Assessing the accuracy of quantitative molecular microbial profiling

This is the final version of the article. Available from MDPI via the DOI in this record.The application of high-throughput sequencing in profiling microbial communities is providing an unprecedented ability to investigate microbiomes. Such studies typically apply one of two methods: amplicon sequencing using PCR to target a conserved orthologous sequence (typically the 16S ribosomal RNA gene) or whole (meta)genome sequencing (WGS). Both methods have been used to catalog the microbial taxa present in a sample and quantify their respective abundances. However, a comparison of the inherent precision or bias of the different sequencing approaches has not been performed. We previously developed a metagenomic control material (MCM) to investigate error when performing different sequencing strategies. Amplicon sequencing using four different primer strategies and two 16S rRNA regions was examined (Roche 454 Junior) and compared to WGS (Illumina HiSeq). All sequencing methods generally performed comparably and in good agreement with organism specific digital PCR (dPCR); WGS notably demonstrated very high precision. Where discrepancies between relative abundances occurred they tended to differ by less than twofold. Our findings suggest that when alternative sequencing approaches are used for microbial molecular profiling they can perform with good reproducibility, but care should be taken when comparing small differences between distinct methods. This work provides a foundation for future work comparing relative differences between samples and the impact of extraction methods. We also highlight the value of control materials when conducting microbial profiling studies to benchmark methods and set appropriate thresholds.The authors acknowledge funding from the European Metrology Research Programme joint research project “INFECT MET” (http://infectmet.lgcgroup.com) (an EMRP project, jointly funded by the EMRP participating countries within EURAMET and the European Union) and the UK National Measurement System for funding of this work and for the support of Thomas Laver by the BBSRC Industrial Case Studentship award BB/H016120/1

Discordant bioinformatic predictions of antimicrobial resistance from whole-genome sequencing data of bacterial isolates: an inter-laboratory study.

Antimicrobial resistance (AMR) poses a threat to public health. Clinical microbiology laboratories typically rely on culturing bacteria for antimicrobial-susceptibility testing (AST). As the implementation costs and technical barriers fall, whole-genome sequencing (WGS) has emerged as a 'one-stop' test for epidemiological and predictive AST results. Few published comparisons exist for the myriad analytical pipelines used for predicting AMR. To address this, we performed an inter-laboratory study providing sets of participating researchers with identical short-read WGS data from clinical isolates, allowing us to assess the reproducibility of the bioinformatic prediction of AMR between participants, and identify problem cases and factors that lead to discordant results. We produced ten WGS datasets of varying quality from cultured carbapenem-resistant organisms obtained from clinical samples sequenced on either an Illumina NextSeq or HiSeq instrument. Nine participating teams ('participants') were provided these sequence data without any other contextual information. Each participant used their choice of pipeline to determine the species, the presence of resistance-associated genes, and to predict susceptibility or resistance to amikacin, gentamicin, ciprofloxacin and cefotaxime. We found participants predicted different numbers of AMR-associated genes and different gene variants from the same clinical samples. The quality of the sequence data, choice of bioinformatic pipeline and interpretation of the results all contributed to discordance between participants. Although much of the inaccurate gene variant annotation did not affect genotypic resistance predictions, we observed low specificity when compared to phenotypic AST results, but this improved in samples with higher read depths. Had the results been used to predict AST and guide treatment, a different antibiotic would have been recommended for each isolate by at least one participant. These challenges, at the final analytical stage of using WGS to predict AMR, suggest the need for refinements when using this technology in clinical settings. Comprehensive public resistance sequence databases, full recommendations on sequence data quality and standardization in the comparisons between genotype and resistance phenotypes will all play a fundamental role in the successful implementation of AST prediction using WGS in clinical microbiology laboratories

Characterisation of the bacterial and fungal communities associated with different lesion sizes of Dark Spot Syndrome occurring in the Coral Stephanocoenia intersepta

The number and prevalence of coral diseases/syndromes are increasing worldwide. Dark Spot Syndrome (DSS) afflicts numerous coral species and is widespread throughout the Caribbean, yet there are no known causal agents. In this study we aimed to characterise the microbial communities (bacteria and fungi) associated with DSS lesions affecting the coral Stephanocoenia intersepta using nonculture molecular techniques. Bacterial diversity of healthy tissues (H), those in advance of the lesion interface (apparently healthy AH), and three sizes of disease lesions (small, medium, and large) varied significantly (ANOSIM R = 0.052 p,0.001), apart from the medium and large lesions, which were similar in their community profile. Four bacteria fitted into the pattern expected from potential pathogens; namely absent from H, increasing in abundance within AH, and dominant in the lesions themselves. These included ribotypes related to Corynebacterium (KC190237), Acinetobacter (KC190251), Parvularculaceae (KC19027), and Oscillatoria (KC190271). Furthermore, two Vibrio species, a genus including many proposed coral pathogens, dominated the disease lesion and were absent from H and AH tissues, making them candidates as potential pathogens for DSS. In contrast, other members of bacteria from the same genus, such as V. harveyii were present throughout all sample types, supporting previous studies where potential coral pathogens exist in healthy tissues. Fungal diversity varied significantly as well, however the main difference between diseased and healthy tissues was the dominance of one ribotype, closely related to the plant pathogen, Rhytisma acerinum, a known causal agent of tar spot on tree leaves. As the corals’ symbiotic algae have been shown to turn to a darker pigmented state in DSS (giving rise to the syndromes name), the two most likely pathogens are R. acerinum and the bacterium Oscillatoria, which has been identified as the causal agent of the colouration in Black Band Disease, another widespread coral disease

G-CSF Prevents the Progression of Structural Disintegration of White Matter Tracts in Amyotrophic Lateral Sclerosis: A Pilot Trial

Background: The hematopoietic protein Granulocyte-colony stimulating factor (G-CSF) has neuroprotective and regenerative properties. The G-CSF receptor is expressed by motoneurons, and G-CSF protects cultured motoneuronal cells from apoptosis. It therefore appears as an attractive and feasible drug candidate for the treatment of amyotrophic lateral sclerosis (ALS). The current pilot study was performed to determine whether treatment with G-CSF in ALS patients is feasible.Methods: Ten patients with definite ALS were entered into a double-blind, placebo-controlled, randomized trial. Patients received either 10 mu g/kg BW G-CSF or placebo subcutaneously for the first 10 days and from day 20 to 25 of the study. Clinical outcome was assessed by changes in the ALS functional rating scale (ALSFRS), a comprehensive neuropsychological test battery, and by examining hand activities of daily living over the course of the study (100 days). The total number of adverse events (AE) and treatment-related AEs, discontinuation due to treatment-related AEs, laboratory parameters including leukocyte, erythrocyte, and platelet count, as well as vital signs were examined as safety endpoints. Furthermore, we explored potential effects of G-CSF on structural cerebral abnormalities on the basis of voxel-wise statistics of Diffusion Tensor Imaging (DTI), brain volumetry, and voxel-based morphometry.Results: Treatment was well-tolerated. No significant differences were found between groups in clinical tests and brain volumetry from baseline to day 100. However, DTI analysis revealed significant reductions of fractional anisotropy (FA) encompassing diffuse areas of the brain when patients were compared to controls. On longitudinal analysis, the placebo group showed significant greater and more widespread decline in FA than the ALS patients treated with G-CSF.Conclusions: Subcutaneous G-CSF treatment in ALS patients appears as feasible approach. Although exploratory analysis of clinical data showed no significant effect, DTI measurements suggest that the widespread and progressive microstructural neural damage in ALS can be modulated by G-CSF treatment. These findings may carry significant implications for further clinical trials on ALS using growth factors

Heritable patterns of tooth decay in the permanent dentition: principal components and factor analyses

<p>Abstract</p> <p>Background</p> <p>Dental caries is the result of a complex interplay among environmental, behavioral, and genetic factors, with distinct patterns of decay likely due to specific etiologies. Therefore, global measures of decay, such as the DMFS index, may not be optimal for identifying risk factors that manifest as specific decay patterns, especially if the risk factors such as genetic susceptibility loci have small individual effects. We used two methods to extract patterns of decay from surface-level caries data in order to generate novel phenotypes with which to explore the genetic regulation of caries.</p> <p>Methods</p> <p>The 128 tooth surfaces of the permanent dentition were scored as carious or not by intra-oral examination for 1,068 participants aged 18 to 75 years from 664 biological families. Principal components analysis (PCA) and factor analysis (FA), two methods of identifying underlying patterns without <it>a priori </it>surface classifications, were applied to our data.</p> <p>Results</p> <p>The three strongest caries patterns identified by PCA recaptured variation represented by DMFS index (correlation, r = 0.97), pit and fissure surface caries (r = 0.95), and smooth surface caries (r = 0.89). However, together, these three patterns explained only 37% of the variability in the data, indicating that <it>a priori </it>caries measures are insufficient for fully quantifying caries variation. In comparison, the first pattern identified by FA was strongly correlated with pit and fissure surface caries (r = 0.81), but other identified patterns, including a second pattern representing caries of the maxillary incisors, were not representative of any previously defined caries indices. Some patterns identified by PCA and FA were heritable (h²= 30-65%, p = 0.043-0.006), whereas other patterns were not, indicating both genetic and non-genetic etiologies of individual decay patterns.</p> <p>Conclusions</p> <p>This study demonstrates the use of decay patterns as novel phenotypes to assist in understanding the multifactorial nature of dental caries.</p

Single-cell analyses reveal aberrant pathways for megakaryocyte-biased hematopoiesis in myelofibrosis and identify mutant clone-specific targets

Myelofibrosis is a severe myeloproliferative neoplasm characterized by increased numbers of abnormal bone marrow megakaryocytes that induce fibrosis, destroying the hematopoietic microenvironment. To determine the cellular and molecular basis for aberrant megakaryopoiesis in myelofibrosis, we performed single-cell transcriptome profiling of 135,929 CD34+ lineage− hematopoietic stem and progenitor cells (HSPCs), single-cell proteomics, genomics, and functional assays. We identified a bias toward megakaryocyte differentiation apparent from early multipotent stem cells in myelofibrosis and associated aberrant molecular signatures. A sub-fraction of myelofibrosis megakaryocyte progenitors (MkPs) are transcriptionally similar to healthy-donor MkPs, but the majority are disease specific, with distinct populations expressing fibrosis- and proliferation-associated genes. Mutant-clone HSPCs have increased expression of megakaryocyte-associated genes compared to wild-type HSPCs, and we provide early validation of G6B as a potential immunotherapy target. Our study paves the way for selective targeting of the myelofibrosis clone and illustrates the power of single-cell multi-omics to discover tumor-specific therapeutic targets and mediators of tissue fibrosis

Immunological analysis of a Lactococcus lactis-based DNA vaccine expressing HIV gp120

For reasons of efficiency Escherichia coli is used today as the microbial factory for production of plasmid DNA vaccines. To avoid hazardous antibiotic resistance genes and endotoxins from plasmid systems used nowadays, we have developed a system based on the food-grade Lactococcus lactis and a plasmid without antibiotic resistance genes. We compared the L. lactis system to a traditional one in E. coli using identical vaccine constructs encoding the gp120 of HIV-1. Transfection studies showed comparable gp120 expression levels using both vector systems. Intramuscular immunization of mice with L. lactis vectors developed comparable gp120 antibody titers as mice receiving E. coli vectors. In contrast, the induction of the cytolytic response was lower using the L. lactis vector. Inclusion of CpG motifs in the plasmids increased T-cell activation more when the E. coli rather than the L. lactis vector was used. This could be due to the different DNA content of the vector backbones. Interestingly, stimulation of splenocytes showed higher adjuvant effect of the L. lactis plasmid. The study suggests the developed L. lactis plasmid system as new alternative DNA vaccine system with improved safety features. The different immune inducing properties using similar gene expression units, but different vector backbones and production hosts give information of the adjuvant role of the silent plasmid backbone. The results also show that correlation between the in vitro adjuvanticity of plasmid DNA and its capacity to induce cellular and humoral immune responses in mice is not straight forward

Chronic non-specific low back pain - sub-groups or a single mechanism?

Copyright 2008 Wand and O'Connell; licensee BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.Background: Low back pain is a substantial health problem and has subsequently attracted a considerable amount of research. Clinical trials evaluating the efficacy of a variety of interventions for chronic non-specific low back pain indicate limited effectiveness for most commonly applied interventions and approaches. Discussion: Many clinicians challenge the results of clinical trials as they feel that this lack of effectiveness is at odds with their clinical experience of managing patients with back pain. A common explanation for this discrepancy is the perceived heterogeneity of patients with chronic non-specific low back pain. It is felt that the effects of treatment may be diluted by the application of a single intervention to a complex, heterogeneous group with diverse treatment needs. This argument presupposes that current treatment is effective when applied to the correct patient. An alternative perspective is that the clinical trials are correct and current treatments have limited efficacy. Preoccupation with sub-grouping may stifle engagement with this view and it is important that the sub-grouping paradigm is closely examined. This paper argues that there are numerous problems with the sub-grouping approach and that it may not be an important reason for the disappointing results of clinical trials. We propose instead that current treatment may be ineffective because it has been misdirected. Recent evidence that demonstrates changes within the brain in chronic low back pain sufferers raises the possibility that persistent back pain may be a problem of cortical reorganisation and degeneration. This perspective offers interesting insights into the chronic low back pain experience and suggests alternative models of intervention. Summary: The disappointing results of clinical research are commonly explained by the failure of researchers to adequately attend to sub-grouping of the chronic non-specific low back pain population. Alternatively, current approaches may be ineffective and clinicians and researchers may need to radically rethink the nature of the problem and how it should best be managed

Ipsilateral irradiation for well lateralized carcinomas of the oral cavity and oropharynx: results on tumor control and xerostomia

<p>Abstract</p> <p>Background</p> <p>In head and neck cancer, bilateral neck irradiation is the standard approach for many tumor locations and stages. Increasing knowledge on the pattern of nodal invasion leads to more precise targeting and normal tissue sparing. The aim of the present study was to evaluate the morbidity and tumor control for patients with well lateralized squamous cell carcinomas of the oral cavity and oropharynx treated with ipsilateral radiotherapy.</p> <p>Methods</p> <p>Twenty consecutive patients with lateralized carcinomas of the oral cavity and oropharynx were treated with a prospective management approach using ipsilateral irradiation between 2000 and 2007. This included 8 radical oropharyngeal and 12 postoperative oral cavity carcinomas, with Stage T1-T2, N0-N2b disease. The actuarial freedom from contralateral nodal recurrence was determined. Late xerostomia was evaluated using the European Organization for Research and Treatment of Cancer QLQ-H&N35 questionnaire and the National Cancer Institute Common Terminology Criteria for Adverse Events (CTCAE), version 3.</p> <p>Results</p> <p>At a median follow-up of 58 months, five-year overall survival and loco-regional control rates were 82.5% and 100%, respectively. No local or contralateral nodal recurrences were observed. Mean dose to the contralateral parotid gland was 4.72 Gy and to the contralateral submandibular gland was 15.30 Gy. Mean score for dry mouth was 28.1 on the 0-100 QLQ-H&N35 scale. According to CTCAE v3 scale, 87.5% of patients had grade 0-1 and 12.5% grade 2 subjective xerostomia. The unstimulated salivary flow was > 0.2 ml/min in 81.2% of patients and 0.1-0.2 ml/min in 19%. None of the patients showed grade 3 xerostomia.</p> <p>Conclusion</p> <p>In selected patients with early and moderate stages, well lateralized oral and oropharyngeal carcinomas, ipsilateral irradiation treatment of the primary site and ipsilateral neck spares salivary gland function without compromising loco-regional control.</p