Comprehensive molecular testing for respiratory pathogens in community-acquired pneumonia

Funding: This work was supported by the Chief Scientist Office (grant number ETM/250).Background. The frequent lack of a microbiological diagnosis in community-acquired pneumonia (CAP) impairs pathogen-directed antimicrobial therapy. This study assessed the use of comprehensive multibacterial, multiviral molecular testing, including quantification, in adults hospitalized with CAP. Methods. Clinical and laboratory data were collected for 323 adults with radiologically-confirmed CAP admitted to 2 UK tertiary care hospitals. Sputum (96%) or endotracheal aspirate (4%) specimens were cultured as per routine practice and also tested with fast multiplex real-time polymerase-chain reaction (PCR) assays for 26 respiratory bacteria and viruses. Bacterial loads were also calculated for 8 bacterial pathogens. Appropriate pathogen-directed therapy was retrospectively assessed using national guidelines adapted for local antimicrobial susceptibility patterns. Results. Comprehensive molecular testing of single lower respiratory tract (LRT) specimens achieved pathogen detection in 87% of CAP patients compared with 39% with culture-based methods. Haemophilus influenzae and Streptococcus pneumoniae were the main agents detected, along with a wide variety of typical and atypical pathogens. Viruses were present in 30% of cases; 82% of these were codetections with bacteria. Most (85%) patients had received antimicrobials in the 72 hours before admission. Of these, 78% had a bacterial pathogen detected by PCR but only 32% were culture-positive (P < .0001). Molecular testing had the potential to enable de-escalation in number and/or spectrum of antimicrobials in 77% of patients. Conclusions. Comprehensive molecular testing significantly improves pathogen detection in CAP, particularly in antimicrobial-exposed patients, and requires only a single LRT specimen. It also has the potential to enable early de-escalation from broad-spectrum empirical antimicrobials to pathogen-directed therapy.Publisher PDFPeer reviewe

A New Stalked Filter-Feeder from the Middle Cambrian Burgess Shale, British Columbia, Canada

Burgess Shale-type deposits provide invaluable insights into the early evolution of body plans and the ecological structure of Cambrian communities, but a number of species, continue to defy phylogenetic interpretations. Here we extend this list to include a new soft-bodied animal, Siphusauctum gregarium n. gen. and n. sp., from the Tulip Beds (Campsite Cliff Shale Member, Burgess Shale Formation) of Mount Stephen (Yoho National Park, British Columbia). With 1,133 specimens collected, S. gregarium is clearly the most abundant animal from this locality

The complete mitochondrial genome of the foodborne parasitic pathogen Cyclospora cayetanensis

Cyclospora cayetanensis is a human-specific coccidian parasite responsible for several food and water-related outbreaks around the world, including the most recent ones involving over 900 persons in 2013 and 2014 outbreaks in the USA. Multicopy organellar DNA such as mitochondrion genomes have been particularly informative for detection and genetic traceback analysis in other parasites. We sequenced the C. cayetanensis genomic DNA obtained from stool samples from patients infected with Cyclospora in Nepal using the Illumina MiSeq platform. By bioinformatically filtering out the metagenomic reads of non-coccidian origin sequences and concentrating the reads by targeted alignment, we were able to obtain contigs containing Eimeria-like mitochondrial, apicoplastic and some chromosomal genomic fragments. A mitochondrial genomic sequence was assembled and confirmed by cloning and sequencing targeted PCR products amplified from Cyclospora DNA using primers based on our draft assembly sequence. The results show that the C. cayetanensis mitochondrion genome is 6274 bp in length, with 33% GC content, and likely exists in concatemeric arrays as in Eimeria mitochondrial genomes. Phylogenetic analysis of the C. cayetanensis mitochondrial genome places this organism in a tight cluster with Eimeria species. The mitochondrial genome of C. cayetanensis contains three protein coding genes, cytochrome (cytb), cytochrome C oxidase subunit 1 (cox1), and cytochrome C oxidase subunit 3 (cox3), in addition to 14 large subunit (LSU) and nine small subunit (SSU) fragmented rRNA genes

Developmental Regulation of KCC2 Phosphorylation Has Long-Term Impacts on Cognitive Function

GABAA receptor-mediated currents shift from excitatory to inhibitory during postnatal brain development in rodents. A postnatal increase in KCC2 protein expression is considered to be the sole mechanism controlling the developmental onset of hyperpolarizing synaptic transmission, but here we identify a key role for KCC2 phosphorylation in the developmental EGABA shift. Preventing phosphorylation of KCC2 in vivo at either residue serine 940 (S940), or at residues threonine 906 and threonine 1007 (T906/T1007), delayed or accelerated the postnatal onset of KCC2 function, respectively. Several models of neurodevelopmental disorders including Rett syndrome, Fragile × and Down’s syndrome exhibit delayed postnatal onset of hyperpolarizing GABAergic inhibition, but whether the timing of the onset of hyperpolarizing synaptic inhibition during development plays a role in establishing adulthood cognitive function is unknown; we have used the distinct KCC2-S940A and KCC2-T906A/T1007A knock-in mouse models to address this issue. Altering KCC2 function resulted in long-term abnormalities in social behavior and memory retention. Tight regulation of KCC2 phosphorylation is therefore required for the typical timing of the developmental onset of hyperpolarizing synaptic inhibition, and it plays a fundamental role in the regulation of adulthood cognitive function

Low-pathogenicity Mycoplasma spp. alter human monocyte and macrophage function and are highly prevalent among patients with ventilator-acquired pneumonia.

BACKGROUND: Ventilator-acquired pneumonia (VAP) remains a significant problem within intensive care units (ICUs). There is a growing recognition of the impact of critical-illness-induced immunoparesis on the pathogenesis of VAP, but the mechanisms remain incompletely understood. We hypothesised that, because of limitations in their routine detection, Mycoplasmataceae are more prevalent among patients with VAP than previously recognised, and that these organisms potentially impair immune cell function. METHODS AND SETTING: 159 patients were recruited from 12 UK ICUs. All patients had suspected VAP and underwent bronchoscopy and bronchoalveolar lavage (BAL). VAP was defined as growth of organisms at >10(4) colony forming units per ml of BAL fluid on conventional culture. Samples were tested for Mycoplasmataceae (Mycoplasma and Ureaplasma spp.) by PCR, and positive samples underwent sequencing for speciation. 36 healthy donors underwent BAL for comparison. Additionally, healthy donor monocytes and macrophages were exposed to Mycoplasma salivarium and their ability to respond to lipopolysaccharide and undertake phagocytosis was assessed. RESULTS: Mycoplasmataceae were found in 49% (95% CI 33% to 65%) of patients with VAP, compared with 14% (95% CI 9% to 25%) of patients without VAP. Patients with sterile BAL fluid had a similar prevalence to healthy donor BAL fluid (10% (95% CI 4% to 20%) vs 8% (95% CI 2% to 22%)). The most common organism identified was M. salivarium. Blood monocytes from healthy volunteers incubated with M. salivarium displayed an impaired TNF-α response to lipopolysaccharide (p=0.0003), as did monocyte-derived macrophages (MDMs) (p=0.024). MDM exposed to M. salivarium demonstrated impaired phagocytosis (p=0.005). DISCUSSION AND CONCLUSIONS: This study demonstrates a high prevalence of Mycoplasmataceae among patients with VAP, with a markedly lower prevalence among patients with suspected VAP in whom subsequent cultures refuted the diagnosis. The most common organism found, M. salivarium, is able to alter the functions of key immune cells. Mycoplasmataceae may contribute to VAP pathogenesis.This study was funded by the Hospital Infection Society, Wellcome Trust/Department of Health Health Innovation Challenge Fund (HICF)(0510/078) and Sir Jules Thorn Charitable Trust (03/JTA).This is the final version of the article. It first appeared from BMJ Publishing Group via http://dx.doi.org/10.1136/thoraxjnl-2015-20805

A soft-bodied mollusc with radula from the Middle Cambrian Burgess Shale

Author Posting. © Nature Publishing Group, 2006. This is the author's version of the work. It is posted here by permission of Nature Publishing Group for personal use, not for redistribution. The definitive version was published in Nature 442 (2006): 159-163, doi:10.1038/nature04894.Odontogriphus omalus was originally described as a problematic non-biomineralized lophophorate organism. Here we reinterpret Odontogriphus based on 189 new specimens including numerous exceptionally well-preserved individuals from the Burgess Shale collections of the Royal Ontario Museum. This additional material provides compelling evidence that the feeding apparatus in Odontogriphus is a radula of molluscan architecture comprising two primary bipartite tooth rows attached to a radular membrane and showing replacement by posterior addition. Further characters supporting molluscan affinity include a broad foot bordered by numerous ctenidia located in a mantle groove and a stiffened cuticular dorsum. Odontogriphus has a radula similar to Wiwaxia corrugata but lacks a scleritome. We interpret these animals to be members of an early stem-group mollusc lineage that likely originated in the Neoproterozoic Ediacaran Period, providing support for the retention of a biomat-based grazing community from the late Precambrian until at least the Middle Cambrian.Our research was in part supported by a Post-Doctoral Natural Sciences and Engineering Research Council of Canada grant (to JBC-2005) and by a Swedish Research Council grant (to CS)

Molecular architecture of potassium chloride co-transporter KCC2

KCC2 is a neuron specific K+-Cl− co-transporter that controls neuronal chloride homeostasis, and is critically involved in many neurological diseases including brain trauma, epilepsies, autism and schizophrenia. Despite significant accumulating data on the biology and electrophysiological properties of KCC2, structure-function relationships remain poorly understood. Here we used calixarene detergent to solubilize and purify wild-type non-aggregated and homogenous KCC2. Specific binding of inhibitor compound VU0463271 was demonstrated using surface plasmon resonance (SPR). Mass spectrometry revealed glycosylations and phosphorylations as expected from functional KCC2. We show by electron microscopy (EM) that KCC2 exists as monomers and dimers in solution. Monomers are organized into “head” and “core” domains connected by a flexible “linker”. Dimers are asymmetrical and display a bent “S-shape” architecture made of four distinct domains and a flexible dimerization interface. Chemical crosslinking in reducing conditions shows that disulfide bridges are involved in KCC2 dimerization. Moreover, we show that adding a tag to the C-terminus is detrimental to KCC2 function. We postulate that the conserved KCC2 C-ter may be at the interface of dimerization. Taken together, our findings highlight the flexible multi-domain structure of KCC2 with variable anchoring points at the dimerization interface and an important C-ter extremity providing the first in-depth functional architecture of KCC2

The rationale and plan for creating a World Antimalarial Resistance Network (WARN)

Drug resistant malaria was a major factor contributing to the failure of a worldwide campaign to eradicate malaria in the last century, and now threatens the large investment being made by the global community in the rollout of effective new drug combinations to replace failed drugs. Four related papers in this issue of Malaria Journal make the case for creating the World Antimalarial Resistance Network (WARN), which will consist of four linked open-access global databases containing clinical, in vitro, molecular and pharmacological data, and networks of reference laboratories that will support these databases and related surveillance activities. WARN will serve as a public resource to guide antimalarial drug treatment and prevention policies and to help confirm and characterize the new emergence of new resistance to antimalarial drugs and to contain its spread

An early Cambrian agglutinated tubular lophophorate with brachiopod characters.

The morphological disparity of lophotrochozoan phyla makes it difficult to predict the morphology of the last common ancestor. Only fossils of stem groups can help discover the morphological transitions that occurred along the roots of these phyla. Here, we describe a tubular fossil Yuganotheca elegans gen. et sp. nov. from the Cambrian (Stage 3) Chengjiang Lagerstätte (Yunnan, China) that exhibits an unusual combination of phoronid, brachiopod and tommotiid (Cambrian problematica) characters, notably a pair of agglutinated valves, enclosing a horseshoe-shaped lophophore, supported by a lower bipartite tubular attachment structure with a long pedicle with coelomic space. The terminal bulb of the pedicle provided anchorage in soft sediment. The discovery has important implications for the early evolution of lophotrochozoans, suggesting rooting of brachiopods into the sessile lophotrochozoans and the origination of their bivalved bauplan preceding the biomineralization of shell valves in crown brachiopods