Points to consider for prioritizing clinical genetic testing services: a European consensus process oriented at accountability for reasonableness.

Given the cost constraints of the European health-care systems, criteria are needed to decide which genetic services to fund from the public budgets, if not all can be covered. To ensure that high-priority services are available equitably within and across the European countries, a shared set of prioritization criteria would be desirable. A decision process following the accountability for reasonableness framework was undertaken, including a multidisciplinary EuroGentest/PPPC-ESHG workshop to develop shared prioritization criteria. Resources are currently too limited to fund all the beneficial genetic testing services available in the next decade. Ethically and economically reflected prioritization criteria are needed. Prioritization should be based on considerations of medical benefit, health need and costs. Medical benefit includes evidence of benefit in terms of clinical benefit, benefit of information for important life decisions, benefit for other people apart from the person tested and the patient-specific likelihood of being affected by the condition tested for. It may be subject to a finite time window. Health need includes the severity of the condition tested for and its progression at the time of testing. Further discussion and better evidence is needed before clearly defined recommendations can be made or a prioritization algorithm proposed. To our knowledge, this is the first time a clinical society has initiated a decision process about health-care prioritization on a European level, following the principles of accountability for reasonableness. We provide points to consider to stimulate this debate across the EU and to serve as a reference for improving patient management.

Estimation of reference curves for the urinary steroid metabolome in the first year of life in healthy children : Tracing the complexity of human postnatal steroidogenesis.

CONTEXT: Complex steroid disorders such as P450 oxidoreductase deficiency or apparent cortisone reductase deficiency may be recognized by steroid profiling using chromatographic mass spectrometric methods. These methods are highly specific and sensitive, and provide a complete spectrum of steroid metabolites in a single measurement of one sample which makes them superior to immunoassays. The steroid metabolome during the fetal-neonatal transition is characterized by (a) the metabolites of the fetal-placental unit at birth, (b) the fetal adrenal androgens until its involution 3-6 months postnatally, and (c) the steroid metabolites produced by the developing endocrine organs. All these developmental events change the steroid metabolome in an age- and sex-dependent manner during the first year of life. OBJECTIVE: The aim of this study was to provide normative values for the urinary steroid metabolome of healthy newborns at short time intervals in the first year of life. METHODS: We conducted a prospective, longitudinal study to measure 67 urinary steroid metabolites in 21 male and 22 female term healthy newborn infants at 13 time-points from week 1 to week 49 of life. Urine samples were collected from newborn infants before discharge from hospital and from healthy infants at home. Steroid metabolites were measured by gas chromatography-mass spectrometry (GC-MS) and steroid concentrations corrected for urinary creatinine excretion were calculated. RESULTS: 61 steroids showed age and 15 steroids sex specificity. Highest urinary steroid concentrations were found in both sexes for progesterone derivatives, in particular 20α-DH-5α-DH-progesterone, and for highly polar 6α-hydroxylated glucocorticoids. The steroids peaked at week 3 and decreased by ∼80% at week 25 in both sexes. The decline of progestins, androgens and estrogens was more pronounced than of glucocorticoids whereas the excretion of corticosterone and its metabolites and of mineralocorticoids remained constant during the first year of life. CONCLUSION: The urinary steroid profile changes dramatically during the first year of life and correlates with the physiologic developmental changes during the fetal-neonatal transition. Thus detailed normative data during this time period permit the use of steroid profiling as a powerful diagnostic tool.

Molecular Characteristics of Neuroblastoma with Special Reference to Novel Prognostic Factors and Diagnostic Applications

Molecular Characteristics of Neuroblastoma with Special Reference to Novel Prognostic Factors and Diagnostic Applications Department of Medical Biochemistry and Genetics Annales Universitatis Turkuensis, Medica-Odontologica, 2009, Turku, Finland Painosalama Oy, Turku, Finland 2009 Background: Neuroblastoma, which is the most common and extensively studied childhood solid cancer, shows a great clinical and biological heterogeneity. Most of the neuroblastoma patients older than one year have poor prognosis despite intensive therapies. The hallmark of neuroblastoma, biological heterogeneity, has hindered the discovery of prognostic tumour markers. At present, few molecular markers, such as MYCN oncogene status, have been adopted into clinical practice. Aims: The aim of the study was to improve the current prognostic methodology of neuroblastoma, especially by taking cognizance of the biological heterogeneity of neuroblastoma. Furthermore, unravelling novel molecular characteristics which associate with neuroblastoma tumour progression and cell differentiation was an additional objective. Results: A new strictly defined selection of neuroblastoma tumour spots of highest proliferation activity, hotspots, appeared to be representative and reliable in an analysis of MYCN amplification status using a chromogenic in situ hybridization technique (CISH). Based on the hotspot tumour tissue microarray immunohistochemistry and high-resolution oligo-array-based comparative genomic hybridization, which was integrated with gene expression and in silico analysis of existing transcriptomics, a polysialylated neural cell adhesion molecule (NCAM) and poorly characterized amplicon at 12q24.31 were discovered to associate with outcome. In addition, we found that a previously considered new neuroblastoma treatment target, the mutated c-kit receptor, was not mutated in neuroblastoma samples. Conclusions: Our studies indicate polysialylated NCAM and 12q24.31 amplicon to be new molecular markers with important value in prognostic evaluation of neuroblastoma. Moreover, the presented hotspot tumour tissue microarray method together with the CISH technique of the MYCN oncogene copy number is directly applicable to clinical use. Key words: neuroblastoma, polysialic acid, neural cell adhesion molecule, MYCN, c-kit, chromogenic in situ hybridization, hotspot

Reference values for lung function tests: I. Static volumes

Static lung volume (LV) measurements have a number of clinical and research applications; however, no previous studies have provided reference values for such tests using a healthy sample of the adult Brazilian population. With this as our main purpose, we prospectively evaluated 100 non-smoking subjects (50 males and 50 females), 20 to 80 years old, randomly selected from more than 8,000 individuals. Gender-specific linear prediction equations were developed by multiple regression analysis with total lung capacity (TLC), functional residual capacity (FRC), residual volume (RV), RV/TLC ratio and inspiratory capacity (IC) as dependent variables, and with age, height, weight, lean body mass and indexes of physical fitness as independent ones. Simpler demographic and anthropometric variables were as useful as more complex measurements in predicting LV values, independent of gender and age (R2 values ranging from 0.49 to 0.78, P<0.001). Interestingly, prediction equations from North American and European studies overestimated the LV at low volumes and underestimated them at high volumes (P<0.05). Our results, therefore, provide a more appropriate frame of reference to evaluate the normalcy of static lung volume values in Brazilian males and females aged 20 to 80 years.

Reference values for lung function tests: II. Maximal respiratory pressures and voluntary ventilation

The strength of the respiratory muscles can be evaluated from static measurements (maximal inspiratory and expiratory pressures, MIP and MEP) or inferred from dynamic maneuvers (maximal voluntary ventilation, MVV). Although these data could be suitable for a number of clinical and research applications, no previous studies have provided reference values for such tests using a healthy, randomly selected sample of the adult Brazilian population. With this main purpose, we prospectively evaluated 100 non-smoking subjects (50 males and 50 females), 20 to 80 years old, selected from more than 8,000 individuals. Gender-specific linear prediction equations for MIP, MEP and MVV were developed by multiple regression analysis: age and, secondarily, anthropometric measurements explained up to 56% of the variability of the dependent variables. The most cited previous studies using either Caucasian or non-Caucasian samples systematically underestimated the observed values of MIP (P<0.05). Interestingly, the self-reported level of regular physical activity and maximum aerobic power correlates strongly with both respiratory and peripheral muscular strength (knee extensor peak torque) (P<0.01). Our results, therefore, provide a new frame of reference to evaluate the normalcy of some useful indexes of respiratory muscle strength in Brazilian males and females aged 20 to 80.

Reference values for lung function tests: III. Carbon monoxide diffusing capacity (transfer factor)

Carbon monoxide diffusing capacity (DLCO) or transfer factor (TLCO) is a particularly useful test of the appropriateness of gas exchange across the lung alveolocapillary membrane. With the purpose of establishing predictive equations for DLCO using a non-smoking sample of the adult Brazilian population, we prospectively evaluated 100 subjects (50 males and 50 females aged 20 to 80 years), randomly selected from more than 8,000 individuals. Gender-specific linear prediction equations were developed by multiple regression analysis with single breath (SB) absolute and volume-corrected (VA) DLCO values as dependent variables. In the prediction equations, age (years) and height (cm) had opposite effects on DLCOSB (ml min-1 mmHg-1), independent of gender (-0.13 (age) + 0.32 (height) - 13.07 in males and -0.075 (age) + 0.18 (height) + 0.20 in females). On the other hand, height had a positive effect on DLCOSB but a negative one on DLCOSB/VA (P<0.01). We found that the predictive values from the most cited studies using predominantly Caucasian samples were significantly different from the actually measured values (P<0.05). Furthermore, oxygen uptake at maximal exercise (VO2max) correlated highly to DLCOSB (R = 0.71, P<0.001); this variable, however, did not maintain an independent role to explain the VO2max variability in the multiple regression analysis (P>0.05). Our results therefore provide an original frame of reference for either DLCOSB or DLCOSB/VA in Brazilian males and females aged 20 to 80 years, obtained from the standardized single-breath technique.

A study of the abilities in oral language comprehension of the Boston Diagnostic Aphasia Examination - Portuguese version: a reference guide for the Brazilian population

We analyzed the performance of 162 normal subjects, subdivided into groups according to age and schooling, in the oral comprehension tasks of the Boston Diagnostic Aphasia Examination translated and adapted to Brazilian Portuguese to obtain a profile of performance for the Brazilian population, as well as cut-off scores for each task, and to determine the best combination of tasks that distinguish normal from aphasic subjects, as a guide for clinicians. The normal subjects were compared to 69 aphasics. Age alone influenced the performance in the designation of actions (subjects above 70 years showing the worst performance); schooling alone influenced the comprehension of forms, colors and numbers (subjects with less than four years of education showing a poorer performance). Both age and schooling influenced the performance in Body Part Identification (BPI) and Complex Ideational Material (CIM) with mean values of 70.5 ± 3.3 (Word Discrimination, WD), 18.9 ± 1.4 (BPI), 14.7 ± 0.9 (Commands), and 10.3 ± 1.7 (CIM) for the whole sample; the cut-off scores obtained were 65 (WD), 17.5 (BPI), 14 (Commands), and 9.5 (CIM) for the whole sample. Logistic regression showed that the combination of BPI + Commands + CIM was the most efficient in differentiating normal subjects from aphasics, with 72.5% sensitivity and 97.6% specificity. However, for low-education subjects, BPI and Commands were sufficient for this differentiation (75.7% sensitivity and 84.7% specificity). The main contribution of this study was to provide reference values that are far more representative of our population to be used by health professionals in Brazil, taking into account cultural differences.

Efficacy of the FEV1/FEV6 ratio compared to the FEV1/FVC ratio for the diagnosis of airway obstruction in subjects aged 40 years or over

The present cross-sectional, population-based study was designed to evaluate the performance of the FEV1/FEV6 ratio for the detection of airway-obstructed subjects compared to the FEV1/FVC <0.70 fixed ratio test, as well as the lower limit of normality (LLN) for 1000 subjects ³40 years of age in the metropolitan area of São Paulo, SP, Brazil. After the exclusion of 37 (3.7%) spirometries, a total of 963 pre-bronchodilator (BD) and 918 post-BD curves were constructed. The majority of the post-BD curves (93.1%) were of very good quality and achieved grade A (762 curves) or B (93 curves). The FEV1/FEV6 and FEV1/FVC ratios were highly correlated (r² = 0.92, P < 0.000). Two receiver operator characteristic curves were constructed in order to express the imbalance between the sensitivity and specificity of the FEV1/FEV6 ratio compared to two FEV1/FVC cut-off points for airway obstruction: equal to 70 (area under the curve = 0.98, P < 0.0001) and the LLN (area under the curve = 0.97, P < 0.0001), in the post-BD curves. According to an FEV1/FVC <0.70, the cut-off point for the FEV1/FEV6 ratio with the highest sum for sensitivity and specificity was 0.75. The FEV1/FEV6 ratio can be considered to be a good alternative to the FEV1/FVC ratio for the diagnosis of airway obstruction, both using a fixed cut-off point or below the LLN as reference. The FEV1/FEV6 ratio has the additional advantage of being an easier maneuver for the subjects and for the lung function technicians, providing a higher reproducibility than traditional spirometry maneuvers.

Anthropometric midarm measurements can detect systemic fat-free mass depletion in patients with chronic obstructive pulmonary disease

Our objective was to determine whether anthropometric measurements of the midarm (MA) could identify subjects with whole body fat-free mass (FFM) depletion. Fifty-five patients (31% females; age: 64.6 ± 9.3 years) with mild/very severe chronic obstructive pulmonary disease (COPD), 18 smokers without COPD (39% females; age: 49.0 ± 7.3 years) and 23 never smoked controls (57% females; age: 48.2 ± 9.6 years) were evaluated. Spirometry, muscle strength and MA circumference were measured. MA muscle area was estimated by anthropometry and MA cross-sectional area by computerized tomography (CT) scan. Bioelectrical impedance was used as the reference method for FFM. MA circumference and MA muscle area correlated with FFM and biceps and triceps strength. Receiver operating characteristic curve analysis showed that MA circumference and MA muscle area cut-off points presented sensitivity and specificity >82% to discriminate FFM-depleted subjects. CT scan measurements did not provide improved sensitivity or specificity. For all groups, there was no significant statistical difference between MA muscle area [35.2 (29.3-45.0) cm²] and MA cross-sectional area values [36.4 (28.5-43.3) cm²] and the linear correlation coefficient between tests was r = 0.77 (P < 0.001). However, Bland-Altman plots revealed wide 95% limits of agreement (-14.7 to 15.0 cm²) between anthropometric and CT scan measurements. Anthropometric MA measurements may provide useful information for identifying subjects with whole body FFM depletion. This is a low-cost technique and can be used in a wider patient population to identify those likely to benefit from a complete body composition evaluation.

Reference value of brachial-ankle pulse wave velocity for the eastern Chinese population and potential influencing factors

The present study was conducted to obtain reference values for brachial-ankle pulse wave velocity (baPWV) and to evaluate influencing factors of baPWV according to gender. Using automatic devices, baPWV was measured simultaneously in 2095 subjects. A total of 647 healthy subjects, none of whom presented atherosclerotic risk factors, were analyzed in the present study. Two different statistical methods were used to obtain reference values for baPWV according to subject gender and age. The association between baPWV value and gender, as well as other features, were analyzed. For male subjects, multiple stepwise analysis showed that age, systolic blood pressure (SBP), heart rate (HR), and plasma levels of triglycerides (TG) were independent predictors of baPWV. For female subjects, age, SBP, HR, and plasma levels of uric acid (UA) were independent predictors of baPWV. In male subjects, the upper limits of baPWV values were 1497.43/1425.00, 1518.67/1513.25, 1715.97/1726.50, 1925.20/1971.90, and 2310.18/2115.00 cm/s, obtained using two different statistical methods for the age ranges of 30-39, 40-49, 50-59, 60-69, and 70 and older, respectively. For females, the upper limits of baPWV values were 1426.70/1411.13, 1559.15/1498.95, 1733.50/1739.00, 1958.63/1973.78, and 2720.80/2577.00 cm/s for the age ranges of 30-39, 40-49, 50-59, 60-69, and 70 and older, respectively. Aging is the most important influencing factor for baPWV value and its effect is more prominent in females. The reference values of baPWV according to age and gender may be useful for the clinical diagnosis and preventive therapy of cardiovascular diseases.

Antiretroviral therapy-associated dyslipidemia in patients from a reference center in Brazil

The aim of this study was to determine the impact of antiretroviral therapy on the lipid profile of human immunodeficiency virus (HIV) patients before and after the initiation of highly active antiretroviral therapy (HAART). This was a cross-sectional analysis of patients receiving HAART at a reference center in Belo Horizonte, Brazil, on the basis of medical records from 2002 to 2006. Patients were included if they had at least one lipid test or a clinical or laboratory diagnosis of dyslipidemia/lipodystrophy. Among the 692 patients, 620 met the eligibility criteria. The majority were males (66.5%), middle age (average 39 years), had a low educational level (60.4%), and low income (51.0%). HAART duration ranged from 11 days to 4.6 years, with a mean of 28.6 months (SD = ± 470.19 days). The prevalence of dyslipidemia/lipodystrophy nearly tripled (11.3% pre- and 32.4% post-HAART). Dyslipidemia was associated with older age (P = 0.007), nucleoside reverse transcriptase inhibitor (NRTI) + protease inhibitor (PI) regimens (P = 0.04), NRTI + non-NRTI (NNRTI) regimens (P = 0.026), the use of stavudine (d4T) in any regimen (P = 0.002) or in NRTI-based regimens (P = 0.006), and longer exposure to HAART (P < 0.000). In addition, there was no correlation between dyslipidemia and gender (P = 0.084). Only 2.0% of the patients received treatment for dyslipidemia during the trial. These results show a need for continuous monitoring of patients under antiretroviral therapy, particularly those using NRTI-based regimens, especially when combined with d4T and PIs. Secondly, interventions should be developed to correct metabolic changes.

Role of a GenoType MTBDRplus line probe assay in early detection of multidrug-resistant tuberculosis at a Brazilian reference center

Resistance to Mycobacterium tuberculosis is a reality worldwide, and its diagnosis continues to be difficult and time consuming. To face this challenge, the World Health Organization has recommended the use of rapid molecular tests. We evaluated the routine use (once a week) of a line probe assay (Genotype MTBDRplus) for early diagnosis of resistance and for assessment of the main related risk factors over 2 years. A total of 170 samples were tested: 15 (8.8%) were resistant, and multidrug resistance was detected in 10 (5.9%). The sensitivity profile took 3 weeks (2 weeks for culture and 1 week for rapid testing). Previous treatment for tuberculosis and the persistence of positive acid-fast smears after 4 months of supervised treatment were the major risk factors observed. The use of molecular tests enabled early diagnosis of drug-resistant bacilli and led to appropriate treatment of the disease. This information has the potential to interrupt the transmission chain of resistant M. tuberculosis.

Développement durable: quelques points litigieux

Ouvrant la réflexion sur les enjeux de la perte massive de la biodiversité, l'article s'appuie sur la question élémentaire de l'importance de la diversité biologique pour l'homme, à la fois sur le plan économique, biologique et éthique. La maîtrise de la nature par l'homme se révèle être une illusion. La réalité étant celle de l'interaction, il est permis de dire que les sociétés humaines agissent sur leurs propres conditions en modifiant les équilibres biologiques pour satisfaire leurs besoins, sans pour autant intégrer cette interaction fondamentale dans l'équation d'un rapport viable entre l'homme et la nature. L'approche récente par le biais des analyses des services écologiques constitue à cet égard un progrès sensible. Il aboutit néanmoins au constat de l'impossibilité pour l'humanité de se substituer aux mécanismes naturels permettant de tirer les services de la nature nécessaires à sa survie. Plutôt que de prétendre contrôler et maîtriser la nature à tout prix, l’homme devrait apprendre à se reconnaître comme une partie consciente de la nature et à vivre avec la vie qui l’entoure. Cet apprentissage soulève des défis scientifiques, humains et de gouvernance. L'article prône la nécessité d'une science de la biodiversité intégrée et non plus éclatée, condition essentielle pour l'élaboration de politiques économiques et sociales qui intègrent la viabilité de l'utilisation des ressources naturelles et des services qu'elles procurent.

Points quantiques : caractérisation et applications en sciences pharmaceutiques

L’imagerie médicale a longtemps été limitée à cause des performances médiocres des fluorophores organiques. Récemment la recherche sur les nanocristaux semi-conducteurs a grandement contribué à l’élargissement de la gamme d’applications de la luminescence dans les domaines de l’imagerie et du diagnostic. Les points quantiques (QDs) sont des nanocristaux de taille similaire aux protéines (2-10 nm) dont la longueur d’onde d’émission dépend de leur taille et de leur composition. Le fait que leur surface peut être fonctionnalisée facilement avec des biomolécules rend leur application particulièrement attrayante dans le milieu biologique. Des QDs de structure « coeur-coquille » ont été synthétisés selon nos besoins en longueur d’onde d’émission. Dans un premier article nous avons modifié la surface des QDs avec des petites molécules bi-fonctionnelles portant des groupes amines, carboxyles ou zwitterions. L’effet de la charge a été analysé sur le mode d’entrée des QDs dans deux types cellulaires. À l’aide d’inhibiteurs pharmacologiques spécifiques à certains modes d’internalisation, nous avons déterminé le mode d’internalisation prédominant. L’endocytose par les radeaux lipidiques représente le mode d’entrée le plus employé pour ces QDs de tailles similaires. D’autres modes participent également, mais à des degrés moindres. Des disparités dans les modes d’entrée ont été observées selon le ligand de surface. Nous avons ensuite analysé l’effet de l’agglomération de différents QDs sur leur internalisation dans des cellules microgliales. La caractérisation des agglomérats dans le milieu de culture cellulaire a été faite par la technique de fractionnement par couplage flux-force (AF4) associé à un détecteur de diffusion de la lumière. En fonction du ligand de surface et de la présence ou non de protéines du sérum, chacun des types de QDs se sont agglomérés de façon différente. À l'aide d’inhibiteur des modes d’internalisation, nous avons corrélé les données de tailles d’agglomérats avec leur mode d’entrée cellulaire. Les cellules microgliales sont les cellules immunitaires du système nerveux central (CNS). Elles répondent aux blessures ou à la présence d’inflammagènes en relâchant des cytokines pro-inflammatoires. Une inflammation non contrôlée du CNS peut conduire à la neurodégénérescence neuronale et est souvent observée dans les cas de maladies chroniques. Nous nous sommes intéressés au développement d’un nanosenseur pour mesurer des biomarqueurs du début de l’inflammation. Les méthodes classiques pour étudier l’inflammation consistent à mesurer le niveau de protéines ou molécules relâchées par les cellules stressées (par exemple monoxyde d’azote, IL-1β). Bien que précises, ces méthodes ne mesurent qu’indirectement l’activité de la caspase-1, responsable de la libération du l’IL-1β. De plus ces méthode ne peuvent pas être utilisées avec des cellules vivantes. Nous avons construit un nanosenseur basé sur le FRET entre un QD et un fluorophore organique reliés entre eux par un peptide qui est spécifiquement clivé par la caspase-1. Pour induire l’inflammation, nous avons utilisé des molécules de lipopolysaccharides (LPS). La molécule de LPS est amphiphile. Dans l’eau le LPS forme des nanoparticules, avec des régions hydrophobes à l’intérieure. Nous avons incorporé des QDs dans ces régions ce qui nous a permis de suivre le cheminement du LPS dans les cellules microgliales. Les LPS-QDs sont internalisés spécifiquement par les récepteurs TLR-4 à la surface des microglies. Le nanosenseur s’est montré fonctionnel dans la détermination de l’activité de la caspase-1 dans cellules microgliales activées par le LPS. Éventuellement, le senseur permettrait d’observer en temps réel l’effet de thérapies ciblant l’inflammation, sur l’activité de la caspase-1.

Nutrient Dynamics in the EEZ of the West Coast of India with Special Reference to the Oxygen Minimum Zone and Denitrification

The present work aims at deciphering the processes that control the nutrient distribution along the EEZ (Exclusive Economic Zone of India) of the west coast of India and to bring out its linkage with primary and secondary productivity. This work assume utmost importance as very few studies have hitherto focused entirely on the EEZ of the west coast of India to address the biochemical responses brought about by monsoons. The present study examines the seasonal variations in physicochemical parameters and associated primary biological responses along the west coast of India. This study targets to measure and understand the shelf ocean exchange in a typical coastal upwelling region of the southeast Arabian Sea, and the influence of convective mixing along the northern part of the west coast of India. The study focuses more directly on coastal upwelling along the southwest coast of India, within the EEZ. The effects of coastal upwelling, eddy formation and the offshore advection are apparent in the present investigation. This has consequences to fisheries and climate, in energy transfer to the food chain and the increased sequestering of carbon in the ocean. The study also focuses on the Oxygen Minimum Zone (OMZ) and dentrification observed along the EEZ of the west coast of India on a seasonal scale. In the study, an attempt is also made to demarcate the geographical boundaries of the denitrification zone in the EEZ of India and on the nature and magnitude of these variations, on a seasonal and inter annual scales