during a 6-month, randomized, double-blind, active-controlled trial. The primary endpoint was the mean percent decrease in total serum alkaline phosphatase (total ALP) from baseline at 6 months. RESULTS: There were no significant differences in total ALP between groups during the 6-month period. There was a higher incidence of clinical AEs in the ALN 280 mg OW OBS (79%) vs. the ALN 40 mg/day tablet group (67%), including drug related AEs (48% and 10%, respectively), which led to study discontinuation (19.0% and 10%, respectively). CONCLUSIONS: Although ALN 280 mg OW OBS was similarly effective as ALN 40 mg/day in reducing total ALP in patients with PDB, the ALN 40 mg/day tablet appears to be better tolerated than ALN 280 mg OW OBS.

Identification of the first vancomycin intermediate-resistant Staphylococcus aureus (VISA) isolate from a hospital in Portugal.

A clinical isolate of methicillin-resistant Staphylococcus aureus (MRSA) with intermediate resistance to vancomycin (minimal inhibitory concentration [MIC] of 4 mug/ml) was isolated in 2006 from a surgical wound of a patient hospitalized at the orthopedics ward of Hospital de São Marcos--Braga, in the town of Braga. A combination of molecular typing methods, including pulsed-field gel electrophoresis (PFGE), spa typing, multilocus sequence typing, and staphylococcal chromosomal cassette mec typing, identified the vancomycin intermediate-resistant S. aureus VISA-BRAGA as a derivative of the epidemic MRSA (EMRSA)-15 clone, which has been isolated with increasing frequency from several Portuguese hospitals recently. Compared to another EMRSA-15 isolate with the same genetic background (including PFGE subtype) the VISA-BRAGA isolate exhibited relatively high oxacillin MIC, slow growth, loss of hemolytic activity, and increased resistance to vancomycin and to daptomycin although neither of these two antibiotics was used in therapy. The VISA-BRAGA isolate described here appears to represent the first S. aureus with decreased susceptibility to vancomycin identified in a Portuguese hospital.

Portuguese recommendations for the prevention, diagnosis and management of primary osteoporosis - 2018 update.

BACKGROUND: Advances in osteoporosis (OP)case definition, treatment options, optimal therapy duration and pharmacoeconomic evidence in the national context motivated the Portuguese Society of Rheumatology (SPR) to update the Portuguese recommendations for the diagnosis and management of osteoporosis published in 2007. METHODS: SPR bone diseases' working group organized meetings involving 55 participants (rheumatologists, rheumatology fellows and one OP specialist nurse) to debate and develop the document. First, the working group selected 11 pertinent clinical questions for the diagnosis and management of osteoporosis in standard clinical practice. Then, each question was investigated through literature review and draft recommendations were built through consensus. When insufficient evidence was available, recommendations were based on experts' opinion and on good clinical practice. At two national meetings, the recommendations were discussed and updated. A draft of the recommendations full text was submitted to critical review among the working group and suggestions were incorporated. A final version was circulated among all Portuguese rheumatologists before publication and the level of agreement was anonymously assessed using an online survey. RESULTS: The 2018 SPR recommendations provide comprehensive guidance on osteoporosis prevention, diagnosis, fracture risk assessment, pharmacological treatment initiation, therapy options and duration of treatment, based on the best available evidence. They attained desirable agreement among Portuguese rheumatologists. As more evidence becomes available, periodic revisions will be performed. Target audience and patient population: The target audience for these guidelines includes all clinicians. The target patient population includes adult Portuguese people. Intended use: These recommendations provide general guidance for typical cases. They may not be appropriate in all situations - clinicians are encouraged to consider this information together with updated evidence and their best clinical judgment in individual cases.

Evaluation of nanoparticles loaded with benzopsoralen in rat peritoneal exudate cells.

Psoralens are widely used for the treatment of hyperproliferative skin disease. In this work, we prepared nanoparticles (NP) containing a benzopsoralen (3-ethoxy carbonyl-2H-benzofuro[3,2-f]-1-benzopiran-2-one) by the solvent evaporation technique. We evaluated important NP parameters such as particle size, drug encapsulation efficiency, effect of the encapsulation process over the drug's photochemistry, zeta potential, external morphology, and in vitro release behavior. We also investigated the nanoparticle as a drug delivery system (DDS), as well as its target delivery to the action site, which is a very important parameter to increase the therapeutic use of psoralens and to reduce their side effects. The uptake of benzopsoralen-loaded PLGA nanoparticles by different kinds of cells found in rat peritoneal exudates was also studied. The photodamage promoted by irradiation with UV light revealed morphological characteristics of cell damage such as cytoplasmic vesiculation, mitochondrial damage, and swelling of both the granular endoplasmatic reticulum and nuclear membrane. This encapsulation method maintained the drug's properties and improved drug delivery to the target cell.

A salivary malignant myoepithelioma in a dog.

Salivary tumours are uncommon in domestic animals and there are no known previous confirmed reports of salivary tumours of myoepithelial origin in dogs. A 12-year-old female mixed breed dog was presented with a lobulated mass, composed of white-yellowish tissues, extending from soft palate to epiglottis. Histological examination revealed a neoplastic lesion consisting of a dense population of cells showing moderate pleomorphism, with pale cytoplasm and large oval nuclei, arranged in solid lobules. Mitotic activity was very high. Tumoral cells were negative for both periodic acid-Schiff reaction and Alcian blue stain and displayed strong immunohistochemical reactivity for pan-cytokeratin, muscle specific actin and myosin and focal positivity for cytokeratin 14. On the basis of the morphological, histochemical and immunohistochemical findings a diagnosis of malignant tumour of myoepithelial origin (malignant myoepithelioma) was made.

Detection of lymph node micrometastases in malignant mammary tumours in dogs by cytokeratin immunostaining.

A series of 131 local and regional lymph nodes from 40 dogs with malignant mammary tumours were evaluated by staining with haematoxylin and eosin and immunohistochemically for antibodies to pancytokeratin (AE1/AE3) and cytokeratin 14. The immunohistochemical tests detected occult micrometastases in 9.2 per cent of the lymph nodes that were negative by haematoxylin and eosin staining. Under the modified TNM classification of canine mammary tumours, these results raised the clinical stage of 12.5 per cent of the affected dogs. However, if the latest TNM classification of human breast cancer had been applied, none of the animals would have been reclassified.

A femorotibial joint swelling with popliteal lymph node enlargement in a Rottweiler.

A 9-year-old male Rottweiler was presented to the Veterinary Medical Hospital Montenegro, Porto, Portugal with a large mass medial to the left stifle and radiographic signs of bone lysis involving the proximal tibia, fibula, and distal femur. A fine-needle aspiration was obtained from left popliteal lymph node, which was markedly enlarged. Cytologic examination revealed a highly cellular sample consisting of pleomorphic cells with marked anisocytosis and anisokaryosis and other criteria of malignancy. Some cells contained intracytoplasmic, granular, dark brown material, consistent with hemosiderin. Histologic evaluation of the surgically-excised lymph node revealed a neoplastic proliferation of histiocytic cells, with marked pleomorphism; occasional cells were erythrophagocytic. Mitotic figures were frequently observed, and many were atypical. Histologic findings were consistent with malignant histiocytosis. Necropsy examination confirmed the diagnosis of malignant histiocytosis with systemic involvement affecting most organs examined. This case of malignant histiocytosis case had an atypical clinical presentation, mimicking a musculoskeletal disorder. It underlines the importance of cytology as a simple, inexpensive, rapid and noninvasive complementary exam in routine clinical practice that can permit early diagnosis of the disease and timely selection of the most adequate therapy.

Molecular and epidemiologic study of Clostridium difficile reveals unusual heterogeneity in clinical strains circulating in different regions in Portugal.

Clostridium difficile infection (CDI) represents a great healthcare burden in developed countries. The emergence of the epidemic PCR ribotype (RT) 027 and its acquired fluoroquinolones resistance have accentuated the need for an active surveillance of CDI. Here we report the first countrywide study of CDI in Portugal with the characterization of 498 C. difficile clinical isolates from 20 hospitals in four regions in Portugal regarding RT, virulence factors and antimicrobial susceptibility. We identified 96 RTs with marked variations between and within regions, as only six RTs appeared in all four regions. RT027 was the most frequent RT overall (18.5%) and among healthcare facility-associated isolates (19.6%), while RT014 was the most common among community-associated isolates (12%). The north showed a high RT diversity among isolates and a low moxifloxacin (MXF) resistance rate (11.9%), being the only region in which RT027 was not predominant. In contrast, the isolates from the centre presented the highest RT027 frequency, and 53.4% were resistant to MXF. Overall, MXF resistance (33.2%) was associated (p <0.001) with the presence of binary toxin genes and mutations in tcdC regardless of the RT. Both traits appeared in almost 30% of the strains. RT027 showed a reduced susceptibility to metronidazole (p <0.01), and RT126 had higher minimum inhibitory concentrations to vancomycin (p = 0.03) compared to other RTs. The present study highlights an unusual heterogeneity of RTs in Portugal, with a high frequency of hypervirulent RTs and the emergence of virulence factors in non-027 RTs, emphasizing the need for a surveillance system for CDI in Portugal.