Biological and technical variables affecting immunoassay recovery of cytokines from human serum and simulated vaginal fluid: A multicenter study

The increase of proinflammatory cytokines in vaginal secretions may serve as a surrogate marker of unwanted inflammatory reaction to microbicide products topically applied for the prevention of sexually transmitted diseases, including HIV-1. Interleukin (IL)-1β and IL-6 have been proposed as indicators of inflammation and increased risk of HIV-1 transmission; however, the lack of information regarding detection platforms optimal for vaginal fluids and interlaboratory variation limit their use for microbicide evaluation and other clinical applications. This study examines fluid matrix variants relevant to vaginal sampling techniques and proposes a model for interlaboratory comparisons across current cytokine detection technologies. IL-1β and IL-6 standards were measured by 12 laboratories in four countries, using 14 immunoassays and four detection platforms based on absorbance, chemiluminescence, electrochemiluminescence, and fluorescence. International reference preparations of cytokines with defined biological activity were spiked into (1) a defined medium simulating the composition of human vaginal fluid at pH 4.5 and 7.2, (2) physiologic salt solutions (phosphate-buffered saline and saline) commonly used for vaginal lavage sampling in clinical studies of cytokines, and (3) human blood serum. Assays were assessed for reproducibility, linearity, accuracy, and significantly detectable fold difference in cytokine level. Factors with significant impact on cytokine recovery were determined by Kruskal−Wallis analysis of variance with Dunn’s multiple comparison test and multiple regression models. All assays showed acceptable intra-assay reproducibility; however, most were associated with significant interlaboratory variation. The smallest reliably detectable cytokine differences (P < 0.05) derived from pooled interlaboratory data varied from 1.5- to 26-fold depending on assay, cytokine, and matrix type. IL-6 but not IL-1β determinations were lower in both saline and phosphate-buffered saline as compared to vaginal fluid matrix, with no significant effect of pH. The (electro)chemiluminescence-based assays were most discriminative and consistently detected <2-fold differences within each matrix type. The Luminex-based assays were less discriminative with lower reproducibility between laboratories. These results suggest the need for uniform vaginal sampling techniques and a better understanding of immunoassay platform differences and cross-validation before the biological significance of cytokine variations can be validated in clinical trials. This investigation provides the first standardized analytic approach for assessing differences in mucosal cytokine levels and may improve strategies for monitoring immune responses at the vaginal mucosal interface

Follow-up mission in the framework of GRIPAVI "FSP Ecologie et épidémiologie de la grippe aviaire dans les pays du Sud" : Ethiopia, 2-5 June 2009

La présente mission a été effectuée dans le cadre du projet "Ecologie et épidémiologie de la grippe aviaire dans les pays du Sud" visant à étudier les dynamiques des pestes aviaires - influenza aviaire hautement pathogène et maladie de Newcastle - et à renforcer les méthodes de surveillance et de contrôle de plusieurs pays africains. L'Ethiopie, bien qu'indemne de grippe aviaire hautement pathogène due au virus H5N1, est un haut lieu stratégique de surveillance de cette maladie du fait de la présence de nombreux oiseaux migrateurs dans les lacs de la Vallée du rift. L'aviculture villageoise en Ethiopie joue un rôle essentiel dans la réduction de la pauvreté et dans le maintient de la sécurité alimentaire des familles rurales. Ainsi l'introduction et/ou la diffusion de maladies aviaires hautement pathogènes dans un pays comme l'Ethiopie peuvent avoir des conséquences dévastatrices non seulement sur l'industrie de la volaille mais également dans le domaine de la santé publique. Cette mission avait pour objectif premier de clarifier la situation au près du NADHIC concernant la Thèse d'Université du Dr Hassen Chaka qui a débuté au 1er Janvier 2009 mais qui a pris du retard dans sa mise en route. Plusieurs réunions individuelles avec le Dr Mesfin (le directeur du NAHDIC), Dr Melesse (coordinateur du projet), Dr Chaka et E. Vallée (étudiante du Master SEAPS en Stage) ont permis au Dr Monicat (CIRAD) de prendre contact avec nos collaborateurs éthiopiens, de rencontrer le thésard et de se rendre compte de la situation de terrain. La troisième convention, pour les analyses de laboratoire, a été discutée au cours de la mission. Il a été décidé que le NAHDIC effectuait 300 PCR et 50 isolements viraux pour l'année 2009 à partir des prélèvements qui seront effectués au cours de la thèse de H. Chaka, et qu'un fond de 9000 Euros seront mis à disposition du labo en cas de suspicion (AI ou maladie de Newcastle) dans la zone d'étude du projet. (Résumé d'auteur

Enabling community-based metrology for wood-degrading fungi

Background: Lignocellulosic biomass could support a greatly-expanded bioeconomy. Current strategies for using biomass typically rely on single-cell organisms and extensive ancillary equipment to produce precursors for downstream manufacturing processes. Alternative forms of bioproduction based on solid-state fermentation and wood-degrading fungi could enable more direct means of manufacture. However, basic methods for cultivating wood-degrading fungi are often ad hoc and not readily reproducible. Here, we developed standard reference strains, substrates, measurements, and methods sufficient to begin to enable reliable reuse of mycological materials and products in simple laboratory settings. Results: We show that a widely-available and globally-regularized consumer product (Pringles™) can support the growth of wood-degrading fungi, and that growth on Pringles™-broth can be correlated with growth on media made from a fully-traceable and compositionally characterized substrate (National Institute of Standards and Technology Reference Material 8492 Eastern Cottonwood Whole Biomass Feedstock). We also establish a Relative Extension Unit (REU) framework that is designed to reduce variation in quantification of radial growth measurements. So enabled, we demonstrate that five laboratories were able to compare measurements of wood-fungus performance via a simple radial extension growth rate assay, and that our REU-based approach reduced variation in reported measurements by up to ~ 75%. Conclusions: Reliable reuse of materials, measures, and methods is necessary to enable distributed bioproduction processes that can be adopted at all scales, from local to industrial. Our community-based measurement methods incentivize practitioners to coordinate the reuse of standard materials, methods, strains, and to share information supporting work with wood-degrading fungi

20th annual meeting of the WEFTA Working Group on Analytical Methods in Fish and Fishery Products and 3rd annual meeting of the WEFTA Working Group on Microbiology. A retrospect on 20 years

After 20 annual meetings it is worth to have a look back and to see how it has started. There has been very little collaboration on research projects between member institutes under the auspices of WEFTA, co-operation in more neutral areas of common interest was developed at an early stage. The area which has proved very fruitful is methodology. It was agreed that probably the best way to make progress was to arrange meetings at each laboratory in turn where experienced, practising scientists could describe in detail how they carried out analyses. In this way, difficulties could be demonstrated or uncovered, and the accuracy, precision, efficiency and cost of the methods used in different laboratories could be compared