Multicentre validation study of nucleic acids extraction from FFPE tissues.

In most pathology laboratories worldwide, formalin-fixed paraffin embedded (FFPE) samples are the only tissue specimens available for routine diagnostics. Although commercial kits for diagnostic molecular pathology testing are becoming available, most of the current diagnostic tests are laboratory-based assays. Thus, there is a need for standardized procedures in molecular pathology, starting from the extraction of nucleic acids. To evaluate the current methods for extracting nucleic acids from FFPE tissues, 13 European laboratories, participating to the European FP6 program IMPACTS (www.impactsnetwork.eu), isolated nucleic acids from four diagnostic FFPE tissues using their routine methods, followed by quality assessment. The DNA-extraction protocols ranged from homemade protocols to commercial kits. Except for one homemade protocol, the majority gave comparable results in terms of the quality of the extracted DNA measured by the ability to amplify differently sized control gene fragments by PCR. For array-applications or tests that require an accurately determined DNA-input, we recommend using silica based adsorption columns for DNA recovery. For RNA extractions, the best results were obtained using chromatography column based commercial kits, which resulted in the highest quantity and best assayable RNA. Quality testing using RT-PCR gave successful amplification of 200 bp-250 bp PCR products from most tested tissues. Modifications of the proteinase-K digestion time led to better results, even when commercial kits were applied. The results of the study emphasize the need for quality control of the nucleic acid extracts with standardised methods to prevent false negative results and to allow data comparison among different diagnostic laboratories.

Performance evaluation of a new fourth-generation HIV combination antigen-antibody assay.

Education and diagnostic tests capable of early detection represent our most effective means of preventing transmission of human immunodeficiency virus (HIV). The importance of early detection is underlined by studies demonstrating increased life expectancy following early initiation of antiviral treatment. The Elecsys(®) HIV combi PT assay is a fourth-generation antigen-antibody combination assay developed to allow earlier detection of seroconversion, and to have increased sensitivity and improved specificity. We aimed to determine how early the assay could detect infection compared with existing assays; whether all HIV variants could be detected; and the assay's specificity using samples from blood donors, routine specimens, and patients with potential cross-reacting factors. Samples were identified as positive by the Elecsys(®) assay 4.9 days after a positive polymerase chain reaction result (as determined by the panel supplier), which was earlier than the 5.3-7.1 days observed with comparators. The analytical sensitivity of the Elecsys(®) HIV combi PT assay for the HIV-1 p24 antigen was 1.05 IU/mL, which compares favorably with the comparator assays. In addition, the Elecsys(®) assay identified all screened HIV subtypes and displayed greater sensitivity to HIV-2 homologous antigen and antibodies to HIV-1 E and O and HIV-2 than the other assays. Overall, the specificity of the Elecsys(®) assay was 99.88 % using samples from blood donors and 99.81 % when analyzing unselected samples. Potential cross-reacting factors did not interfere with assay performance. The Elecsys(®) HIV combi PT assay is a sensitive and specific assay that has been granted the CE mark according to Directive 2009/886/EC.

Brief DISCERN, six questions for the evaluation of evidence-based content of health-related websites.

OBJECTIVE: To extract and to validate a brief version of the DISCERN which could identify mental health-related websites with good content quality. METHOD: The present study is based on the analysis of data issued from six previous studies which used DISCERN and a standardized tool for the evaluation of content quality (evidence-based health information) of 388 mental health-related websites. After extracting the Brief DISCERN, several psychometric properties (content validity through a Factor analysis, internal consistency by the Cronbach's alpha index, predictive validity through the diagnostic tests, concurrent validity by the strength of association between the Brief DISCERN and the original DISCERN scores) were investigated to ascertain its general applicability. RESULTS: A Brief DISCERN composed of two factors and six items was extracted from the original 16 items version of the DISCERN. Cronbach's alpha coefficients were more than acceptable for the complete questionnaire (alpha=0.74) and for the two distinct domains: treatments information (alpha=0.87) and reliability (alpha=0.83). Sensibility and specificity of the Brief DISCERN cut-off score > or =16 in the detection of good content quality websites were 0.357 and 0.945, respectively. Its predictive positive and negative values were 0.98 and 0.83, respectively. A statistically significant linear correlation was found between the total scores of the Brief DISCERN and those of the original DISCERN (r=0.84 and p<0.0005). CONCLUSION: The Brief DISCERN seems to be a reliable and valid instrument able to discriminate between websites with good and poor content quality. PRACTICE IMPLICATIONS: The Brief DISCERN is a simple tool which could facilitate the identification of good information on the web by patients and general consumers.

Combinations of prognostic tools for identification of high-risk normotensive patients with acute symptomatic pulmonary embolism.

Background: In haemodynamically stable patients with acute symptomatic pulmonary embolism (PE), studies have not evaluated the usefulness of combining the measurement of cardiac troponin, transthoracic echocardiogram (TTE), and lower extremity complete compression ultrasound (CCUS) testing for predicting the risk of PE-related death. Methods: The study assessed the ability of three diagnostic tests (cardiac troponin I (cTnI), echocardiogram, and CCUS) to prognosticate the primary outcome of PE-related mortality during 30 days of follow-up after a diagnosis of PE by objective testing. Results: Of 591 normotensive patients diagnosed with PE, the primary outcome occurred in 37 patients (6.3%; 95% CI 4.3% to 8.2%). Patients with right ventricular dysfunction (RVD) by TTE and concomitant deep vein thrombosis (DVT) by CCUS had a PE-related mortality of 19.6%, compared with 17.1% of patients with elevated cTnI and concomitant DVT and 15.2% of patients with elevated cTnI and RVD. The use of any two-test strategy had a higher specificity and positive predictive value compared with the use of any test by itself. A combined three-test strategy did not further improve prognostication. For a subgroup analysis of high-risk patients, according to the pulmonary embolism severity index (classes IV and V), positive predictive values of the two-test strategies for PE-related mortality were 25.0%, 24.4% and 20.7%, respectively. Conclusions: In haemodynamically stable patients with acute symptomatic PE, a combination of echocardiography (or troponin testing) and CCUS improved prognostication compared with the use of any test by itself for the identification of those at high risk of PE-related death.

Antibiotic-induced modifications of the stiffness of bacterial membranes.

In the latest years the importance of high resolution analysis of the microbial cell surface has been increasingly recognized. Indeed, in order to better understand bacterial physiology and achieve rapid diagnostic and treatment techniques, a thorough investigation of the surface modifications induced on bacteria by different environmental conditions or drugs is essential. Several instruments are nowadays available to observe at high resolution specific properties of microscopic samples. Among these, AFM can routinely study single cells in physiological conditions, measuring the mechanical properties of their membrane at a nanometric scale (force volume). Such analyses, coupled with high resolution investigation of their morphological properties, are increasingly used to characterize the state of single cells. In this work we exploit such technique to characterize bacterial systems. We have performed an analysis of the mechanical properties of bacteria (Escherichia coli) exposed to different conditions. Such measurements were performed on living bacteria, by changing in real-time the liquid environment: standard phosphate buffered saline, antibiotic (ampicillin) in PBS and growth medium. In particular we have focused on the determination of the membrane stiffness modifications induced by these solutions, in particular between stationary and replicating phases and what is the effect of the antibiotic on the bacterial structure.

Hyponatrémie: une histoire en deux temps... [Hyponatremia: an unusual case report]

Hyponatremia is a frequent finding and asks for a rapid diagnostic evaluation. We report a case of recurrent hyponatremia secondary to an adrenal insufficiency of medicamentous and tuberculous origin. This case illustrates the importance of a rapid etiologic diagnosis of hyponatremia and allows us to review adrenal insufficiency of tuberculous origin. It also stresses the danger of potential drug interactions in case of corticosteroid substitution.

Urinalysis and pre-renal acute kidney injury: time to move on.

Urinary indices are classically believed to allow differentiation of transient (or pre-renal) acute kidney injury (AKI) from persistent (or acute tubular necrosis) AKI. However, the data validating urinalysis in critically ill patients are weak. In the previous issue of Critical Care, Pons and colleagues demonstrate in a multicenter observational study that sodium and urea excretion fractions as well as urinary over plasma ratios performed poorly as diagnostic tests to separate such entities. This study confirms the limited diagnostic and prognostic ability of urine testing. Together with other studies, this study raises more fundamental questions about the value, meaning and pathophysiologic validity of the pre-renal AKI paradigm and suggests that AKI (like all other forms of organ injury) is a continuum of injury that cannot be neatly divided into functional (pre-renal or transient) or structural (acute tubular necrosis or persistent).

Etiology of community-acquired pneumonia in hospitalized children based on WHO clinical guidelines.

Community-acquired pneumonia (CAP) is a major cause of death in developing countries and of morbidity in developed countries. The objective of the study was to define the causative agents among children hospitalized for CAP defined by WHO guidelines and to correlate etiology with clinical severity and surrogate markers. Investigations included an extensive etiological workup. A potential causative agent was detected in 86% of the 99 enrolled patients, with evidence of bacterial (53%), viral (67%), and mixed (33%) infections. Streptococcus pneumoniae was accounted for in 46% of CAP. Dehydration was the only clinical sign associated with bacterial pneumonia. CRP and PCT were significantly higher in bacterial infections. Increasing the number of diagnostic tests identifies potential causes of CAP in up to 86% of children, indicating a high prevalence of viruses and frequent co-infections. The high proportion of pneumococcal infections re-emphasizes the importance of pneumococcal immunization.

Persistent palinopsia following ingestion of lysergic acid diethylamide (LSD).

OBJECTIVE: To identify a distinctive chronic visual complication of lysergic acid diethylamide (LSD) use. DESIGN: Description of the clinical findings in three patients with this disorder. SETTING: A neuro-ophthalmology referral center. RESULTS: All three patients experienced prolonged afterimages (palinopsia) during LSD intoxication and have continued to be symptomatic up to 3 years after they ceased to ingest the drug. Results of neuro-ophthalmologic and neurologic examinations and neuroimaging and electrophysiologic studies were normal. CONCLUSIONS: We have described three patients in whom persistent palinopsia developed following ingestion of LSD. Clinicians should inquire about past LSD use in all patients who initially have seemingly spontaneous, isolated palinopsia. Recognition of this distinctive clinical syndrome associated with LSD use might avoid unnecessary anxiety and excessive diagnostic tests for patients with this disorder.

Bilan préopératoire en chirurgie viscérale [Preoperative assessment in visceral surgery]

The purpose of preoperative assessment is to evaluate the patient's health status, to address known or unidentified co-morbidities and to perform adequate complementary exams if necessary. On the other hand, it allows to prepare and protect the patient in order to reduce perioperative risk. The assessment consists of patient's history and physical examination, both focusing on cardiovascular and respiratory assessment. Complementary exams have to be chosen selectively depending on the patient's risk factors and the type of surgery. They are indicated if their result leads to a potential patient's benefit only, either by a modification in anesthetic and/or surgical management or by introduction of a pharmacological strategy, adequate and maximal if necessary, especially for cardioprotection.

Recommendations for malaria prevention in moderate to low risk areas: travellers' choice and risk perception.

BACKGROUND: The considerable malaria decline in several countries challenges the strategy of chemoprophylaxis for travellers visiting moderate- to low-risk areas. An international consensus on the best strategy is lacking. It is essential to include travellers' opinions in the decision process. The preference of travellers regarding malaria prevention for moderate- to low-risk areas, related to their risk perception, as well as the reasons for their choices were investigated. METHODS: Prior to pre-travel consultation in the Travel Clinic, a self-administered questionnaire was given to travellers visiting moderate- to low-risk malaria areas. Four preventive options were proposed to the traveller, i.e., bite prevention only, chemoprophylaxis, stand-by emergency treatment alone, and stand-by emergency treatment with rapid diagnostic test. The information was accompanied by a risk scale for incidence of malaria, anti-malarial adverse drug reactions and other travel-related risks, inspired by Paling palettes from the Risk Communication Institute. RESULTS: A total of 391 travellers were included from December 2012 to December 2013. Fifty-nine (15%) opted for chemoprophylaxis, 116 (30%) for stand-by emergency treatment, 112 (29%) for stand-by emergency treatment with rapid diagnostic test, 100 (26%) for bite prevention only, and four (1%) for other choices. Travellers choosing chemoprophylaxis justified their choice for security reasons (42%), better preventive action (29%), higher efficacy (15%) and easiness (15%). The reasons for choosing stand-by treatment or bite prevention only were less medication consumed (29%), less adverse drug reactions (23%) and lower price (9%). Those who chose chemoprophylaxis were more likely to have used it in the past (OR = 3.0 (CI 1.7-5.44)), but were not different in terms of demographic, travel characteristics or risk behaviour. CONCLUSIONS: When travelling to moderate- to low-risk malaria areas, 85% of interviewees chose not to take chemoprophylaxis as malaria prevention, although most guidelines recommend it. They had coherent reasons for their choice. New recommendations should include shared decision-making to take into account travellers' preferences.

Added value of acute multimodal CT-based imaging (MCTI) : a comprehensive analysis

Introduction: MCTI is used to assess acute ischemic stroke (AIS) patients.We postulated that use of MCTI improves patient outcome regardingindependence and mortality.Methods: From the ASTRAL registry, all patients with an AIS and a non-contrast-CT (NCCT), angio-CT (CTA) or perfusion-CT (CTP) within24 h from onset were included. Demographic, clinical, biological, radio-logical, and follow-up caracteristics were collected. Significant predictorsof MCTI use were fitted in a multivariate analysis. Patients undergoingCTA or CTA&CTP were compared with NCCT patients with regards tofavourable outcome (mRS ≤ 2) at 3 months, 12 months mortality, strokemechanism, short-term renal function, use of ancillary diagnostic tests,duration of hospitalization and 12 months stroke recurrence.

Screening and overdiagnosis : public health implications

Overdiagnosis is the diagnosis of an abnormality that bears no substantial health hazard and no benefit for patients to be aware of. Resulting mainly from the use of increasingly sensitive screening and diagnostic tests, as well as broadened definitions of conditions requiring an intervention, overdiagnosis is a growing but still largely misunderstood public health issue. Fear of missing a diagnosis or of litigation, financial incentives or patient's need of reassurance are further causes of overdiagnosis. The main consequence of overdiagnosis is overtreatment. Treating an overdiagnosed condition bears no benefit but can cause harms and generates costs. Overtreatment also diverts health professionals from caring for those most severely ill. Recognition of overdiagnosis due to screening is challenging since it is rarely identifiable at the individual level and difficult to quantify precisely at the population level. Overdiagnosis exists even for screening of proven efficacy and efficiency. Measures to reduce overdiagnosis due to screening include heightened sensitization of health professionals and patients, active surveillance and deferred treatment until early signs of disease progression and prognosis estimation through biomarkers (including molecular) profiling. Targeted screening and balanced information on its risk and benefits would also help limit overdiagnosis. Research is needed to assess the the public health burden and implications of overdiagnosis due to screening activity.

Role of immune and angiogenic markers in the pathology of endometriosis

Background: Endometriosis is an estrogen-dependent, pro-inflammatory, pro-angiogenic condition that affects 5 to 10% of women of reproductive age. Its defining feature is the presence of endometrium-like tissue in sites outside the uterine cavity, primarily on the pelvic peritoneum and ovaries. The main clinical features are chronic pain, pain during intercourse and infertility. In patients with endometriosis, inflammatory and immune responses, angiogenesis and apoptosis are altered in favour of the survival and replenishment of endometriotic tissue. These basic pathological processes depend on the excessive formation of estrogen and prostaglandins. Recently, new cellular and molecular mechanisms for the resolution of inflammation have been discovered, revealing key roles for lipid mediators such as lipoxins, resolvins and protectins. It is possible that disequilibrium in the expression of these molecules exists in endometriosis. Objective: To compare the expression of two proteins involved in the synthe sis and in the function of lipid mediators; the Arachidonate 15-lipoxygenase (ALOXI5), implicated in the synthesis of lipoxins A4 and B4 and the Formyl peptide receptor 1 (FPRLI), the specific receptor for Lipoxin A4 and B4, between women who suffer from endometriosis and a control group. We wish to demonstrate the cellular localisation of these two molecules and to investigate if their expression is alteted in this pathology. Methods and Materials Using immunohistochemistry we will compare ALOXI5 and FPRLI staining, in endometrium, normal peritoneum and endometriotic lesions. The samples are being collected in the department of Gynaecology and Obstetrics at the Centre Hospitalier Universitaire Lausanne (CHUV). Women attending the department for laparoscopic investigation of pain/infertility, suspected endometriosis or for a hysterectomy, are invited to participate. Approval of the ethics committee (Commission d'Ethique de la recherché clinique) was obtained in March 2009. Clinical samples will only be obtained from subjects having consented. Expected results and interpretation: No published studies investigating the expression of these two molecules in endometriotic lesions exist. A better understanding of the mechanisms underlying this disease will result in the development of new medical therapies and new diagnostic tests, with the aim of ameliorating the quality of life of endometriosis patients.