Prescription-free consultation: A cross-sectional study in general practice.

PURPOSE: In 2005, 10% of consultations in France ended without a prescription. In 2019, a review of the literature found 30 to 70% of prescription-free consultations in Northern Europe and 10 to 22% in Southern Europe and underlined the scarcity of quantitative data. Different factors contribute to this heterogeneity, such as product availability and status, modes of management, distribution channels, clinical practice recommendations, public policies targeting certain classes, etc. The main objective of our study was to quantify the rate of prescription-free consultations in general practice in France in 2021. The secondary objective was to characterize prescription-free consultations and analyze their determinants. METHODS: This was a quantitative observational study conducted using self-questionnaires among patients in medical practices in Auvergne. RESULTS: Out of 540 questionnaires, the rate of prescription-free consultations was 24% (95% CI [20.11-27.41]). Prescription-free consultations were for prevention, administrative problems, and gestures. The limiting factors are "feeling a need for a medication" (OR=0,006), "not knowing if a medication is needed" (OR=0.11) and "consultations for acute reasons" (OR=0.33). CONCLUSION: Acute consultations limit prescription-free consultations. General practitioners (GPs) probably overestimate patients' expectation of drug prescription. The French GP must be supported in their decision to not prescribe drugs. This is a long-term investment of time, to educate patients and avoid new consultations for acute reasons. A tool to help doctors manage non-prescription during acute consultations will be created in a future study in France.

Comparison between radiometry and spectrophotometry for the determination of angiotensin-converting enzyme activity in cerebrospinal fluid.

Determination of angiotensin-converting enzyme (ACE) activity in cerebrospinal fluid (CSF) can help for establishing the diagnosis of neurosarcoidosis. We investigated the performance characteristics of two assays for ACE determination in 57 CSF, radiometry with [glycine-1-14C] benzoyl-L-histidyl-L-leucine and spectrophotometry with furylacryloyl-phenylalanyl-L-glycyl-L-glycine (FAPGG) as substrates. We compared both kinetic assays to an ELISA specific for human ACE. Within run and between run imprecisions were 14-17% for radiometry, 6-19% for spectrophotometry and 5-8% for ELISA. The limit of detection was 0.04 U/L for radiometry, 1.0 U/L for spectrophotometry and 0.156 µg/L for ELISA. The limit of quantification was 0.06 U/L for radiometry, 1.5 U/L for spectrophotometry, but not known for ELISA. The domain for quantification was 0.06-4.0 U/L for radiometry, 1.5-24 U/L for spectrophotometry and 0.156-10 µg/L for ELISA. Deming regression and Bland-Altman plots show good correlations between the three assays, but with high slopes, because both kinetic assays use different substrates and ELISA measures ACE molecule but not activity. Radiometry was more sensitive than spectrophotometry, which has a limit of detection above most pathological levels. ELISA could be an alternative to radiometry but only after complete evaluation, determination of normal values and assessment of its clinical value. We claim for standardization of ACE determination as well as in serum as in other biological fluids, in particular CSF.

Autoxidation Kinetics of Tetrahydrobiopterin-Giving Quinonoid Dihydrobiopterin the Consideration It Deserves.

In humans, tetrahydrobiopterin (H4Bip) is the cofactor of several essential hydroxylation reactions which dysfunction cause very serious diseases at any age. Hence, the determination of pterins in biological media is of outmost importance in the diagnosis and monitoring of H4Bip deficiency. More than half a century after the discovery of the physiological role of H4Bip and the recent advent of gene therapy for dopamine and serotonin disorders linked to H4Bip deficiency, the quantification of quinonoid dihydrobiopterin (qH2Bip), the transient intermediate of H4Bip, has not been considered yet. This is mainly due to its short half-life, which goes from 0.9 to 5 min according to previous studies. Based on our recent disclosure of the specific MS/MS transition of qH2Bip, here, we developed an efficient HPLC-MS/MS method to achieve the separation of qH2Bip from H4Bip and other oxidation products in less than 3.5 min. The application of this method to the investigation of H4Bip autoxidation kinetics clearly shows that qH2Bip's half-life is much longer than previously reported, and mostly longer than that of H4Bip, irrespective of the considered experimental conditions. These findings definitely confirm that an accurate method of H4Bip analysis should include the quantification of qH2Bip.

Quantification of monoamine biomarkers in cerebrospinal fluid: Comparison of a UHPLC-MS/MS method with a UHPLC coupled to fluorescence detection method.

Inborn errors of monoamine neurotransmitter metabolism are rare genetic diseases classified as catecholamine and serotonin metabolism disorders or neurotransmitter transportopathies. To diagnose these orphan diseases, monoamine metabolites have been identified and validated as cerebrospinal fluid (CSF) biomarkers: 5-hydroxy-tryptophane, 5-hydroxy-indol-acetic acid, 3-ortho-methyl-DOPA, homovanillic acid, and 3-methoxy-4-hydroxyphenylglycol. The present work presents a UHPLC-MS/MS method developed for the quantification of these metabolites in CSF and compares it with a previously described UHPLC with fluorescence detection (UHPLC-FD) method. MS/MS detection was performed in positive electrospray ionization and multiple reaction monitoring mode. The UHPLC-MS/MS and UHPLC-FD methods were validated in terms of accuracy, linearity, precision and matrix effect. The lower limits of quantification (LLOQ) ranged between 0.5 and 10 nm and between 1 and 5 nm for the UHPLC-MS/MS method and the UHPLC-FD one, respectively. We verified the applicability of both methods by analyzing 30 CSF samples. The measured concentrations were comparable with the reference values described in the literature. The two methods allowed pathological samples to be distinguished from healthy ones for clinical diagnosis. UHPLC-MS/MS and UHPLC-FD methods exhibited very close LLOQs. As the UHPLC-MS/MS method is more selective, it allows faster analysis with a run time of 6 min per run vs. 10 min for the UHPLC-FD method.

Identification of biomarker panels as predictors of severity in coronary artery disease.

Matrix metalloproteinases (MMPs) are implicated in atherosclerotic plaque rupture and recondition. Specific tissue inhibitors (TIMPs) control MMP functions. Both MMPs and TIMPs are potential biomarkers of plaque instability. Elevated Apo-CII and CIII and Apo-E levels are recognized as cardiovascular disease risk factors. We aimed to establish the best blood biomarker panel to evaluate the coronary artery disease (CAD) severity. Plasma levels of MMP-3 and MMP-9, TIMP-1 and TIMP-2, Apo-CII, Apo-CIII and Apo-E were measured in 472 patients with CAD evaluated by coronary angiography and electrocardiography, and in 285 healthy controls. MMP-3 and MMP-9 plasma levels in CAD patients were significantly increased (P < 0.001) compared to controls (3.54- and 3.81-fold, respectively). Furthermore, these increments are modulated by CAD severity as well as for Apo-CII and Apo-CIII levels (P < 0.001). TIMPs levels were decreased in CAD versus controls (P < 0.001) and in inverse correlation to MMPs. Standard ROC curve approach showed the importance of panels of biomarkers, including MMP-3, MMP-9, TIMP-1, TIMP-2, Apo-CII and Apo-CIII, for disease aggravation diagnosis. A high area under curve (AUC) value (0.995) was reached for the association of MMP-9, TIMP-2 and Apo-CIII. The unbalance between MMPs and TIMPs in vascular wall and dyslipidaemia creates favourable conditions for plaque disruption. Our study suggests that the combination of MMP-9, TIMP-2 and Apo-CIII values ('CAD aggravation panel') characterizes the severity of CAD, that is electrophysiological state, number of involved vessels, stent disposal and type of stent.

[Measurement of angiotensin-I-converting enzyme in the blood: help for method validation]. / Dosage de l'enzyme de conversion de l'angiotensine-I sanguine : aide à la validation de méthode.

Blood angiotensin-converting enzyme (ACE) assay is now realized by the determination of enzyme activity on synthetic substrate, mostly furylacryloyl-phenylalanyl-L-glycyl-L-glycine (FAPGG). The matrix can be serum or heparin-plasma, with or without a separator; the assay developed on serum or plasma is not adapted to other matrix such as cerebrospinal fluid where the ACE activity is much lower. This assay has been adapted on a number of automated biochemistry analyzers with the specifications of the supplier of reagents, sometimes with modification of volumes or times for analysis. Samples can be stored at +4̊C for at least for one week, freezing at -20̊C is possible but refreezing is not advised. The assay is linear from 10 to 200 UI/L. Fidelity is excellent after calibration of the assay. Accuracy can be calculated from IQA and EQA results, and the analytical uncertainty is between 2% and 5% in function of the serum ACE value. Usual values will be soon available from studies on age brackets and sex, because ACE activity seems to be more elevated in boys during adolescence. At signature, it is interesting to have medical information on the diagnosis of sarcoidosis or its treatment including ACE inhibitors as a proof of intake; we can give a commentary on elevation of serum ACE activity from other causes than sarcoidosis and the causes for low activities.

Direct liquid chromatography tandem mass spectrometry analysis of amino acids in human plasma.

Here we describe a new HPLC-MS/MS method using a mixed mode stationary phase and a binary gradient of elution for the rapid separation and quantification of AAs in human plasma without derivatization or ion pairing reagent addition. The sample preparation procedure consists in a single dilution step after protein precipitation with sulfosalicylic acid. The proposed method allows for the unambiguous identification and analysis of 52 AAs and related compounds including the separation of isomers and isobars in an 18 min chromatographic run including the conditioning and the equilibration times. AAs were detected by selective reaction monitoring. Internal calibration was used for the quantification of 37 AAs, including 25 using the corresponding isotopically labeled internal standards. External calibration (no internal standard) was used for five additional analytes. Qualitative detection was achieved for the remaining compounds. Validation studies evaluated accuracy, linearity, within- and between-run precision, lower limits of detection and quantification for 37 amino acids present in commonly used quality control samples. For within-run precision CVs averaged 3.8 % (n = 30) for all compounds. For between-run precision, CVs averaged 8.6 % for all compounds (n = 20). Correlation with the common standard ion-exchange chromatography with post-column derivatization method was also performed for 32 plasma samples. While the proposed method is at least 50 times more sensitive, the data showed good correlation with slopes equal or higher than 0.9 and correlation coefficients mostly higher than 0.90. The method was successfully applied for analysis of plasma samples for detection of inherited disorders of amino acid metabolism.

[Examples of accreditation of serum and urinary proteins electrophoresis]. / Expériences d'accréditation des électrophorèses sériques et urinaires.

Serum proteins and urinary proteins electrophoresis are useful biological tests. They are often prescribed for the screening of monoclonal gammopathys and also during follow-up for treatment response. This test can be accredited according to standard NF ISO 15189 since laboratories use analysers and adapted reagents, but there are numerous protocols for the method validation. This paper present the results of a survey proposed in 2019 to biologists by CNBH and SFBC. The aim of this survey is to give biologists a choice among several protocols that have been positively evaluated by COFRAC.

Multi-centre evaluation of recent troponin assays for the diagnosis of NSTEMI.

OBJECTIVES: We aimed to compare the use of nine different cardiac troponin (cTn) assays (2 cTnT and 7 cTnI) for the diagnosis of NSTEMI in a single multi-centre population. DESIGN AND METHODS: One hundred and fifty-eight patients were included (mean age 60 years, SD 17 years), including 23 patients (14%) with NSTEMI. RESULTS: The analytical comparison highlighted a large heterogeneity of cTn assays, as reflected by percentages of patients with detectable cTn, correlation coefficients, Passing-Bablok comparisons and concordance coefficients. Correlations within cTnI assays were good and correlation within cTnT assays was excellent. Diagnostic performances demonstrated that each cTn assay has specific threshold values. Furthermore, some assays (HS-cTnI and T, cTnI-Pathfast and cTnI-Centaur) indicated high sensitivity and negative predictive value using the limit of detection (LoD) diagnostic strategy. For the latter assays, a significant increase in specificity was found when using the 99th percentile or the H0-H3 strategies, in comparison to the LoD strategy. When applying the European Society of Cardiology H0-H3 algorithm, comparable diagnostic performances were obtained. CONCLUSION: All 9 cTn assays indicated overall good diagnostic performances for the diagnosis of NSTEMI in emergency departments when the recommended algorithm based on the variation of cTn value between two measurements at admission and 3 h later was used.

Diagnostic accuracy of the Coopscore© to predict liver fibrosis in human immunodeficiency virus/hepatitis B virus co-infection.

Background Non-invasive methods for assessing liver fibrosis are increasingly used as an alternative to liver biopsy. Recently, a score-based biochemical blood test (Coopscore©) was developed in a cohort of patients chronically infected with hepatitis C virus, showing higher diagnostic performances than Fibrometer®, Fibrotest®, Hepascore® and Fibroscan™. Here, we assess its performance in patients co-infected with the human immunodeficiency virus and hepatitis B virus. Methods Ninety-seven human immunodeficiency virus/hepatitis B virus co-infected patients with liver biopsies were included from a previously described cohort. Histological fibrosis staging using METAVIR criteria was used as the reference. Coopscore©, Fibrotest®, Fibrometer®, Hepascore® and Zeng score were computed and compared with the Coopscore© using the Obuchowski index and area under the receiving operator characteristic curves. Results The distribution of liver fibrosis levels was as follows: F0-F1 ( n = 42), F2 ( n = 25), F3 ( n = 15) and F4 ( n = 15). The Obuchowski index was higher for Coopscore© (0.774) than Fibrometer® (0.668), Hepascore® (0.690) and Zeng scores (0.704) ( P < 0.05), reflecting a better ability to discriminate between fibrosis stages. Similarly, when predicting significant fibrosis (≥F2), the AUROC was significantly greater for the Coopscore© (0.836) than the Hepascore® (0.727) and Zeng scores (0.746), but not for the Fibrotest® (0.778, P = 0.14) or Fibrometer® (0.790, P = 0.19). The Coopscore© did not show a higher capacity than other scores to predict advanced fibrosis (≥F3) or cirrhosis (F4). Conclusions This study supports the diagnostic value of the Coospcore© in fibrosis staging among human immunodeficiency virus/hepatitis B virus co-infected patients, especially to predict significant fibrosis.

Search for biomarkers of neurosarcoidosis by proteomic analysis of cerebrospinal fluid.

Sarcoidosis is a systemic granulomatous disease, which mostly affects lung. Central nervous system can be affected causing a neurosarcoidosis in 5 to 15% of all sarcoidosis patients. The definitive diagnosis is established on histological examination of brain granulomas. Angiotensin converting enzyme is currently the most relevant biomarker to confirm a probable diagnosis; however, it lacks sensitivity and specificity. We aim to find novel biomarkers of neurosarcoidosis in cerebrospinal fluid (CSF) by proteomic analysis, combining two-dimension electrophoresis (2-DE) and mass spectrometry. We performed CSF proteomic profile of both patients (group S) and control subjects (group H). The statistical analysis of 2-D gels highlighted 42 spots significantly different between the two groups. Twenty-five spots were subjected to tryptic digestion; the peptides were analyzed by MALDI-TOF and MALDI-TOF-TOF, giving rise to 10 identifications. Among the identified proteins, low-molecular-mass-kininogen and vitamin-D-binding-protein were increased, while transthyretin was decreased. These proteins have probably an intrathecal source and could be interesting candidates. This study led to the identification of several proteins which can be used for the diagnosis and/or monitoring of neurosarcoidosis. These putative biomarkers have to be confirmed on a larger cohort and assessed for their sensitivity and specificity.

Relevance of diagnostic investigations in patients with uveitis: Retrospective cohort study on 300 patients.

OBJECTIVE: The diagnostic workup of uveitis is a challenge due to the wide range of diagnoses and the lack of a well-codified diagnostic procedure. We aimed to evaluate the relevance of diagnostic investigations for the etiological diagnosis of uveitis. METHODS: Retrospective cohort study of patients referred for etiological diagnosis of uveitis. Uveitis related to ophthalmological diseases or occurring during the course of previously diagnosed diseases were not included. RESULTS: Three hundred patients were included. Chest CT-scan was suggestive of sarcoidosis in 83 (29%). Features associated with abnormal CT-scan were: snowballs and/or peripheral multifocal choroiditis (PMC) upon ocular examination (P=0.004), blood lymphopenia (P<0.0001), angiotensin converting enzyme (ACE) level>1.5 ULN (P=0.0003). Bronchoscopy showed granuloma in 18 (11%) while alveolar lymphocytosis suggestive of sarcoidosis was reported in 45 (27%). Presence of granuloma on bronchial biopsies was always associated with chest CT-scan abnormalities, whereas 31% of patients with alveolar lymphocytosis had normal CT-scans. Features associated with contributive bronchoscopy were: snowballs and/or PMC (P=0.003), ACE>1.5 ULN (P=0.007), abnormal chest-CT scan (P<0.0001). Salivary gland biopsy revealed granuloma in 12 patients (5%). Cerebral MRI was abnormal in 15 patients (9%) who mostly presented with snowballs and/or retinal vasculitis. Finally, the main causes of uveitis were latent tuberculosis (25%) and sarcoidosis (22%), but 34% remained of undetermined origin. Uveitis relapses were observed in 31% and did not differ between patients with an identified diagnosis and those with idiopathic uveitis. CONCLUSION: Identification of factors associated with abnormal investigations might improve the optimal diagnostic workup adapted to each patient.

Pathological interactions between the endothelin-1 and the angiotensin- converting enzyme among Tunisian coronary patients.

BACKGROUND: The correct understanding of the biochemical and metabolic interactions between coronary risk factors contribute to the exploration of cardiovascular pathophysiology and improves therapeutic care. The aim of this study was to explore the endothelin-1 (ET-1) concentration and the angiotensin converting enzyme (ACE) activity among Tunisian patients with coronary heart disease, and to investigate the metabolic relationships between these two markers,… and to assess the possible relationship between them and the different risk factors. In this present study, ET-1 concentration was determined by an analytical method (High Performance Chromatography, coupled by Mass Spectrometry), ACE activity was measured by a kinetic method for patients and healthy controls. These subjects (157 patients and 142 controls) beneficed also by a biochemical exploration (lipid, apolipoproteins and glucose profiles) to quantify cardiovascular risk. RESULTS: A statistically significant increase of the ET-1 concentration was found among patients compared to healthy controls (15.2 ± 5.3 nM vs 7.1 ± 2.7 nM, p < 0,00001). For the ACE activity, in spite the treatment of the majority of patients (97%) with ACE inhibitors, this activity was statistically elevated in patients compared to healthy subjects (86.7 ± 25.4 IU/L vs 42.8 ± 12.1 IU/L, p < 0.00001). Furthermore, a statistically positive correlation was identified between these two cardiac markers (r = 0.68 p < 0.00001). CONCLUSION: The study of the metabolic relationship between the ET-1 and ACE among coronary patients reveals other therapeutics targets.

Serum proteomic signatures as biomarkers of primary biliary cirrhosis diagnosis and prognosis.

Primary biliary cirrhosis (PBC) is a chronic cholestatic liver disease considered as an autoimmune disease. To identify new biomarkers of PBC, serum profiling analysis using Surface-enhanced laser desorption/ionization time-of-flight mass spectrometry (SELDI-TOF-MS) was employed. Twelve patients with either asymptomatic PBC (group 1, n=6) or PBC with a poor response to UDCA (group 2, n=6), were compared to healthy controls (group 3, n=6). Analysing the 18 sera by using four SELDI-TOF arrays under various conditions, we found four biomarkers of PBC at 5.9, 8.6, 8.9 and 9.0 kDa. The combination of the two arrays IMAC-40/Zn2+ and CM-10/pH 7 improved the positive diagnosis of this disease. We also found a biomarker of severity of PBC at 95.2 kDa on LSAX-30 array which characterized patients with a bad prognosis. In conclusion, our study identified several serum proteomics signatures as potential biomarkers of PBC for its diagnosis or prognosis.

Relationship between the A(8002)G intronic polymorphism of pre-pro-endothelin-1 gene and the endothelin-1 concentration among Tunisian coronary patients.

BACKGROUND: Acute coronary syndromes (ACS) are complex and polygenic diseases which are a real problem of public health. These syndromes require multidisciplinary studies to understand the pathogenesis mechanisms. Our study aims to evaluate the endothelin-1 (ET-1) serum concentration in Tunisian coronary compared to controls healthy, as well as the study of the impact of an intronic polymorphism A (8002) G of pre-pro-endothelin-1 Gene (inactive precursor of ET-1) on the change in serum endothelin-1 and in the susceptibility to Acute coronary syndrome (SCA). METHODS: Our samples were subdivided into coronary patients (157) and healthy subjects (142). The quantification of the ET-1 concentration was performed by high performance liquid chromatography, the identification of the different genotypes of the polymorphism A(8002)G was made by PCR-RFLP. The association between the ET-1 concentration and identified genotypes was realized by adapted software for descriptive statistics, Statistical Package for the Sociological Sciences (SPSS v 21.0). RESULTS: Our study showed that the concentration of ET-1 was significantly higher in patients compared to controls and that the mutated allele prevails in patients F (G) = 0.78 and there is a minority in controls F (G) = 0.3. Secondly the homozygous genotype GG is associated with higher concentrations of ET-1 in patients and controls, heterozygous genotype AG is associated with intermediaries' values and AA genotype is related to lower values. CONCLUSION: Although the polymorphism studied is an intronic polymorphism, it is involved in the change in serum concentration of ET-1 and is a candidate gene in susceptibility to SCA. Cardiovascular diseases are "polygenic" pathology and do not obey of the law for transmission of Mendel.

Plasma proprotein convertase subtilisin/kexin type 9 is associated with Lp(a) in type 2 diabetic patients.

AIM: Recent in vitro researches have shown that plasma Lp(a) can be reduced using a proprotein convertase subtilisin/kexin type 9 (PCSK9)-inhibitory monoclonal antibody. In our clinical study we tried to investigate the association between plasma Lp(a) and PCSK9 in Type 2 diabetic patients with elevated plasma Lp(a), and to check whether such an association would be related to LDL-receptor (LDL-R) levels. METHODS: Plasma PCSK9 and LDL-R concentrations were measured by sandwich ELISA methods using recombinant human PCSK9 protein and LDL-R protein as standards in a cohort with type 2 diabetic patients (n=50) compared to an age- and sex-matched control group (n=50). Both clinical and biochemical parameters were determined in all patients. RESULTS: Plasma PCSK9 level was significantly elevated in T2DM patients compared to controls (44.61±14.44 and 33.22±11.79ng/mL, respectively, P<0.0001). However LDL-R levels did not differ between the two groups. Remarkably, plasma PCSK9 levels were positively correlated with Lp(a) levels in whole population (r=+0.227, P=0.03) as well as in T2DM group (r=+0.398, P=0.0061) but not in control group. Multiple linear regression analysis showed that plasma Lp(a) levels were independently associated to those of PCSK9. CONCLUSION: Lp(a) has been proposed as a contributing factor to the accelerated development of macrovascular complications in T2DM. Its synergic effect with PCSK9 may explain the enhanced atherogenicity in T2DM patients.

Metabolic interactions between hyperhomocysteinemia and endothelin-1 among Tunisian patients with acute coronary diseases.

BACKGROUND: Acute coronary syndromes (ACS) are complex and polygenic diseases which are a real problem of public health. These syndromes require multidisciplinary studies to understand the pathogenesis mechanisms and metabolic interactions between different risk factors.This study aimed to explore the variation of two coronary risk parameters not mentioned by Framingham cohorts, hyperhomocysteinemia and endothelin-1 (ET-1) in Tunisian coronary and the study of the variation of these parameters based on various cardiac risk factors and metabolic relationship between them.To 157 coronary and 142 healthy subjects, the concentration of homocysteine was quantified by fluorescence polarization immunoassay; the concentration of ET-1 was measured by an analytical technique, the High Performance Liquid Chromatography (HPLC) coupled with mass spectrometry. RESULTS: Our study showed that homocysteine and ET-1 were significantly higher in patients compared to healthy subjects (24.40 ± 12.5 µmol/L vs 7.44 ± 2.5 µmol/L p <0.00001) for homocysteine and (15.2 ± 5.3 nmol/L vs 7.1 ± 2.7 nmol/L, p <0.00001) for ET-1. On the other hand, homocysteine varies according to tobacco and diabetes while ET-1 depends on the sex, hypertension, smoking, obesity and dyslipidemia and a statistically negative correlation was shown between homocysteine and ET-1 in coronary patients (r = -0.66 p <0.00001). CONCLUSION: The study of the variation of these two parameters in coronary patients and metabolic exploration of the relationship between homocysteine and ET-1 according to various risk factors and the interactions between themselves facilitates the decision of therapeutic treatment.