Regulation of veterinary point-of-care testing in the European Union, the United States of America and Japan.

Point-of-care testing (POCT) is used to detect diseases and other conditions or to monitor therapeutic procedures. In veterinary medicine, POCT not only helps during the prevention, diagnosis and treatment of animal diseases but it also has a direct impact on human health by safeguarding food supplies and preventing zoonoses. Despite its importance, the regulation of the quality, safety and effectiveness of POCT products is rarely discussed. This review reveals that the level of regulatory surveillance of veterinary POCT products in the European Union (EU), the United States of America and Japan is strikingly different, ranging from no regulation (EU) to comprehensive regulation, which is comparable to the procedures for the regulation of human in vitro medical devices (Japan). Details about the licensing procedures in these three locations, discussion of their strengths and weaknesses, and suggestions for possible future development of the regulation of these products are also provided.

Les tests utilisables sur le lieu d'intervention (POCT) permettent de détecter des maladies ou d'autres affections et d'assurer un suivi des procédures thérapeutiques appliquées. En médecine vétérinaire, les POCT apportent un soutien utile lors de la prévention, du diagnostic et du traitement des maladies animales, en plus d'avoir un impact direct sur la santé humaine en participant à la sécurité de l'approvisionnement alimentaire et à la prévention des zoonoses. La réglementation applicable aux produits utilisés pour les POCT, afin d'en garantir la qualité, la sécurité et l'efficacité, fait rarement l'objet de débats, bien qu'il s'agisse d'une question importante. L'examen présenté par les auteurs révèle que le niveau de la surveillance réglementaire exercée sur les produits vétérinaires utilisés pour les POCT dans l'Union européenne (UE), les états-Unis d'Amérique et le Japon est extrêmement variable, allant d'une absence totale de réglementation (UE) à une réglementation exhaustive comparable aux procédures appliquées pour les dispositifs in vitro utilisés en médecine humaine (Japon). Les auteurs décrivent en détail les procédures d'autorisation de mise sur le marché dans ces trois pays, examinent leurs atouts et points faibles respectifs et font quelques propositions pour faire évoluer la réglementation de ces produits à l'avenir.

Las pruebas realizadas en el punto de consulta, o «pruebas de diagnóstico inmediato¼, sirven para detectar enfermedades y otros trastornos o para seguir de cerca los resultados de procedimientos terapéuticos. En medicina veterinaria, estas pruebas no solo ayudan a prevenir, diagnosticar y tratar enfermedades animales, sino que también tienen una influencia directa en la salud humana, en la medida en que permiten proteger los suministros alimentarios y prevenir zoonosis. A pesar de su importancia, rara vez se aborda el tema de la regulación de la calidad, seguridad y eficacia de los productos que contienen pruebas de diagnóstico inmediato. Los autores revelan aquí llamativas disparidades en los regímenes de vigilancia reglamentaria que aplican a las pruebas de diagnóstico veterinario inmediato la Unión Europea (UE), los Estados Unidos de América (EE.UU.) y el Japón, regímenes que van desde la inexistencia de reglamentos (UE) hasta una regulación exhaustiva comparable a los procedimientos aplicados a los dispositivos médicos in vitro (Japón). Los autores también exponen en detalle los procedimientos de obtención de licencia vigentes en estos tres contextos, examinan sus ventajas e inconvenientes y formulan propuestas para el futuro desarrollo de la reglamentación aplicada a los antedichos productos.

Absence of LKM-1 antibody reactivity in autoimmune and hepatitis-C-related chronic liver disease in Sweden. Swedish Internal Medicine Liver club.

BACKGROUND: Type-2 autoimmune hepatitis is a subgroup of chronic hepatitis characterized by the presence of liver/kidney microsomal autoantibodies type 1 (LKM-1). A frequent association with chronic hepatitis C suggests that hepatitis virus might trigger autoimmune reactivity. LKM-1-positive chronic hepatitis is not uncommon in southern Europe but is rarely seen in the USA and the UK. The prevalence in Scandinavia is hitherto unknown. METHODS: We used an automated prototype LKM-1 immunometry-based assay (IMx) to detect LKM-1 antibodies in sera from 350 Swedish patients with chronic liver diseases (100 with primary biliary cirrhosis, 80 with primary sclerosing cholangitis, 100 with hepatitis C, and 70 patients with various forms of chronic hepatitis, including 36 autoimmune cases), and from 17 children with autoimmune hepatitis. Sera reactive in the IMx assay were subjected to immunofluorescence testing. RESULTS: No clearly LKM-reactive sera were detected. Serum samples from 29 patients were borderline reactive in the IMx assay but tested negative in the confirmatory immunofluorescence test. Positive tests in the former assay were likely caused by reactivity against microsomal antigens other than LKM-1/cytochrome P450IID6. CONCLUSIONS: LKM-1-positive type-2 autoimmune hepatitis is very rare in Sweden. Furthermore, chronic hepatitis C did not trigger this type of autoimmune reactivity in our patients, probably owing to genetic insusceptibility.

Concentration time courses of troponin and myosin subunits after acute myocardial infarction.

BACKGROUND: As a result of the limited sensitivity and specificity of creatine kinase and lactate dehydrogenase (LDH) as well as their isoenzymes, there is increasing interest in the use of cardiac contractile proteins for the diagnosis of acute myocardial infarction (AMI) and myocardial damage. METHODS: This study compared the release of creatine kinase, creatine kinase MB, myoglobin, cardiac troponin I (cTnI), cardiac troponin T (cTnT), cardiac myosin light chain-1 (cMLC-1), and beta-type myosin heavy chains (bMHC) in serial blood samples from 13 patients (10 men, three women; median age 54 years, range 40-74 years) with first-time AMI (11 Q-wave, two non-Q-wave AMI; three anterior and 10 inferior wall AMI). All but one patient received intravenous thrombolytic treatment. RESULTS: Myoglobin was the first marker to increase in blood after AMI and showed the earliest peak levels, whereas bMHC increased latest, showing the latest peak levels. cTnI and cTnT increased significantly earlier than cMLC-1 and bMHC. cTnI and cTnT increased and reached peak levels parallel to each other, but the latter tended to stay increased longer. cTnT time courses were biphasic in the majority of AMI patients, unlike cTnI time courses. cMLC-1 release was mostly biphasic. cMLC-1 allows diagnosis during the acute phase as well as several days after the onset of AMI. The time courses of bMHC were usually monophasic. Its delayed appearance makes it useful for the diagnosis of remote infarction. In contrast to cTnI and cTnT, cMLC-1 and bMHC time courses were not significantly influenced by early reperfusion. CONCLUSION: Our results demonstrate the impact of the intracellular compartmentation of an intramyocardial protein (cytosolic, structurally bound, or structurally bound with soluble pool) on its concentration time course after AMI, particularly on the rapidity of its release.

Measurement of liver microsomal cytochrome p450 (CYP2D6) activity using [O-methyl-14C]dextromethorphan.

The activity of human liver microsomal cytochrome P4502D6 (CYP2D6) is readily estimated by following the O-demethylation of [O-methyl-14C]dextromethorphan. The basis of the assay is the quantitative measurement of [14C]formaldehyde (0.05-4.0 microM) after addition of NaOH to the microsomal incubates and extraction with methylene chloride. The assay is relatively simple, sensitive (limit of detection is approximately 5.0 pmol HCHO/h/mg microsomal protein) and does not require the use of HPLC or an internal standard. Formation of radiolabeled formaldehyde in human liver microsomes is linear for 20 min, up to a final protein concentration of 1.0 mg/ml. Furthermore, the O-demethylase activity in a panel of microsomes prepared from a series of human livers was significantly correlated with the immunochemically determined levels of CYP2D6 protein (r = 0.925, p < 0.001), and was inhibited (> 89%) by quinidine and lobeline. In addition, [O-methyl-14C]-dextromethorphan O-demethylation was exclusively catalyzed by cDNA-expressed CYP2D6 in microsomes prepared from human B-lymphoblast cells. The method is suitable for rapid screening of compounds as potential CYP2D6 cosubstrates and/or inhibitors.

Precursor-specific requirements for SecA, SecB, and delta muH+ during protein export of Escherichia coli.

We compare translocation into inside-out plasma membrane vesicles (INV) of the in vitro synthesized outer membrane proteins LamB and OmpA and the periplasmic protein Skp of Escherichia coli and demonstrate a precursor-specific dependence on the export factors SecA, SecB, and the proton-motive force (delta mu H+). A partial reduction in soluble SecA caused a 50% decrease in translocation of preLamB. In contrast, removal of INV-bound SecA by urea extraction was required to see a decrease in translocation of preOmpA and preSkp, with 8% of preSkp still being translocated into urea-treated INV. Translocation of the three precursors into INV showed a corresponding differential sensitivity toward dissipation of delta mu H+ following removal of the F1-ATPase from the INV. While depletion of both F1 and SecA or simply lowering of the reaction temperature resulted in an inhibition of complete transmembrane translocation, it interfered less severely with signal sequence cleavage, indicating the formation of translocation intermediates under these conditions. The relative amounts of intermediate obtained were also different for the three preproteins correlating a low requirement for SecA and delta mu H+ with a facilitated initiation of translocation. Whereas preSkp was translocated independently of SecB, preLamB was not even targeted to the INV in its absence. Functional targeting of preOmpA required the presence of SecB during incubation of the precursor with INV and not during its synthesis. SecB, exogenously added during the period of synthesis, did not prevent the formation of translocation-incompetent preLamB. The latter results are consistent with an important targeting function of SecB, which so far has mostly been described as a molecular chaperone. The findings are discussed with respect to current models of bacterial protein export usually derived from the analysis of a single precursor.