False-positive PCR results linked to administration of seasonal influenza vaccine

False-positive PCR results usually occur as a consequence of specimen-to-specimen or amplicon-to-specimen contamination within the laboratory. Evidence of contamination at time of specimen collection linked to influenza vaccine administration in the same location as influenza sampling is described. Clinical, circumstantial and laboratory evidence was gathered for each of five cases of influenza-like illness (ILI) with unusual patterns of PCR reactivity for seasonal H1N1, H3N2, H1N1 (2009) and influenza B viruses. Two 2010 trivalent influenza vaccines and environmental swabs of a hospital influenza vaccination room were also tested for influenza RNA. Sequencing of influenza A matrix (M) gene amplicons from the five cases and vaccines was undertaken. Four 2009 general practitioner (GP) specimens were seasonal H1N1, H3N2 and influenza B PCR positive. One 2010 GP specimen was H1N1 (2009), H3N2 and influenza B positive. PCR of 2010 trivalent vaccines showed high loads of detectable influenza A and B RNA. Sequencing of the five specimens and vaccines showed greatest homology with the M gene sequence of Influenza A/Puerto Rico/8/1934 H1N1 virus (used in generation of influenza vaccine strains). Environmental swabs had detectable influenza A and B RNA. RNA detection studies demonstrated vaccine RNA still detectable for at least 66 days. Administration of influenza vaccines and clinical sampling in the same room resulted in the contamination with vaccine strains of surveillance swabs collected from patients with ILI. Vaccine contamination should therefore be considered, particularly where multiple influenza virus RNA PCR positive signals (e.g. H1N1, H3N2 and influenza B) are detected in the same specimen.

Isolation of Alternaria alternata from an emollient cream:implications for public health

A specimen of emollient cream, which was observed to be contaminated peripherally with a filamentous fungus was examined for the presence of fungi and the resulting fungal colonies were examined phenotypically and genotypically. Subsequent DNA extraction and PCR amplification of the large internal transcribed spacer region [ITS1-5.8S-ITS2] yielded an amplicon of 512 bp. Sequence analysis identified this as Alternaria alternata at the 100% homology level with all 512/512 bases called. This organism has been previously reported as a cause of opportunistic infections involving skin and immunocompromised patients. This is the first report of an emollient cream as a source of this organism. It highlights the need for proper management of such preparations in order to minimize the potential spread of fungi to susceptible patient populations.

Inflammatory bowel disease, gut bacteria and probiotic therapy

Crohn's disease (CD) and ulcerative colitis (UC) are the two major forms of inflammatory bowel disease (IBD) and both diseases lead to high morbidity and health care costs. Complex interactions between the immune system, enteric commensal bacteria and host genotype are thought to underlie the development of IBD although the precise aetiology of this group of diseases is still unknown. The understanding of the composition and complexity of the normal gut microbiota has been greatly aided by the use of molecular methods and is likely to be further increased with the advent of metagenomics and metatranscriptomics approaches, which will allow an increasingly more holistic assessment of the microbiome with respect to both diversity and function of the commensal gut microbiota. Studies thus far have shown that the intestinal microbiota drives the development of the gut immune system and can induce immune homeostasis as well as contribute to the development of IBD. Probiotics which deliver some of the beneficial immunomodulatory effects of the commensal gut microbiota and induce immune homeostasis have been proposed as a suitable treatment for mild to moderate IBD. This review provides an overview over the current understanding of the commensal gut microbiota, its interactions with the mucosal immune system and its capacity to induce both gut homeostasis as well as dysregulation of the immune system. Bacterial-host events, including interactions with pattern recognition receptors (PRRs) expressed on epithelial cells and dendritic cells (DCs) and the resultant impact on immune responses at mucosal surfaces will be discussed. (C) 2009 Elsevier GmbH. All rights reserved.

The Interlaboratory Robustness Of Next-Generation Sequencing (IRON) Study Phase II: Deep-Sequencing Analyses Of Hematological Malignancies Performed In 8,867 Cases By An International Network Involving 27 Laboratories

Introduction: Amplicon deep-sequencing using second-generation sequencing technology is an innovative molecular diagnostic technique and enables a highly-sensitive detection of mutations. As an international consortium we had investigated previously the robustness, precision, and reproducibility of 454 amplicon next-generation sequencing (NGS) across 10 laboratories from 8 countries (Leukemia, 2011;25:1840-8).

Aims: In Phase II of the study, we established distinct working groups for various hematological malignancies, i.e. acute myeloid leukemia (AML), acute lymphoblastic leukemia (ALL), chronic lymphocytic leukemia (CLL), chronic myelogenous leukemia (CML), myelodysplastic syndromes (MDS), myeloproliferative neoplasms (MPN), and multiple myeloma. Currently, 27 laboratories from 13 countries are part of this research consortium. In total, 74 gene targets were selected by the working groups and amplicons were developed for a NGS deep-sequencing assay (454 Life Sciences, Branford, CT). A data analysis pipeline was developed to standardize mutation interpretation both for accessing raw data (Roche Amplicon Variant Analyzer, 454 Life Sciences) and variant interpretation (Sequence Pilot, JSI Medical Systems, Kippenheim, Germany).

Results: We will report on the design, standardization, quality control aspects, landscape of mutations, as well as the prognostic and predictive utility of this assay in a cohort of 8,867 cases. Overall, 1,146 primer sequences were designed and tested. In detail, for example in AML, 924 cases had been screened for CEBPA mutations. RUNX1 mutations were analyzed in 1,888 cases applying the deep-sequencing read counts to study the stability of such mutations at relapse and their utility as a biomarker to detect residual disease. Analyses of DNMT3A (n=1,041) were focused to perform landscape investigations and to address the prognostic relevance. Additionally, this working group is focusing on TET2, ASXL1, and TP53 analyses. A novel prognostic model is being developed allowing stratification of AML into prognostic subgroups based on molecular markers only. In ALL, 1,124 pediatric and adult cases have been screened, including 763 assays for TP53 mutations both at diagnosis and relapse of ALL. Pediatric and adult leukemia expert labs developed additional content to study the mutation incidence of other B and T lineage markers such as IKZF1, JAK2, IL7R, PAX5, EP300, LEF1, CRLF2, PHF6, WT1, JAK1, PTEN, AKT1, IL7R, NOTCH1, CREBBP, or FBXW7. Further, the molecular landscape of CLL is changing rapidly. As such, a separate working group focused on analyses including NOTCH1, SF3B1, MYD88, XPO1, FBXW7 and BIRC3. Currently, 922 cases were screened to investigate the range of mutational burden of NOTCH1 mutations for their prognostic relevance. In MDS, RUNX1 mutation analyses were performed in 977 cases. The prognostic relevance of TP53 mutations in MDS was assessed in additional 327 cases, including isolated deletions of chromosome 5q. Next, content was developed targeting genes of the cellular splicing component, e.g. SF3B1, SRSF2, U2AF1, and ZRSR2. In BCR-ABL1-negative MPN, nine genes of interest (JAK2, MPL, TET2, CBL, KRAS, EZH2, IDH1, IDH2, ASXL1) have been analyzed in a cohort of 155 primary myelofibrosis cases searching for novel somatic mutations and addressing their relevance for disease progression and leukemia transformation. Moreover, an assay was developed and applied to CMML cases allowing the simultaneous analysis of 25 leukemia-associated target genes in a single sequencing run using just 20 ng of starting DNA. Finally, nine laboratories are studying CML, applying ultra-deep sequencing of the BCR-ABL1 tyrosine kinase domain. Analyses were performed on 615 cases investigating the dynamics of expansion of mutated clones under various tyrosine kinase inhibitor therapies.

Conclusion: Molecular characterization of hematological malignancies today requires high diagnostic sensitivity and specificity. As part of the IRON-II study, a network of laboratories analyzed a variety of disease entities applying amplicon-based NGS assays. Importantly, the consortium not only standardized assay design for disease-specific panels, but also achieved consensus on a common data analysis pipeline for mutation interpretation. Distinct working groups have been forged to address scientific tasks and in total 8,867 cases had been analyzed thus far.

Multiplexing with three-primer PCR for rapid and economical microsatellite validation.

The next generation sequencing revolution has enabled rapid discovery of genetic markers, however, development of fully functioning new markers still requires a long and costly process of marker validation. This study reports a rapid and economical approach for the validation and deployment of polymorphic microsatellite markers obtained from a 454 pyrosequencing library of Atlantic cod, Gadus morhua, Linnaeus 1758. Primers were designed from raw reads to amplify specific amplicon size ranges, allowing effective PCR multiplexing. Multiplexing was combined with a three-primer PCR approach using four universal tails to label amplicons with separate fluorochromes. A total of 192 primer pairs were tested, resulting in 73 polymorphic markers. Of these, 55 loci were combined in six multiplex panels each containing between six and eleven markers. Variability of the loci was assessed on G. morhua from the Celtic Sea (n 46) and the Scotian Shelf (n 46), two locations that have shown genetic differentiation in previous studies. Multilocus FST between the two samples was estimated at 0.067 (P 0.001). After three loci potentially under selection were excluded, the global FST was estimated at 0.043 (P 0.001). Our technique combines three- primer and multiplex PCR techniques, allowing simultaneous screening and validation of relatively large numbers of microsatellite loci.

A primer for use of genetic tools in selecting and testing the suitability of set-aside sites protected from deep-sea seafloor massive sulfide mining activities

Seafloor massive sulfide (SMS) mining will likely occur at hydrothermal systems in the near future. Alongside their mineral wealth, SMS deposits also have considerable biological value. Active SMS deposits host endemic hydrothermal vent communities, whilst inactive deposits support communities of deep water corals and other suspension feeders. Mining activities are expected to remove all large organisms and suitable habitat in the immediate area, making vent endemic organisms particularly at risk from habitat loss and localised extinction. As part of environmental management strategies designed to mitigate the effects of mining, areas of seabed need to be protected to preserve biodiversity that is lost at the mine site and to preserve communities that support connectivity among populations of vent animals in the surrounding region. These "set-aside" areas need to be biologically similar to the mine site and be suitably connected, mostly by transport of larvae, to neighbouring sites to ensure exchange of genetic material among remaining populations. Establishing suitable set-asides can be a formidable task for environmental managers, however the application of genetic approaches can aid set-aside identification, suitability assessment and monitoring. There are many genetic tools available, including analysis of mitochondrial DNA (mtDNA) sequences (e.g. COI or other suitable mtDNA genes) and appropriate nuclear DNA markers (e.g. microsatellites, single nucleotide polymorphisms), environmental DNA (eDNA) techniques and microbial metagenomics. When used in concert with traditional biological survey techniques, these tools can help to identify species, assess the genetic connectivity among populations and assess the diversity of communities. How these techniques can be applied to set-aside decision making is discussed and recommendations are made for the genetic characteristics of set-aside sites. A checklist for environmental regulators forms a guide to aid decision making on the suitability of set-aside design and assessment using genetic tools. This non-technical primer document represents the views of participants in the VentBase 2014 workshop.

A novel method of microsatellite genotyping-by-sequencing using individual combinatorial barcoding

This study examines the potential of next-generation sequencing based ‘genotyping-by-sequencing’ (GBS) of microsatellite loci for rapid and cost-effective genotyping in large-scale population genetic studies. The recovery of individual genotypes from large sequence pools was achieved by PCR-incorporated combinatorial barcoding using universal primers. Three experimental conditions were employed to explore the possibility of using this approach with existing and novel multiplex marker panels and weighted amplicon mixture. The GBS approach was validated against microsatellite data generated by capillary electrophoresis. GBS allows access to the underlying nucleotide sequences that can reveal homoplasy, even in large datasets and facilitates cross laboratory transfer. GBS of microsatellites, using individual combinatorial barcoding, is potentially faster and cheaper than current microsatellite approaches and offers better and more data.

A rapid staining-assisted wood sampling method for PCR-based detection of pine wood nematode Bursaphelenchus xylophilus in Pinus massoniana wood tissue

For reasons of unequal distribution of more than one nematode species in wood, and limited availability of wood samples required for the PCR-based method for detecting pinewood nematodes in wood tissue of Pinus massoniana, a rapid staining-assisted wood sampling method aiding PCR-based detection of the pine wood nematode Bursaphelenchus xylophilus (Bx) in small wood samples of P. massoniana was developed in this study. This comprised a series of new techniques: sampling, mass estimations of nematodes using staining techniques, and lowest limit Bx nematode mass determination for PCR detection. The procedure was undertaken on three adjoining 5-mg wood cross-sections, of 0.5 · 0.5 · 0.015 cm dimension, that were cut from a wood sample of 0.5 · 0.5 · 0.5 cm initially, then the larger wood sample was stained by acid fuchsin, from which two 5-mg wood cross-sections (that adjoined the three 5-mg wood cross-sections, mentioned above) were cut. Nematode-staining-spots (NSSs) in each of the two stained sections were counted under a microscope at 100· magnification. If there were eight or more NSSs present, the adjoining three sections were used for PCR assays. The B. xylophilus – specific amplicon of 403 bp (DQ855275) was generated by PCR assay from 100.00% of 5-mg wood cross-sections that contained more than eight Bx NSSs by the PCR assay. The entire sampling procedure took only 10 min indicating that it is suitable for the fast estimation of nematode numbers in the wood of P. massonina as the prelimary sample selections for other more expensive Bx-detection methods such as PCR assay.

Ecological and biotechnological potential of sponge microbiome

Marine sponges harbor microbial communities of immense ecological and biotechnological importance. Recently, they have been focus of heightened attention due to the wide range of biologically active compounds with potential application, particularly, in chemical, cosmetic and pharmaceutical industries. However, we still lack fundamental knowledge of their microbial ecology and biotechnological potential. The development of high-throughput sequencing technologies has given rise to a new range of tools that can help us explore the biotechnological potential of sponges with incredible detail. Metagenomics, in particular, has the power to revolutionize the production of bioactive compounds produced by unculturable microorganisms. It can offer the identification of biosynthetic genes or gene clusters that can be heterologously expressed on a cultivable and suitable host. This review focus on the exploration of the biotechnological potential of sponge-associated microorganisms, and integration of molecular approaches, whose increasing efficiency can play an essential role on achieving a sustainable source of natural products.

Metagenomic systems biology: frameworks for modeling and characterizing the gut microbiome

Thesis (Ph.D.)--University of Washington, 2014

La régulation génique chez Solanum chacoense : de la pollinisation jusqu’à l’embryogenèse

Chez les végétaux supérieurs, l’embryogenèse est une phase clé du développement au cours de laquelle l’embryon établit les principales structures qui formeront la future plante et synthétise et accumule des réserves définissant le rendement et la qualité nutritionnelle des graines. Ainsi, la compréhension des évènements moléculaires et physiologiques menant à la formation de la graine représente un intérêt agronomique majeur. Toutefois, l'analyse des premiers stades de développement est souvent difficile parce que l'embryon est petit et intégré à l'intérieur du tissu maternel. Solanum chacoense qui présente des fleurs relativement grande facilitant l’isolation des ovules, a été utilisée pour l’étude de la biologie de la reproduction plus précisément la formation des gamètes femelles, la pollinisation, la fécondation et le développement des embryons. Afin d'analyser le programme transcriptionnel induit au cours de la structuration de ces étapes de la reproduction sexuée, nous avons mis à profit un projet de séquençage de 7741 ESTs (6700 unigènes) exprimés dans l’ovule à différents stades du développement embryonnaire. L’ADN de ces ESTs a été utilisé pour la fabrication de biopuces d’ADN. Dans un premier temps, ces biopuces ont été utilisé pour comparer des ADNc issus des ovules de chaque stade de développement embryonnaire (depuis le zygote jusqu’au embryon mature) versus un ovule non fécondé. Trois profils d’expression correspondant au stade précoce, intermédiaire et tardive ont été trouvés. Une analyse plus approfondie entre chaque point étudié (de 0 à 22 jours après pollinisation), a permis d'identifier des gènes spécifiques caractérisant des phases de transition spécifiques. Les annotations Fonctionnelles des gènes differentiellement exprimés nous ont permis d'identifier les principales fonctions cellulaires impliquées à chaque stade de développement, révélant que les embryons sont engagés dans des actifs processus de différenciation. Ces biopuces d’ADN ont été par la suite utilisé pour comparer différent types de pollinisation (compatible, incompatible, semi-compatible et inter-espèce) afin d’identifier les gènes répondants à plusieurs stimuli avant l'arrivé du tube pollinique aux ovules (activation à distance). Nous avons pu démontrer que le signal perçu par l’ovaire était différent et dépend de plusieurs facteurs, incluant le type de pollen et la distance parcourue par le pollen dans le style. Une autre analyse permettant la comparaison des différentes pollinisations et la blessure du style nous a permis d’identifier que les programmes génétiques de la pollinisation chevauchent en partie avec ceux du stress. Cela était confirmé en traitant les fleurs par une hormone de stress, méthyle jasmonate. Dans le dernier chapitre, nous avons utilisé ces biopuces pour étudier le changement transcriptionnel d’un mutant sur exprimant une protéine kinase FRK2 impliqué dans l’identité des ovules. Nous avons pu sélectionner plusieurs gènes candidat touchés par la surexpression de cette kinase pour mieux comprendre la voie se signalisation. Ces biopuces ont ainsi servi à déterminer la variation au niveau transcriptionnelle des gènes impliqués lors de différents stades de la reproduction sexuée chez les plantes et nous a permis de mieux comprendre ces étapes.

Identification of a novel herpesvirus associated with cutaneous ulcers in a fisher (Martes pennanti).

On December 8, 2008, a male fisher (Martes pennanti) housed in a quarantine enclosure at the St-Félicien Zoo was found dead with multiple skin ulcers on the muzzle and plantar pads. At necropsy, no major findings were found, and a specific cause of death was not determined microscopically. However, at the borders of ulcerated sites, there were increased numbers of koilocytes, with perinuclear vacuolation and nuclear enlargement. A pan-herpesvirus nested polymerase chain reaction (PCR) assay was conducted, and an expected PCR product of 230 nucleotides was obtained within tissues collected from around the skin ulcers. Other tissues, including intestines and pool of lung, liver, and kidney, tested negative. The obtained PCR amplicon was sequenced and was highly related to the partial viral DNA polymerase (DPOL) gene of Mustelid herpesvirus 1. Virus isolation was negative, and no virion was detected by electron microscopy. The pathogenic potential of this novel herpesvirus and its role in the death of the fisher are unknown.

Sexage et phylogénie, à partir des gènes CHD (-Z et -W) et COX-1, des oiseaux de proie du Québec et de perroquets d’attrait vétérinaire

Connaître le sexe d’un oiseau est important pour divers domaines notamment pour les vétérinaires, les écologistes ainsi que pour les éleveurs d’oiseaux qui veulent former des couples qui serviront à la reproduction. Plusieurs espèces d’oiseaux, juvéniles et adultes, n’ont pas de dimorphisme sexuel. L’utilisation de l’ADN est une façon rapide de déterminer le sexe à partir d’un échantillon de sang, de muscle, de plumes ou de fèces. Par contre, la méthode devrait être validée pour chaque espèce et idéalement, standardisée. Le premier objectif de cette étude est de développer une méthode de sexage par séquençage des oiseaux à partir des séquences du gène CHD, en utilisant les oiseaux de proie et les perroquets vus en clinique au Québec. Un deuxième objectif est de faire l’identification de l’espèce à sexer, à partir du gène mitochondrial COX-1 et aussi à partir des séquences CHD-Z et CHD-W, utilisés pour le sexage. Un troisième objectif est d’évaluer les séquences sorties (CHD-Z, CHD-W et COX-1) en vue d’une étude phylogénique. Une extraction d’ADN a été effectuée chez 27 espèces de perroquets, 34 espèces d’oiseaux de proie, une corneille (Corvus brachyrhynchos) et un poulet (Gallus gallus). Une amplification par PCR a été exécutée pour les exons partiels 23 et 24 du gène CHD. Le séquençage de cet amplicon permettait de savoir s’il s’agissait d’un mâle (séquence simple CHD-Z) ou d’une femelle (séquences CHD-Z et CHD-W qui se chevauchent). Afin d’avoir des séquences CHD-W distinctes, un sous-clonage a été fait chez les femelles de chaque espèce. De cette manière, les séquences partielles du gène CHD, Z et W, ont été trouvées pour les espèces échantillonnées. Une étude phylogénique a été effectuée avec les séquences de COX-1, CHD-Z et CHD-W grâce au site « Clustal-Omega ». La méthode de sexage des oiseaux par séquençage du gène CHD est standard et efficace. Le gène COX-1 permet une meilleure identification des espèces parentes et le gène CHD-Z est le plus utile pour étudier la phylogénie profonde.

Biofilm Forming Multi Drug Resistant Staphylococcus spp. Among Patients with Conjunctivitis

Biofilm forming multidrug resistant Staphylococcus spp. are major reservoirs for transmission of ophthalmic infections. They were isolated from ocular patients suffering from conjunctivitis. In this study we analyzed biofilm forming ability, antibiotic resistance profile of the Staphylococcus spp. isolated from clinical ocular patients, and their phylogenetic relationship with other community MRSA. Sixty Staphylococcus spp. strains isolated from clinical subjects were evaluated for their ability to form biofilm and express biofilm encoding ica gene. Among them 93% were slime producers and 87% were slime positive. Staphylococcus aureus and S. epidermidis were dominant strains among the isolates obtained from ocular patients. The strains also exhibited a differential biofilm formation quantitatively. Antibiotic susceptibility of the strains tested with Penicillin G, Ciprofloxacin, Ofloxacin, Methicillin, Amikacin, and Gentamicin indicated that they were resistant to more than one antibiotic. The amplicon of ica gene of strong biofilm producing S. aureus strains, obtained by polymerase chain reaction, was sequenced and their close genetic relationship with community acquired MRSA was analyzed based on phylogenetic tree. Our results indicate that they are genetically close to other community acquired MRSA