An external quality assessment feasibility study; cross laboratory comparison of haemagglutination inhibition assay and microneutralization assay performance for seasonal influenza serology testing: A FLUCOP study

Introduction: External Quality Assessment (EQA) schemes are designed to provide a snapshot of laboratory proficiency, identifying issues and providing feedback to improve laboratory performance and inter-laboratory agreement in testing. Currently there are no international EQA schemes for seasonal influenza serology testing. Here we present a feasibility study for conducting an EQA scheme for influenza serology methods. Methods: We invited participant laboratories from industry, contract research organizations (CROs), academia and public health institutions who regularly conduct hemagglutination inhibition (HAI) and microneutralization (MN) assays and have an interest in serology standardization. In total 16 laboratories returned data including 19 data sets for HAI assays and 9 data sets for MN assays. Results: Within run analysis demonstrated good laboratory performance for HAI, with intrinsically higher levels of intra-assay variation for MN assays. Between run analysis showed laboratory and strain specific issues, particularly with B strains for HAI, whilst MN testing was consistently good across labs and strains. Inter-laboratory variability was higher for MN assays than HAI, however both assays showed a significant reduction in inter-laboratory variation when a human sera pool is used as a standard for normalization. Discussion: This study has received positive feedback from participants, highlighting the benefit such an EQA scheme would have on improving laboratory performance, reducing inter laboratory variation and raising awareness of both harmonized protocol use and the benefit of biological standards for seasonal influenza serology testing.publishedVersio

Genetic analysis of plasmid-encoded mcr-1 resistance in Enterobacteriaceae derived from poultry meat in the Netherlands

BACKGROUND: Colistin is classified as the highest priority and critically important antimicrobial for human medicine by WHO as it is the last resort agent for treatment of carbapenem-resistant Enterobacteriaceae in humans. Additional research is necessary to elucidate the genetic structure of mcr-1 resistance genes, commonly found on plasmids, using WGS. OBJECTIVES: To map and compare the genetic characteristics of 35 mcr-1-mediated colistin-resistant Enterobacteriaceae isolated from chicken meat to highlight the genetic variation of the mcr-1-containing plasmids. METHODS: Sequencing was performed using Illumina HiSeq2500, Novaseq6000 and ONT’s GridION. GridION data was locally basecalled and demultiplexed using ONT’s Albacore 2.3.4 followed by Porechop 2.3. Quality filtering was performed using Filtlong 2.0. Hybrid Assembly was performed using Unicycler 4.7. Plasmids were compared with reference sequences in plasmid-RefSeq and pATLAS. RESULTS: A total of 35 mcr-1 positive Enterobacteriaceae were investigated, which resulted in 34 qualitatively robust hybrid assemblies of 2 Klebsiella pneumoniae and 32 Escherichia coli. mcr-1.1 was present in 33/34 isolates. One isolate contained an mcr-1.1-like resistance gene, due to a deletion of one codon. Two mcr-1.1 genes were located on the chromosome, while the majority of the mcr-1 genes were found on IncX4 type plasmids (n = 19). Almost all plasmids identified in this study were highly similar to plasmids found in human-derived strains. CONCLUSIONS: The mcr-1.1-containing plasmids from retail chicken show high sequence similarity to human mcr-1.1 plasmids, suggesting that this may be a contributor to the presence of colistin resistance in humans

Development and international validation trial of an advanced, multi-locus DNA metabarcoding metho to identify endangered species in complex samples

Background: DNA metabarcoding, which involves Next-Generation Sequencing (NGS) of DNA barcodes, holds great promise for species identification in complex samples such as food supplements and Traditional Medicines (TMs). Such method would aid CITES (the Convention on International Trade in Endangered Species of Wild Fauna and Flora) enforcement officers to combat wildlife crime by preventing illegal trade of endangered plant and animal species. The objective of this research was to develop a multi-locus DNA metabarcoding method for wildlife forensic species identification and to evaluate the applicability and reproducibility of the this approach across different laboratories. Results: The DNA metabarcoding method developed in this study makes use of 12 DNA barcode markers that have demonstrated universal applicability across a wide range of plant and animal taxa, and that facilitate the identification of plant and animal species in highly processed samples containing degraded DNA. The DNA metabarcoding method was developed on the basis of NGS data generated for 15 well-defined experimental mixtures using Illumina MiSeq technology, for which a bioinformatics pipeline with user-friendly web interface was developed. The performance of the DNA metabarcoding method was assessed in an international validation trial by 16 laboratories, in which the method was found to be highly reproducible and sensitive enough to identify species present in a mixture at 1% dry weight content. Conclusion: The advanced, multi-locus DNA metabarcoding method assessed in this study provides reliable and detailed data on the composition of complex food products, including information on the presence of CITES species. The method provides improved resolution for species identification, while verifying species with multiple DNA barcodes contributes to enhanced quality assurance

Development and international validation trial of an advanced, multi-locus DNA metabarcoding metho to identify endangered species in complex samples

Background: DNA metabarcoding, which involves Next-Generation Sequencing (NGS) of DNA barcodes, holds great promise for species identification in complex samples such as food supplements and Traditional Medicines (TMs). Such method would aid CITES (the Convention on International Trade in Endangered Species of Wild Fauna and Flora) enforcement officers to combat wildlife crime by preventing illegal trade of endangered plant and animal species. The objective of this research was to develop a multi-locus DNA metabarcoding method for wildlife forensic species identification and to evaluate the applicability and reproducibility of the this approach across different laboratories. Results: The DNA metabarcoding method developed in this study makes use of 12 DNA barcode markers that have demonstrated universal applicability across a wide range of plant and animal taxa, and that facilitate the identification of plant and animal species in highly processed samples containing degraded DNA. The DNA metabarcoding method was developed on the basis of NGS data generated for 15 well-defined experimental mixtures using Illumina MiSeq technology, for which a bioinformatics pipeline with user-friendly web interface was developed. The performance of the DNA metabarcoding method was assessed in an international validation trial by 16 laboratories, in which the method was found to be highly reproducible and sensitive enough to identify species present in a mixture at 1% dry weight content. Conclusion: The advanced, multi-locus DNA metabarcoding method assessed in this study provides reliable and detailed data on the composition of complex food products, including information on the presence of CITES species. The method provides improved resolution for species identification, while verifying species with multiple DNA barcodes contributes to enhanced quality assurance

Development and international validation trial of an advanced, multi-locus DNA metabarcoding metho to identify endangered species in complex samples

Background: DNA metabarcoding, which involves Next-Generation Sequencing (NGS) of DNA barcodes, holds great promise for species identification in complex samples such as food supplements and Traditional Medicines (TMs). Such method would aid CITES (the Convention on International Trade in Endangered Species of Wild Fauna and Flora) enforcement officers to combat wildlife crime by preventing illegal trade of endangered plant and animal species. The objective of this research was to develop a multi-locus DNA metabarcoding method for wildlife forensic species identification and to evaluate the applicability and reproducibility of the this approach across different laboratories. Results: The DNA metabarcoding method developed in this study makes use of 12 DNA barcode markers that have demonstrated universal applicability across a wide range of plant and animal taxa, and that facilitate the identification of plant and animal species in highly processed samples containing degraded DNA. The DNA metabarcoding method was developed on the basis of NGS data generated for 15 well-defined experimental mixtures using Illumina MiSeq technology, for which a bioinformatics pipeline with user-friendly web interface was developed. The performance of the DNA metabarcoding method was assessed in an international validation trial by 16 laboratories, in which the method was found to be highly reproducible and sensitive enough to identify species present in a mixture at 1% dry weight content. Conclusion: The advanced, multi-locus DNA metabarcoding method assessed in this study provides reliable and detailed data on the composition of complex food products, including information on the presence of CITES species. The method provides improved resolution for species identification, while verifying species with multiple DNA barcodes contributes to enhanced quality assurance

Lipid exchanges drove the evolution of mutualism during plant terrestrialization

Symbiosis with arbuscular mycorrhizal fungi (AMF) improves plant nutrition in most land plants, and its contribution to the colonization of land by plants has been hypothesized. Here, we identify a conserved transcriptomic response to AMF among land plants, including the activation of lipid metabolism. Using gain of function, we show the transfer of lipids from the liverwort Marchantia paleacea to AMF and its direct regulation by the transcription factor WRINKLED (WRI). Arbuscules, the nutrient-exchange structures, were not formed in loss-of-function wri mutants in M. paleacea, leading to aborted mutualism. Our results show the orthology of the symbiotic transfer of lipids across land plants and demonstrate that mutualism with arbuscular mycorrhizal fungi was present in the most recent ancestor of land plants 450 million years ago

Lipid exchanges drove the evolution of mutualism during plant terrestrialization

Symbiosis with arbuscular mycorrhizal fungi (AMF) improves plant nutrition in most land plants, and its contribution to the colonization of land by plants has been hypothesized. Here, we identify a conserved transcriptomic response to AMF among land plants, including the activation of lipid metabolism. Using gain of function, we show the transfer of lipids from the liverwort Marchantia paleacea to AMF and its direct regulation by the transcription factor WRINKLED (WRI). Arbuscules, the nutrient-exchange structures, were not formed in loss-of-function wri mutants in M. paleacea, leading to aborted mutualism. Our results show the orthology of the symbiotic transfer of lipids across land plants and demonstrate that mutualism with arbuscular mycorrhizal fungi was present in the most recent ancestor of land plants 450 million years ago

Development and validation of a multi-locus DNA metabarcoding method to identify endangered species in complex samples

DNA metabarcoding provides great potential for species identification in complex samples such as food supplements and traditional medicines. Such a method would aid Convention on International Trade in Endangered Species of Wild Fauna and Flora (CITES) enforcement officers to combat wildlife crime by preventing illegal trade of endangered plant and animal species. The objective of this research was to develop a multi-locus DNA metabarcoding method for forensic wildlife species identification and to evaluate the applicability and reproducibility of this approach across different laboratories. A DNA metabarcoding method was developed that makes use of 12 DNA barcode markers that have demonstrated universal applicability across a wide range of plant and animal taxa and that facilitate the identification of species in samples containing degraded DNA. The DNA metabarcoding method was developed based on Illumina MiSeq amplicon sequencing of well-defined experimental mixtures, for which a bioinformatics pipeline with user-friendly web-interface was developed. The performance of the DNA metabarcoding method was assessed in an international validation trial by 16 laboratories, in which the method was found to be highly reproducible and sensitive enough to identify species present in a mixture at 1% dry weight content. The advanced multi-locus DNA metabarcoding method assessed in this study provides reliable and detailed data on the composition of complex food products, including information on the presence of CITES-listed species. The method can provide improved resolution for species identification, while verifying species with multiple DNA barcodes contributes to an enhanced quality assurance