Faecal calprotectin determination: impact of preanalytical sample treatment and stool consistency on within- and between-method variability

Introduction: We assessed the differences in faecal calprotectin (FC) concentrations measured by two assays depending on the stool consistency and extraction method. Materials and methods: Stool samples were extracted using the EliA Stool Extraction Kit, Calex® Cap extraction device and respective weighing methods, while FC concentrations were measured using the EliATM Calprotectin and Bühlmann fCAL® Turbo method and checked for within- and between-method variability with regard to extraction method and stool consistency category. Extraction yield was evaluated for impact of different sample incubation time (10 min and 1 h) in extraction buffer for both methods and for impact of different initial sample dilutions (1:50, 1:100, 1:500) for fCAL® Turbo method. Results: Results determined from Calex® Cap extracts were higher compared to weighing method extracts (mean bias 33.3%; P < 0.001), while no significant difference was found between results obtained with EliA Stool Extraction Kit and weighing method (mean bias 0.1%; P = 0.484), in both cases irrespective of stool consistency. Bühlmann fCAL® Turbo results were higher than EliATM Calprotectin results (mean bias 32.3%, P = 0.025 weighing method; and mean bias 53.9%, P < 0.001 extraction devices), the difference is dependent on stool consistency and FC concentration. Significantly higher FC extraction yield was obtained with longer sample incubation time for both methods (P = 0.019 EliATM Calprotectin; P < 0.001 fCAL® Turbo) and with increasing initial sample dilution for fCAL® Turbo method (P < 0.001). Conclusion: Preanalytical stool sample handling proved to be a crucial factor contributing to within- and between-FC assay variability. Standardization is urgently needed in order to assure comparable and reliable FC results

The freshwater Sponge Ephydatia Fluviatilis harbours diverse pseudomonas species (Gammaproteobacteria, Pseudomonadales) with broad-spectrum antimicrobial activity

Bacteria are believed to play an important role in the fitness and biochemistry of sponges (Porifera). Pseudomonas species (Gammaproteobacteria, Pseudomonadales) are capable of colonizing a broad range of eukaryotic hosts, but knowledge of their diversity and function in freshwater invertebrates is rudimentary. We assessed the diversity, structure and antimicrobial activities of Pseudomonas spp. in the freshwater sponge Ephydatia fluviatilis. Polymerase Chain Reaction - Denaturing Gradient Gel Electrophoresis (PCR-DGGE) fingerprints of the global regulator gene gacA revealed distinct structures between sponge-associated and free-living Pseudomonas communities, unveiling previously unsuspected diversity of these assemblages in freshwater. Community structures varied across E. fluviatilis specimens, yet specific gacA phylotypes could be detected by PCR-DGGE in almost all sponge individuals sampled over two consecutive years. By means of whole-genome fingerprinting, 39 distinct genotypes were found within 90 fluorescent Pseudomonas isolates retrieved from E. fluviatilis. High frequency of in vitro antibacterial (49%), antiprotozoan (35%) and anti-oomycetal (32%) activities was found among these isolates, contrasting less-pronounced basidiomycetal (17%) and ascomycetal (8%) antagonism. Culture extracts of highly predation-resistant isolates rapidly caused complete immobility or lysis of cells of the protozoan Colpoda steinii. Isolates tentatively identified as P. jessenii, P. protegens and P. oryzihabitans showed conspicuous inhibitory traits and correspondence with dominant sponge-associated phylotypes registered by cultivation-independent analysis. Our findings suggest that E. fluviatilis hosts both transient and persistent Pseudomonas symbionts displaying antimicrobial activities of potential ecological and biotechnological value.European Regional Development Fund (ERDF) through the COMPETE (Operational Competitiveness Programme); national funds through FCT (Foundation for Science and Technology) [PEst-C/MAR/LA0015/2011]; FCT-funded project [PTDC/BIA-MIC/3865/2012]; Federation of European Microbiological Societies (FEMS)info:eu-repo/semantics/publishedVersio