Biofilm formation by staphylococci on fresh, fresh-frozen and processed human and bovine bone grafts.

Biofilm formation is a multi-step process influenced by surface properties. We investigated early and mature biofilm of Staphylococcus aureus on 4 different biological calcium phosphate (CaP) bone grafts used for filling bone defects. We investigated standardised cylinders of fresh and fresh-frozen human bone grafts were harvested from femoral heads; processed humanand bovine bone grafts were obtained preformed. Biofilm formation was done in tryptic soy broth (TSB) using S. aureus (ATCC 29213) with static conditions. Biofilm density after 3 h (early biofilm) and 24 h (mature biofilm) was investigated by sonication and microcalorimetry. After 3 h, bacterial density was highest on fresh-frozenandfresh bone grafts. After 24 h, biofilm density was lowest on freshbone grafts (p < 0.001) compared to the other 3 materials, which did not differ quantitatively (p > 0.05). The lowest increase in bacterial density was detected on fresh bone grafts (p < 0.001). Despite normal shaped colonies, we found additional small colonies on the surface of the fresh and fresh-frozen samples by sonication. This was also apparent in microcalorimetric heat-flow curves. The four investigated CaP bone grafts showed minor structural differences in architecture but marked differences concerning serum coverage and the content of bone marrow, fibrous tissue and bone cells. These variations resulted in a decreased biofilm density on freshand fresh-frozenbone grafts after 24 h, despite an increased early biofilm formation and might also be responsible for the variations in colony morphology (small colonies). Detection of small colony variants by microcalorimetry might be a new approach to improve the understanding of biofilm formation.

Embolization for multicompartmental bleeding in patients in hemodynamically unstable condition: prognostic factors and outcome.

PURPOSE: To determine prognostic factors and evaluate outcomes of transcatheter arterial embolization in severely injured patients in hemodynamically unstable condition with multicompartmental bleeding.¦MATERIALS AND METHODS: Between June 2000 and May 2008, 36 consecutive patients treated with transcatheter arterial embolization for major retroperitoneal bleeding associated with at least one additional source of bleeding were retrospectively reviewed. Mean Injury Severity Score (ISS) was 49.4 ± 15.8. Univariate and multivariate analyses were performed to identify parameters associated with failure of embolization, need for additional surgery to control bleeding, and fatal outcome at 30 d.¦RESULTS: Embolization was technically successful in 35 of 36 patients (97.2%) and resulted in immediate and sustained (> 24 h) hemodynamic improvement in 29 (80.5%). Additional hemostatic surgery was necessary after embolization in six patients (16.6%). Fifteen patients (41.6%) died within 30 d. Failure to restore hemodynamic stability was correlated with the rate of administration of packed red blood cells (P = .014), rate of administration of fresh frozen plasma (FFP; P = .031), and systolic blood pressure (SBP) immediately before embolization (P = .002). The need for additional surgery was correlated with FFP administration rate before embolization (P = .0002) and hemodynamic success (P = .003). Death was correlated with Glasgow Coma Scale score at admission (P = .001), ISS (P = .014), New Injury Severity Score (P = .016), number of injured sites (P = .012), SBP before embolization (P = .042), need for vasopressive drugs before embolization (P = .037), and hemodynamic success (P = .0004).¦CONCLUSIONS: In patients in hemodynamically unstable condition, transcatheter arterial embolization effectively controls bleeding and improves hemodynamic stability. Immediate survival is related to hemodynamic condition before embolization, and 30-d mortality is mainly related to associated brain trauma.

Molecular risk assessment of BIG 1-98 participants by expression profiling using RNA from archival tissue.

Background: The purpose of the work reported here is to test reliable molecular profiles using routinely processed formalin-fixed paraffin-embedded (FFPE) tissues from participants of the clinical trial BIG 1-98 with a median follow-up of 60 months. Methods: RNA from fresh frozen (FF) and FFPE tumor samples of 82 patients were used for quality control, and independent FFPE tissues of 342 postmenopausal participants of BIG 1-98 with ER-positive cancer were analyzed by measuring prospectively selected genes and computing scores representing the functions of the estrogen receptor (eight genes, ER_8), the progesterone receptor (five genes, PGR_5), Her2 (two genes, HER2_2), and proliferation (ten genes, PRO_10) by quantitative reverse transcription PCR (qRT-PCR) on TaqMan Low Density Arrays. Molecular scores were computed for each category and ER_8, PGR_5, HER2_2, and PRO_10 scores were combined into a RISK_25 score. Results: Pearson correlation coefficients between FF- and FFPE-derived scores were at least 0.94 and high concordance was observed between molecular scores and immunohistochemical data. The HER2_2, PGR_ 5, PRO_10 and RISK_25 scores were significant predictors of disease free-survival (DFS) in univariate Cox proportional hazard regression. PRO_10 and RISK_25 scores predicted DFS in patients with histological grade II breast cancer and in lymph node positive disease. The PRO_10 and PGR_ 5 scores were independent predictors of DFS in multivariate Cox regression models incorporating clinical risk indicators; PRO_10 outperformed Ki-67 labeling index in multivariate Cox proportional hazard analyses. Conclusions: Scores representing the endocrine responsiveness and proliferation status of breast cancers were developed from gene expression analyses based on RNA derived from FFPE tissues. The validation of the molecular scores with tumor samples of participants of the BIG 1-98 trial demonstrates that such scores can serve as independent prognostic factors to estimate disease free survival (DFS) in postmenopausal patients with estrogen receptor positive breast cancer.

Three years of haemovigilance in a general university hospital.

The aim of this study is to describe a newly implemented haemovigilance system in a general university hospital. We present a series of short cases, highlighting particular aspects of the reports, and an overview of all reported incidents between 1999 and 2001. Incidents related to transfusion of blood products were reported by the clinicians using a standard preformatted form, giving a synopsis of the incident. After analysis, we distinguished, on the one hand, transfusion reactions, that are transfusions which engendered signs or symptoms, and, on the other hand, the incidents where management errors and/or dysfunctions took place. Over 3 years, 233 incidents were reported, corresponding to 4.2 events for 1000 blood products delivered. Of the 233, 198 (85%) were acute transfusion reactions and 35 (15%) were management errors and/or dysfunctions. Platelet units gave rise to statistically (P < 0.001) more transfusion reactions (10.7 per thousand ) than red blood cells (3.5 per thousand ) and fresh frozen plasma (0.8 per thousand ), particularly febrile nonhaemolytic transfusion reactions and allergic reactions. A detailed analysis of some of the transfusion incident reports revealed complex deviations and/or failures of the procedures in place in the hospital, allowing the implementation of corrective and preventive measures. Thus, the haemovigilance system in place in the 'Centre Hospitalier Universitaire Vaudois, CHUV' appears to constitute an excellent instrument for monitoring the security of blood transfusion.

Effects of KRAS, BRAF, NRAS, and PIK3CA mutations on the efficacy of cetuximab plus chemotherapy in chemotherapy-refractory metastatic colorectal cancer: a retrospective consortium analysis.

Background Following the discovery that mutant KRAS is associated with resistance to anti-epidermal growth factor receptor (EGFR) antibodies, the tumours of patients with metastatic colorectal cancer are now profiled for seven KRAS mutations before receiving cetuximab or panitumumab. However, most patients with KRAS wild-type tumours still do not respond. We studied the effect of other downstream mutations on the efficacy of cetuximab in, to our knowledge, the largest cohort to date of patients with chemotherapy-refractory metastatic colorectal cancer treated with cetuximab plus chemotherapy in the pre-KRAS selection era. Methods 1022 tumour DNA samples (73 from fresh-frozen and 949 from formalin-fixed, paraffin-embedded tissue) from patients treated with cetuximab between 2001 and 2008 were gathered from 11 centres in seven European countries. 773 primary tumour samples had sufficient quality DNA and were included in mutation frequency analyses; mass spectrometry genotyping of tumour samples for KRAS, BRAF, NRAS, and PIK3CA was done centrally. We analysed objective response, progression-free survival (PFS), and overall survival in molecularly defined subgroups of the 649 chemotherapy-refractory patients treated with cetuximab plus chemotherapy. Findings 40.0% (299/747) of the tumours harboured a KRAS mutation, 14.5% (108/743) harboured a PIK3CA mutation (of which 68.5% [74/108] were located in exon 9 and 20.4% [22/108] in exon 20), 4.7% (36/761) harboured a BRAF mutation, and 2.6% (17/644) harboured an NRAS mutation. KRAS mutants did not derive benefit compared with wild types, with a response rate of 6.7% (17/253) versus 35.8% (126/352; odds ratio [OR] 0.13, 95% CI 0.07-0.22; p<0.0001), a median PFS of 12. weeks versus 24 weeks (hazard ratio [HR] 1 98, 1.66-2.36; p<0.0001), and a median overall survival of 32 weeks versus 50 weeks (1.75, 1.47-2.09; p<0.0001). In KRAS wild types, carriers of BRAF and NRAS mutations had a significantly lower response rate than did BRAF and NRAS wild types, with a response rate of 8.3% (2/24) in carriers of BRAF mutations versus 38.0% in BRAF wild types (124/326; OR 0.15, 95% CI 0.02-0.51; p=0.0012); and 7.7% (1/13) in carriers of NRAS mutations versus 38.1% in NRAS wild types (110/289; OR 0.14, 0.007-0.70; p=0.013). PIK3CA exon 9 mutations had no effect, whereas exon 20 mutations were associated with a worse outcome compared with wild types, with a response rate of 0.0% (0/9) versus 36.8% (121/329; OR 0.00,0.00-0.89; p=0.029), a median PFS of 11.5 weeks versus 24 weeks (HR 2.52, 1.33-4.78; p=0.013), and a median overall survival of 34 weeks versus 51 weeks (3.29, 1.60-6.74; p=0.0057). Multivariate analysis and conditional inference trees confirmed that, if KRAS is not mutated, assessing BRAF, NRAS, and PIK3CA exon 20 mutations (in that order) gives additional information about outcome. Objective response rates in our series were 24.4% in the unselected population, 36.3% in the KRAS wild-type selected population, and 41.2% in the KRAS, BRAF, NRAS, and PIK3CA exon 20 wild-type population. Interpretation While confirming the negative effect of KRAS mutations on outcome after cetuximab, we show that BRAF, NRAS, and PIK3CA,exon 20 mutations are significantly associated with a low response rate. Objective response rates could be improved by additional genotyping of BRAF, NRAS, and PIK3CA exon 20 mutations in a KRAS wild-type population.

Ex-vivo ultrasound control of resection margins during partial nephrectomy

Introduction: Surgery represents the treatment of choice for localized renal cell neoplasia. Partial nephrectomy (PN) has widened its indications over the past two decades and has shown oncological results equivalent to radical nephrectomy for small tumors. The role of negative surgical margins has been widely debated. Intraoperative fresh frozen section analysis is shown to be unreliable, expensive, time-consuming and not well correlated to final pathology. The goal of the present study was to assess the feasibility of intraoperative ex-vivo ultrasound (US) control of resection margins and its correlation to margin status at definitive pathology in patients undergoing PN.Material and Methods: The study was carried out in our institution from February 2008 to March 2010. Patients undergoing PN for T1-T2 renal tumors were included. Ex vivo US was performed by one single senior radiologist. Considering its availability, not all consecutive eligible patients were included. PN was undertaken in a standardized technique applying the "minimal healthy tissue margin" technique. Once resected, the specimen was kept in a saline solution and ex-vivo US was performed to evaluate the whole tumor pseudocapsule.Results: Twelve patients (five women, age (mean}SD) 65}11 years) were included. Intraoperative ex-vivo US showed negative surgical margin in all cases. US duration ranged from 1 to 4 minutes, with a median time of 1 minute. Definitive histological analysis confirmed the presence of two angiomyolipoma, eight pT1a tumors, of which seven were clear cell carcinoma and one was a type II papillary tumor, one pT1b clear cell carcinoma and one pT2 chromophobe carcinoma (size 2.9}2.3 cm). Final pathology revealed R0 margins.Conclusion: Intraoperative ex-vivo US control of resection margins in patients undergoing PN is feasible, time-efficient and well correlated to definitive pathological examination with regards to margin status.

Rôle de la MGMT et implications cliniques dans les tumeurs cérébrales [Role of MGMT and clinical applications in brain tumours]

Glioblastoma multiforme is the most common and most malignant primary brain tumour with a dismal prognosis. The advent of new chemotherapies with alkylating agents crossing the blood-brain barrier, like temozolomide, have permitted to notably ameliorate the survival of a subgroup of patients. Improved outcome was associated with epigenetic silencing of the MGMT (O6-methylguanin methyltransferase) gene by promotor methylation, thereby blocking its repair capability, thus rendering the alkylating agents more effective. This particularity can be tested by methylation specific PCR on resected tumour tissue, best on fresh frozen biopsies, and allows identification of patients more susceptible to respond favourably to the treatment.

Visceral leishmaniasis and hemophagocytic syndrome

A 11 months old female infant from Portugal, free of family history, consults for apathy, weight loss, tachycardia, tachypnea, petechiae, pallor without icterus and hepatoslenomegaly. Seven months earlier, while being in Portugal, she presented a persistent bluish pimple on her buttock. Laboratory results showed anemia (35 g/l), leucopenia (3.3 G/l), thrombocytopenia (13 G/l), impaired coagulation (INR 1.4, PTT 41 sec.), hyponatremia (124 mmol/l), elevated CRP (139 mg/l), high ferritin (34.775 μg/l) and high triglycerides (5.22 mmol/l). After correction of vital parameters, a bone marrow aspiration and biopsy (BMB) revealed both the etiological diagnosis, namely a visceral leishmaniasis (VL) as well as one of its potential complications, the hemophagocytic syndrome (HS). Transfusions of whole blood, platelets and fresh frozen plasma were immediately started. Dexamethasone (10 mg/m2) and amphotericin B (3 mg/kg/day) have also been administrated. Visceral leishmaniasis is caused by a protozoan (Leishmania donovani) transmitted by the female sandfly. It is endemic in the Mediterranean basin (including France, Italy, Spain and Portugal), South America, sub-Saharan Africa as well as in India and Bangladesh. The parasite infects macrophages and, after several weeks of incubation, the disease occurs by affection of bloodlines (anemia, leucopenia, thrombocytopenia), hepatosplenomegaly, cachexia, gastrointestinal damage. The complications of the disease may lead to death. Liposomal amphotericin B is the currently recommended treatment. HS is caused by the proliferation and activation of macrophages in the marrow in response to a cytokine storm. It may be of primary cause. When it is secondary, it may be related to infections such as leishmaniasis. Patients present with fever and laboratory diagnostic criteria include cytopenia, hypertriglyceridemia, high ferritin and hemophagocytosis in the BMB. The treatment consists among other in the administration of high doses corticosteroids and, in secondary cases, in the treatment of the underlying cause. In conclusion, the clinical and biological features of VL may mimic haematological disorders as leukemia, but an enlargement of the liver and especially of the spleen should remind in this parasitic infection and its potential fatal complication, the HS.

Biomarker analysis of stored blood products: emphasis on pre-analytical issues.

Millions of blood products are transfused every year; many lives are thus directly concerned by transfusion. The three main labile blood products used in transfusion are erythrocyte concentrates, platelet concentrates and fresh frozen plasma. Each of these products has to be stored according to its particular components. However, during storage, modifications or degradation of those components may occur, and are known as storage lesions. Thus, biomarker discovery of in vivo blood aging as well as in vitro labile blood products storage lesions is of high interest for the transfusion medicine community. Pre-analytical issues are of major importance in analyzing the various blood products during storage conditions as well as according to various protocols that are currently used in blood banks for their preparations. This paper will review key elements that have to be taken into account in the context of proteomic-based biomarker discovery applied to blood banking.

A comprehensive analysis of human gene expression profiles identifies stromal immunoglobulin κ C as a compatible prognostic marker in human solid tumors.

PURPOSE: Although the central role of the immune system for tumor prognosis is generally accepted, a single robust marker is not yet available. EXPERIMENTAL DESIGN: On the basis of receiver operating characteristic analyses, robust markers were identified from a 60-gene B cell-derived metagene and analyzed in gene expression profiles of 1,810 breast cancer; 1,056 non-small cell lung carcinoma (NSCLC); 513 colorectal; and 426 ovarian cancer patients. Protein and RNA levels were examined in paraffin-embedded tissue of 330 breast cancer patients. The cell types were identified with immunohistochemical costaining and confocal fluorescence microscopy. RESULTS: We identified immunoglobulin κ C (IGKC) which as a single marker is similarly predictive and prognostic as the entire B-cell metagene. IGKC was consistently associated with metastasis-free survival across different molecular subtypes in node-negative breast cancer (n = 965) and predicted response to anthracycline-based neoadjuvant chemotherapy (n = 845; P < 0.001). In addition, IGKC gene expression was prognostic in NSCLC and colorectal cancer. No association was observed in ovarian cancer. IGKC protein expression was significantly associated with survival in paraffin-embedded tissues of 330 breast cancer patients. Tumor-infiltrating plasma cells were identified as the source of IGKC expression. CONCLUSION: Our findings provide IGKC as a novel diagnostic marker for risk stratification in human cancer and support concepts to exploit the humoral immune response for anticancer therapy. It could be validated in several independent cohorts and carried out similarly well in RNA from fresh frozen as well as from paraffin tissue and on protein level by immunostaining.

Médecine d'urgence [Emergency medicine: updates 2013].

New evidences published this year are susceptible to change the management of several medical emergencies. Combined antiplatelet therapy might be beneficial for the management of TIA or minor stroke and rapid blood pressure lowering might improve the outcome in patients with intracerebral hemorrhage. A restrictive red cell transfusion strategy is indicated in case of upper digestive bleeding and coagulation factors concentrates are superior to fresh frozen plasma for urgent warfarin reversal. Prolonged systemic steroid therapy is not warranted in case of acute exacerbation of BPCO, and iterative physiotherapy is not beneficial after acute whiplash. Finally, family presence during cardiopulmonary resuscitation may reduce post-traumatic stress disorder among relatives.

Angiotensin converting enzyme inhibitor induced angio-oedema: a review of the pathophysiology and risk factors.

Angio-oedema (AE) is a known adverse effect of angiotensin converting enzyme inhibitor (ACE-I) therapy. Over the past several decades, evidence of failure to diagnose this important and potentially fatal reaction is commonly found in the literature. Because this reaction is often seen first in the primary care setting, a review was undertaken to analyse and document the keys to both diagnostic criteria as well as to investigate potential risk factors for ACE-I AE occurrence. A general review of published literature was conducted through Medline, EMBASE, and the Cochrane Database, targeting ACE-I-related AE pathomechanism, diagnosis, epidemiology, risk factors, and clinical decision making and treatment. The incidence and severity of AE appears to be on the rise and there is evidence of considerable delay in diagnosis contributing to significant morbidity and mortality for patients. The mechanism of AE due to ACE-I drugs is not fully understood, but some genomic and metabolomic information has been correlated. Additional epidemiologic data and clinical treatment outcome predictors have been evaluated, creating a basis for future work on the development of clinical prediction tools to aid in risk identification and diagnostic differentiation. Accurate recognition of AE by the primary care provider is essential to limit the rising morbidity associated with ACE-I treatment-related AE. Research findings on the phenotypic indicators relevant to this group of patients as well as basic research into the pathomechanism of AE are available, and should be used in the construction of better risk analysis and clinical diagnostic tools for ACE-I AE.

Médecine d'urgence [Emergency medicine: updates 2013].

New evidences published this year are susceptible to change the management of several medical emergencies. Combined antiplatelet therapy might be beneficial for the management of TIA or minor stroke and rapid blood pressure lowering might improve the outcome in patients with intracerebral hemorrhage. A restrictive red cell transfusion strategy is indicated in case of upper digestive bleeding and coagulation factors concentrates are superior to fresh frozen plasma for urgent warfarin reversal. Prolonged systemic steroid therapy is not warranted in case of acute exacerbation of BPCO, and iterative physiotherapy is not beneficial after acute whiplash. Finally, family presence during cardiopulmonary resuscitation may reduce post-traumatic stress disorder among relatives.

Mycobacterium tuberculosis aortic graft infection with recurrent hemoptysis: a case report.

INTRODUCTION: Mycobacterium tuberculosis may cause a large variety of clinical presentations due to its ability to disseminate by contiguity or hematogenously. Tuberculosis may remain undiagnosed for years due to the chronic course of the disease, with potentially life-threatening long-term complications. CASE PRESENTATION: In this case report, we describe a tuberculous aortic graft infection in a 72-year-old man documented by polymerase chain reaction and cultures. The patient presented with three episodes of hemoptysis following a remote history of miliary tuberculosis. The infection was treated by graft replacement and prolonged antimycobacterial therapy. CONCLUSION: Tuberculous infection of a vascular graft is an uncommon complication, but should be considered in patients with an intravascular device and a history of previous tuberculosis, especially when hematogenous spread may have occurred a few months after surgery, or when an active mycobacterial infection is present in close proximity to the graft.