The Barts Health NHS Trust COVID-19 cohort: characteristics, outcomes and risk scoring of patients in East London.

BACKGROUND: Barts Health National Health Service Trust (BHNHST) serves a diverse population of 2.5 million people in London, UK. We undertook a health services assessment of factors used to evaluate the risk of severe acute respiratory coronavirus 2 (SARS-CoV-2) infection.METHODS: Patients with confirmed polymerase chain reaction (PCR) test results admitted between 1 March and 1 August 2020 were included, alongwith clinician-diagnosed suspected cases. Prognostic factors from the 4C Mortality score and 4C Deterioration scores were extracted from electronic health records and logistic regression was used to quantify the strength of association with 28-day mortality and clinical deterioration using national death registry linkage.RESULTS: Of 2783 patients, 1621 had a confirmed diagnosis, of whom 61% were male and 54% were from Black and Minority Ethnic groups; 26% died within 28 days of admission. Mortality was strongly associated with older age. The 4C mortality score had good stratification of risk with a calibration slope of 1.14 (95% CI 1.01-1.27). It may have under-estimated mortality risk in those with a high respiratory rate or requiring oxygen.CONCLUSION: Patients in this diverse patient cohort had similar mortality associated with prognostic factors to the 4C score derivation sample, but survival might be poorer in those with respiratory failure.

Imperceptible magnetic sensor matrix system integrated with organic driver and amplifier circuits.

Artificial electronic skins (e-skins) comprise an integrated matrix of flexible devices arranged on a soft, reconfigurable surface. These sensors must perceive physical interaction spaces between external objects and robots or humans. Among various types of sensors, flexible magnetic sensors and the matrix configuration are preferable for such position sensing. However, sensor matrices must efficiently map the magnetic field with real-time encoding of the positions and motions of magnetic objects. This paper reports an ultrathin magnetic sensor matrix system comprising a 2 × 4 array of magnetoresistance sensors, a bootstrap organic shift register driving the sensor matrix, and organic signal amplifiers integrated within a single imperceptible platform. The system demonstrates high magnetic sensitivity owing to the use of organic amplifiers. Moreover, the shift register enabled real-time mapping of 2D magnetic field distribution.

Comparative genomics reveals signature regions used to develop a robust and sensitive multiplex TaqMan real-time qPCR assay to detect the genus Dickeya and Dickeya dianthicola.

AIMS: Dickeya species are high consequence plant pathogenic bacteria; associated with potato disease outbreaks and subsequent economic losses worldwide. Early, accurate and reliable detection of Dickeya spp. is needed to prevent establishment and further dissemination of this pathogen. Therefore, a multiplex TaqMan qPCR was developed for sensitive detection of Dickeya spp. and specifically, Dickeya dianthicola. METHODS AND RESULTS: A signature genomic region for the genus Dickeya (mglA/mglC) and unique genomic region for D. dianthicola (alcohol dehydrogenase) were identified using a whole genome-based comparative genomics approach. The developed multiplex TaqMan qPCR was validated using extensive inclusivity and exclusivity panels, and naturally/artificially infected samples to confirm broad range detection capability and specificity. Both sensitivity and spiked assays showed a detection limit of 10 fg DNA. CONCLUSION: The developed multiplex assay is sensitive and reliable to detect Dickeya spp. and D. dianthicola with no false positives or false negatives. It was able to detect mixed infection from naturally and artificially infected plant materials. SIGNIFICANCE AND IMPACT OF THE STUDY: The developed assay will serve as a practical tool for screening of propagative material, monitoring the presence and distribution, and quantification of target pathogens in a breeding programme. The assay also has applications in routine diagnostics, biosecurity and microbial forensics.

Scalable Production of Equine Platelet Lysate for Multipotent Mesenchymal Stromal Cell Culture.

Translation of multipotent mesenchymal stromal cell (MSC)-based therapies is advancing in human and veterinary medicine. One critical issue is the in vitro culture of MSC before clinical use. Using fetal bovine serum (FBS) as supplement to the basal medium is still the gold standard for cultivation of many cell types including equine MSC. Alternatives are being explored, with substantial success using platelet lysate-supplemented media for human MSC. However, progress lags behind in the veterinary field. The aim of this study was to establish a scalable protocol for equine platelet lysate (ePL) production and to test the ePL in equine MSC culture. Whole blood was harvested into blood collection bags from 20 healthy horses. After checking sample materials for pathogen contamination, samples from 19 animals were included. Platelet concentrates were prepared using a buffy coat method. Platelets, platelet-derived growth factor BB, and transforming growth factor ß1 concentrations were increased in the concentrates compared with whole blood or serum (p < 0.05), while white blood cells were reduced (p < 0.05). The concentrates were lysed using freeze/thaw cycles, which eliminated the cells while growth factor concentrations were maintained. Donor age negatively correlated with platelet and growth factor concentrations after processing (p < 0.05). Finally, all lysates were pooled and the ePL was evaluated as culture medium supplement in comparison with FBS, using adipose-derived MSC from four unrelated donor horses. MSC proliferated well in 10% FBS as well as in 10% ePL. However, using 5 or 2.5% ePL entailed highly inconsistent proliferation or loss of proliferation, with significant differences in generation times and confluencies (p < 0.05). MSC expressed the surface antigens CD90, CD44, and CD29, but CD73 and CD105 detection was low in all culture media. Adipogenic and osteogenic differentiation led to similar results in MSC from different culture media. The buffy coat method is useful to produce equine platelet concentrate with increased platelet and reduced white blood cell content in large scales. The ePL obtained supports MSC expansion similar as FBS when used at the same concentration (10%). Further investigations into equine MSC functionality in culture with ePL should follow.

A service evaluation of simultaneous near-patient testing for influenza, respiratory syncytial virus, Clostridium difficile and norovirus in a UK district general hospital.

BACKGROUND: The Cepheid® GeneXpert® (GXP) can simultaneously test for norovirus (NV), Clostridium difficile (CD), influenza A/B (IFA/B) and respiratory syncytial virus (RSV). AIM: To compare centralized multiplex polymerase chain reaction (PCR) testing with localized GXP testing at a district general hospital. METHODS: From December 2017 to December 2018, samples received at Whipps Cross University Hospital (WCUH) were first tested at the local laboratory before transport centrally to the Royal London Hospital (RLH). At the RLH, a non-proprietary multiplex reverse transcriptase (RT) PCR assay was performed, which also tested for gastrointestinal or respiratory pathogens not tested for by the GXP. FINDINGS: A total of 1111 stool and respiratory samples were processed at both sites; 591 were respiratory and 520 were stool samples. Compared to centralized testing, the GXP gave sensitivity, specificity, and NPV all in excess of 97%, with the exception of RSV. The RSV assay had a sensitivity of 66.7% (95% confidence interval (CI) 24.1, 94.0) but an NPV of 99.7% (95% CI 98.6, 99.9). At the RLH, 65 (5.9%) additional respiratory or gastrointestinal viruses were detected, predominantly rhinovirus 35 (3.2%) and adenovirus 11 (1.0%). Compared to centralized testing, the median time saved for local respiratory and gastrointestinal sample testing was 19 h and 46 min and 17 h and 6 min, respectively. CONCLUSIONS: Local GXP testing compared to centralized multiplex PCR testing for IF, NV and CD, demonstrated sensitivities, specificities and NPV between 95% and 100%. Turnaround times were faster, enabling quicker infection prevention and control decision making. In our local setting (WCUH), the GXP demonstrated the potential to reduce NV and IFA/B outbreaks.

Ultra-sensitive detection of papaya ringspot virus using single-tube nested PCR.

Aphid-transmitted papaya ringspot virus (PRSV) is the greatest disease threat to the commercial production of papaya worldwide. Specific ultrasensitive assays are important for the early detection of PRSV in the field. We have developed a single-tube nested PCR (STNP) assay to address this need. Two nested PCR primer sets were designed to target the P3 gene of PRSV. The annealing temperatures and concentrations of both primer pairs were optimized to reduce potential competition between primer sets in STNP. The assay is more sensitive than regular RT-PCR as determined by serial dilutions of cDNA and RNA templates and sample extracts from infected plants. STNP is capable of detecting PRSV in plants 7 days post-inoculation, whereas RT-PCR and ELISA are capable of detecting PRSV 14 to 21 days post-inoculation. This new assay can also detect PRSV from virus infected but asymptomatic plants. This system could assist epidemiological studies in the field and in quarantine protocols by enabling early detection of very low PRSV infection rates in the field and in imported plant samples. Keywords: early detection; quarantine protocols.

Does the presence of a urinary catheter predict severe sepsis in a bacteraemic cohort?

BACKGROUND: Sepsis is a major cause of mortality with an estimated 37,000 deaths in the UK each year. This study aimed to determine host factors that can predict severe sepsis in a bacteraemic cohort. METHODS: From December 2012 to November 2013, demographic, clinical and microbiological data were collected on consecutive patients with bacteraemia at a London teaching hospital. These data were used to categorize patients as having severe or non-severe sepsis. Multi-variate logistic regression was used to determine the association between host factors and severe sepsis. FINDINGS: Five hundred and ninety-four bacteraemic episodes occurred in 500 patients. The majority of cases were in patients aged >50 years (382/594, 64.3%) and in males (346/594, 58.2%). The most common isolates were Escherichia coli (207/594, 34.8%) and meticillin-susceptible Staphylococcus aureus (57/594, 9.6%). In logistic regression multi-variable analysis, site of infection was significantly associated with severe sepsis. For catheter-associated urinary tract infections, the association was significant after adjustment for age, sex, Charlson comorbidity index and where infection was acquired (odds ratio 3.94, 95% confidence interval 1.70-9.11). CONCLUSIONS: Urinary catheters increase the risk of severe sepsis. They should only be used if clinically indicated. If inserted, a care bundle approach should be used and the anticipated removal date should be recorded unless a long-term catheter is required. In the context of sepsis, the presence of a urinary catheter should prompt immediate implementation of 'Sepsis Six' and consideration of transfer to a critical care unit.

A Multiplex PCR Assay for Differentiating Coconut Rhinoceros Beetle (Coleoptera: Scarabaeidae) From Oriental Flower Beetle (Coleoptera: Scarabaeidae) in Early Life Stages and Excrement.

The coconut rhinoceros beetle, Oryctes rhinoceros (L.), is a major pest of coconut and other palm trees. An incipient coconut rhinoceros beetle population was recently discovered on the island of Oahu, Hawaii and is currently the target of a large, mutiagency eradication program. Confounding this program is the widespread presence of another scarab beetle on Oahu, the oriental flower beetle, Protaetia orientalis (Gory and Percheron 1833). Eggs, early life stages, and fecal excrement of coconut rhinoceros beetle and oriental flower beetle are morphologically indistinguishable, thereby creating uncertainty when such specimens are discovered in the field. Here, we report the development of a multiplex PCR assay targeting cytochrome oxidase I of coconut rhinoceros beetle and oriental flower beetle that can rapidly detect and distinguish between these insects. This assay also features an internal positive control to ensure DNA of sufficient quantity and quality is used in the assay, increasing its reliability and reducing the chances of false negative results.

Gram-negative bacteraemia; a multi-centre prospective evaluation of empiric antibiotic therapy and outcome in English acute hospitals.

Increasing antibiotic resistance makes choosing antibiotics for suspected Gram-negative infection challenging. This study set out to identify key determinants of mortality among patients with Gram-negative bacteraemia, focusing particularly on the importance of appropriate empiric antibiotic treatment. We conducted a prospective observational study of 679 unselected adults with Gram-negative bacteraemia at ten acute english hospitals between October 2013 and March 2014. Appropriate empiric antibiotic treatment was defined as intravenous treatment on the day of blood culture collection with an antibiotic to which the cultured organism was sensitive in vitro. Mortality analyses were adjusted for patient demographics, co-morbidities and illness severity. The majority of bacteraemias were community-onset (70%); most were caused by Escherichia coli (65%), Klebsiella spp. (15%) or Pseudomonas spp. (7%). Main foci of infection were urinary tract (51%), abdomen/biliary tract (20%) and lower respiratory tract (14%). The main antibiotics used were co-amoxiclav (32%) and piperacillin-tazobactam (30%) with 34% receiving combination therapy (predominantly aminoglycosides). Empiric treatment was inappropriate in 34%. All-cause mortality was 8% at 7 days and 15% at 30 days. Independent predictors of mortality (p <0.05) included older age, greater burden of co-morbid disease, severity of illness at presentation and inflammatory response. Inappropriate empiric antibiotic therapy was not associated with mortality at either time-point (adjusted OR 0.82; 95% CI 0.35-1.94 and adjusted OR 0.92; 95% CI 0.50-1.66, respectively). Although our study does not exclude an impact of empiric antibiotic choice on survival in Gram-negative bacteraemia, outcome is determined primarily by patient and disease factors.

Outcomes in consecutive hospitalized UK patients with bacteraemia or fungaemia caused by medical devices and procedures.

BACKGROUND: There is a lack of outcome data on hospitalized patients with bacteraemia or fungaemia caused by medical devices or procedures. AIM: To determine the association between death and bacteraemia or fungaemia caused by medical devices and procedures. METHODS: From December 2012 to November 2013, demographic, clinical, and microbiological data were collected on consecutive inpatients with bacteraemia or fungaemia. Multivariate analysis, using generalized estimating equations, was used to define the association. FINDINGS: A total of 594 bacteraemic or fungaemic episodes occurred in 500 patients. Among patients with episodes caused by medical devices or procedures, 7-day and 30-day mortality were 7/167 [4.2%; 95% confidence interval (CI): 1.7-8.4] and 12/167 (7.2%; CI: 3.8-12.2) respectively. After adjustment, the association between death and bacteraemic or fungaemic episodes related to medical devices and procedures was non-significant as 7- and 30-day mortality odds ratios (OR) were 2.86 (95% CI: 0.80-10.12) and 1.72 (95% CI: 0.71-4.16) respectively. The difference between 30-day mortality associated with Escherichia coli and Staphylococcus aureus bacteraemia demonstrated a trend towards significance [6/47 (12.8%; CI: 4.8-25.7) vs 0/24; P = 0.067]. Thirty-day mortality associated with bacteraemia or fungaemia in patients with urinary catheter infections (often E. coli-associated) was significantly higher than intravascular device-associated infections (often S. aureus-associated) [4/51 (7.8%; 95% CI: 2.2-18.8) vs 1/62 (1.6%; 95% CI: 0.0-8.7); P = 0.028]. CONCLUSION: Special attention is required to prevent medical device- or procedure-related bacteraemia caused by E. coli. Greater attention should be placed on preventing infections caused by urinary catheters.

Phylogenetic Diversity of Rhizoctonia solani Associated with Canola and Wheat in Alberta, Manitoba, and Saskatchewan.

Rhizoctonia solani is a damaging soilborne pathogen, which affects most field crops in the Canadian provinces of Alberta, Manitoba, and Saskatchewan. The objective of this study was to conduct a phylogenetic comparison of isolates of R. solani collected from a previous survey in the major canola- and wheat-growing regions of western Canada. A total of 128 multinucleate isolates from a previous survey were identified by internal transcribed spacer (ITS) sequence and compared to anastomosis group (AG) results. The multinucleate isolates of R. solani were grouped into eight distinct clades. Each clade corresponded to a specific AG with the exception of two distinct clades that were observed for isolates classified as AG 2-1 by anastomosis testing. While most isolates of AG 5 clustered together according to ITS sequences, three isolates classified by anastomosis grouping as AG 5 grouped with AG 2-1, AG 4, and a binucleate Rhizoctonia sp. in the phylogenetic analysis. In most instances, the results from AG tests were consistent with ITS sequence, but there were still several cases where isolates were inconsistently classified or failed to undergo anastomosis with any of the tester strains used in this study. This provides support for the use of the ITS region as a valuable tool for rapid identification of R. solani isolates to their respective AGs.

First Report of Capsicum chlorosis virus Infecting Waxflower (Hoya calycina Schlecter) in the United States.

In February 2013, an ornamental waxflower (Hoya calycina Schlecter) with leaves displaying concentric chlorotic and necrotic rings surrounding sunken, necrotic lesions typical of tospovirus infection was observed at a community garden in Honolulu, HI. Symptomatic leaf tissue tested negative for Tomato spotted wilt virus (TSWV), a common tospovirus in Hawaii, using a TSWV ImmunoStrips (AgDia, Elkhart, IN) assay following the manufacturer's instructions. Double-stranded RNAs were isolated from a symptomatic leaf and reverse transcribed using random primers (2). The cDNA was then used as template in a universal tospovirus PCR assay using primers gL3637 and gL4435c, which amplify sequences of the L segment encoding the RNA-dependent RNA polymerase of tospoviruses (1). An ~800-bp product was amplified and cloned using pGEM-T Easy (Promega, Madison, WI). Three clones were selected and found to be identical by dye-terminator sequencing performed at the University of Hawaii's Advanced Studies in Genomics, Proteomics, and Bioinformatics laboratory. Following primer sequence trimming, the 773-bp sequence (GenBank Accession No. KF030938) was found to be 97, 88, and 87% identical to Capsicum chlorosis virus (CaCV; a tentative species in the family Bunyaviridae, genus Tospovirus) strains Ch-Har (GU199334), TwTom1 (HM021140), and AIT (DQ256124), respectively. To confirm the presence of CaCV, the cDNA was also used as template in a universal tospovirus PCR assay with primers 3'T12 and TsMCR2 which amplify a region of the S segment of tospoviruses (3). The amplification product from this assay was cloned and sequenced as described above and found to be 93 to 98% identical to CaCV nucleotide sequences present in GenBank. Attempts to detect the CaCV strain in waxflower using a watermelon silver mottle virus and groundnut bud necrosis virus triple antibody sandwich ELISA (AgDia) were unsuccessful. No other plants in the community garden had typical tospovirus-like symptoms; however, samples from tomato (Solanum lycopersicum L.; two samples), chili pepper (Capsicum spp.; four samples), eggplant (Solanum melongena L.; one sample), and passionfruit (Passiflora edulis Sims; one sample) with virus-like symptoms were collected from the garden and had RNA isolated using a NucleoSpin RNA II kit (Macherey-Nagel, Bethlehem, PA). No tospoviruses were detected in any of these samples with the RT-PCR assay using primers gL3637 and gL4435. The waxflower plant infected with CaCV was immediately removed by community garden members and destroyed, preventing any additional serological or biological assays to be performed. CaCV is transmitted by several species of thrips, including Thrips palmi, which is present in Hawaii. Waxflower is not native to Hawaii and it is unclear whether CaCV entered Hawaii in this plant or whether it was infected by viruliferous thrips. A survey for CaCV in known hosts is essential to determine the geographic distribution of CaCV in Hawaii, as this virus poses a considerable threat to tomato, chili pepper, and phalaenopsis orchid production in Hawaii and the United States. References: (1) F.-H. Chu et al. Phytopathology 91:361, 2001. (2) M. J. Melzer et al. Virus Genes 40:111, 2010. (3) M. Okuda and K. Hanada. J. Virol. Methods 96:149, 2001.

First Report of Taro vein chlorosis virus Infecting Taro (Colocasia esculenta) in the United States.

In March 2013, taro plants (Colocasia esculenta [L.] Schott cv. Iliuaua) with leaves displaying veinal chlorosis and necrosis were observed on the island of Molokai. These symptoms were similar to those of taro vein chlorosis, a disease of taro caused by Taro vein chlorosis virus (TaVCV; family Rhabdoviridae, genus Nucleorhabdovirus). To explore this possibility, RNA was isolated from both symptomatic and asymptomatic taro leaves using the NucleoSpin RNA II extraction kit (Macherey-Nagel, Bethlehem, PA) according to the provided protocol, except that RLT Buffer (Qiagen Inc., Valencia, CA) was used as the initial extraction buffer. The RNAs were converted to cDNA using random primers and MMLV-RT reverse transcriptase (Promega, Madison, WI). The cDNA underwent PCR assays using primer sets Pol2A1/Pol2A2 and Cap2A/Cap2B which target the RNA-dependent RNA polymerase (RdRp) and putative nucleocapsid genes of TaVCV, respectively (1). Amplification products of the correct size were obtained for both primer sets, and these underwent molecular cloning using pGEM-T Easy (Promega). Three clones were selected and their sequences determined by dye-terminator sequencing. After primer sequence removal, the Pol2A1/Pol2A2 product (952 bp; GenBank Accession No. KF921085) and Cap2A/Cap2B product (1,050 bp; KF921086) were found to be 79 and 84% identical to a Fijian strain of TaVCV (AY674964), respectively. Samples from 328 plants with and without taro vein chlorosis symptoms were collected from 35 sites on five of the Hawaiian islands and assayed for TaVCV using the Pol2A1/Pol2A2 primer set as described above. The incidence of TaVCV in these samples was 21.6%, with positive samples coming from each island. Although a very strong association between symptoms and the presence of TaVCV was observed, eight asymptomatic plants were also positive, suggesting the detection assay was able to detect the virus before the onset of symptoms. Conversely, three symptomatic plants were found to be negative, suggesting the Pol2A1/Pol2A2 PCR assay might not detect all strains of TaVCV in Hawaii. A digoxygenin-labeled probe (Roche Applied Science, Indianapolis, IN) derived from the Pol2A1/Pol2A2 amplification product of one sample hybridized with the cDNA of only four of nine TaVCV-infected samples collected from three different islands in a dot-blot hybridization assay performed at high stringency. This probe did not hybridize with the cDNA of five TaVCV-negative samples. TaVCV exhibits a great deal of genetic diversity in the South Pacific nations where it is found; nucleotide divergence of up to 27% in regions of the RdRp gene has been reported (1). The high genetic divergence between the TaVCV isolate characterized in Hawaii and the TaVCV accession in GenBank, as well as the dot blot hybridization assay results support this observation. The widespread distribution of TaVCV in Hawaii suggests it is not a recent introduction. However, the common practice of farmers sharing taro propagules has likely accelerated its spread. An arthropod vector of TaVCV has yet to be identified, so it is unknown whether natural spread is also occurring in Hawaii. Taro has both economic and cultural importance to Hawaii. These findings, representing the first detection of TaVCV in Hawaii and the United States, illustrate the need to develop virus-free germplasm for local, national, and international distribution of this important staple crop. Reference: (1) P. Revill et al. J. Gen Virol. 86:491, 2005.

Increased symplasmic permeability in barley root epidermal cells correlates with defects in root hair development.

It is well known that the process of plant cell differentiation depends on the symplasmic isolation of cells. Before starting the differentiation programme, the individual cell or group of cells should restrict symplasmic communication with neighbouring cells. We tested the symplasmic communication between epidermal cells in the different root zones of parental barley plants Hordeum vulgare L., cv. 'Karat' with normal root hair development, and two root hairless mutants (rhl1.a and rhl1.b). The results clearly show that symplasmic communication was limited during root hair differentiation in the parental variety, whereas in both root hairless mutants epidermal cells were still symplasmically connected in the corresponding root zone. This paper is the first report on the role of symplasmic isolation in barley root cell differentiation, and additionally shows that a disturbance in the restriction of symplasmic communication is present in root hairless mutants.

Chromatin alterations during pollen development in Hordeum vulgare.

The dynamics of posttranslational histone modifications in relation to nuclear architecture has been analyzed during pollen development in Hordeum vulgare L. cv. Igri. Notwithstanding the asymmetry of cytokinesis associated with pollen mitosis I, immunolabeling revealed that the vegetative and generative nuclei initially display identical chromatin modification patterns. Yet, differential chromatin modification patterns between vegetative and generative nuclei emerge with the development of conspicuous differences in nuclear morphology as visualized by 4',6-diamidino-2-phenylindole staining. The temporal and spatial distribution of most histone modifications observed is in agreement with reduced gene activity in the generative nucleus and increased expression in the vegetative nucleus as indicated by immunolabeling of active RNA polymerase II. Signals of trimethylation of histone H3 lysine 27 proved to be particularly enriched in euchromatic domains of subtelomeric regions. In the context of nuclear differentiation in bicellular pollen, this modification became restricted to the vegetative nucleus, indicating a role in activating rather than suppressing gene expression. The presence of acetylated histone H3 at lysine 9 in the cytoplasm of the generative cell is indicative of a more complex, still unknown function of this particular modification.

30-day mortality in UK patients with bacteraemic community-acquired pneumonia.

OBJECTIVES: To determine 7 and 30-day mortality in consecutive patients with bacteraemic community-acquired pneumonia (CAP) and the association between predicted variables and likelihood of death. METHODS: From August 2007 to July 2011, demographic, clinical and microbiological data were prospectively collected on patients with bacteraemic CAP. Patients were followed until death, hospital discharge or recovery from infection. Univariate and multivariate analysis was performed to determine the association between predictor variables and 30-day mortality. RESULTS: 7-day mortality was 61/252 [24.4%, 95% confidence interval (CI) 19.1-30.0%] and by 30 days, this had risen to 77/252 (30.6%, 95% CI 24.9-36.6%). In univariate analysis, factors associated with 30-day mortality were age, speciality within 48 h of admission, blood culture isolate and Charlson co-morbidity index (CCI). In multivariate analysis, age and CCI remained significantly associated. There was also a trend towards significance for meticillin-sensitive Staphylococcus aureus (MSSA) and Pseudomonas aeruginosa blood culture isolates compared to Streptococcus pneumoniae. CONCLUSIONS: Overall, bacteraemic CAP was associated with high inpatient mortality. Because of their association with poor outcomes, patients with MSSA and P. aeruginosa bacteraemic CAP require further study.

Thirty-day mortality in UK patients with community-onset and hospital-acquired meticillin-susceptible Staphylococcus aureus bacteraemia.

BACKGROUND: The difference in mortality between patients with community-onset and hospital-acquired Staphylococcus aureus infections has rarely been described and where it has, results have been conflicting. AIM: To determine 30-day mortality in consecutive patients with meticillin-susceptible Staphylococcus aureus (MSSA) bacteraemia and the association between community-onset infection and outcome. METHODS: From August 2007 to July 2011, demographic, clinical and microbiological data were prospectively collected on patients with MSSA bacteraemia. Patients were followed until death, hospital discharge or recovery from infection. Multivariate logistic regression was used to determine the association between community-onset infection and 30-day mortality. FINDINGS: A total of 403 bacteraemic episodes occurred in 392 patients. Overall, there were 44 deaths (11.2%; 95% confidence interval: 7.9-14.0%) at 7 days and 101 deaths (25.8%; 21.5-30.4%) at 30 days. The difference in 30-day mortality between patients with community-onset and hospital-acquired infection was 71/256 (27.7%) versus 31/147 21.1%). Community-onset infection more frequently caused infective endocarditis (13/14, 92.9%), vertebral osteomyelitis (12/13, 92.3%) and skin and soft tissue infection (61/71, 85.9%) whereas intravascular catheter-associated infections were predominantly hospital-acquired (60/82, 73.2%). Age, Pitt score, Charlson comorbidity index (CCI), specific sites of infection (skin and soft tissue, lower respiratory tract and peripheral joints) and delay in appropriate treatment were strongly associated with 30-day mortality. In multivariate analysis, after adjustment for age, CCI and delay in appropriate treatment, community-onset infection was strongly associated with 30-day mortality (odds ratio: 1.59; 95% confidence interval: 0.91-2.80). CONCLUSIONS: Compared with hospital-acquired MSSA bacteraemic infection, community-onset infection was associated with worse 30-day outcomes. Hospital-acquired MSSA bacteraemic infections were rarely metastatic, frequently associated with medical devices and patients had better outcomes.

Time-lapse imaging of the initiation of pollen embryogenesis in barley (Hordeum vulgare L.).

Pollen embryogenesis provides exciting opportunities in the areas of breeding and biotechnology as well as representing a convenient model for studying the process of plant cell proliferation in general and embryogenesis in particular. A cell culture system was devised in which immature barley pollen could be cultured as a monolayer trapped between the bottom glass-cover slip of a live-cell chamber and a diaphanous PTFE membrane within a liquid medium over a period of up to 28 d, allowing the process of embryogenesis to be tracked in individual pollen. Z-stacks of images were automatically captured every 3min, starting from the unicellular pollen stage up to the development of multicellular, embryogenic structures. The method should prove useful for the elucidation of ultrastructural features and molecular processes associated with pollen embryogenesis.

A highly sensitive single-tube nested PCR assay for the detection of Pineapple mealybug wilt associated virus-2 (PMWaV-2).

An assay was developed for the detection of Pineapple mealybug wilt associated virus-2 (PMWaV-2), an important factor in the etiology of mealybug wilt of pineapple. The assay combines reverse transcription of RNA isolated from pineapple with a specific and very sensitive, single, closed-tube nested polymerase chain reaction (PCR) to amplify a segment of the coat protein gene of the PMWaV-2. The outer primers were designed to anneal at higher temperatures than the nested primers to prevent primer competition in consecutive amplification reactions. To reduce potential competition further, the outer primers were used at one-thousandth the concentration of the nested primers. The specificity and sensitivity of this assay are much greater than PCR using only a single primer-pair. A TaqMan(®) probe was also designed for use in quantitative PCR to detect and quantify the PCR amplification products directly in a single-tube assay. The advantages of the single-tube assays using both conventional and quantitative PCR are reduced handling time and prevention of cross contamination compared to regular nested PCR in which the reactions are carried out in two separate tubes.