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Abstract The sequencing platform and workflow strongly influence microbial community analyses through potential errors at each step. Effective diagnostics and experimental controls are needed to validate data and improve reproducibility. This cross-laboratory study evaluates sources of variability and error at three main steps of a standardized amplicon sequencing workflow (DNA extraction, polymerase chain reaction [PCR], and sequencing) using Oxford Nanopore MinION to analyze agricultural soils and a simple mock community. Variability in sequence results occurs at each step in the workflow with PCR errors and differences in library size greatly influencing diversity estimates. Common bioinformatic diagnostics and the mock community are ineffective at detecting PCR abnormalities. This work outlines several diagnostic checks and techniques to account for sequencing depth and ensure accuracy and reproducibility in soil community analyses. These diagnostics and the inclusion of a reference soil can help ensure data validity and facilitate the comparison of multiple sequencing runs within and between laboratories.

Abstract The past decade has seen an increase in the prevalence of sequence type (ST) 45 methicillin-resistant Staphylococcus aureus (MRSA), yet the underlying drivers for its emergence and spread remain unclear. To better understand the worldwide dissemination of ST45 S. aureus, we performed phylogenetic analyses of Australian isolates, supplemented with a global population of ST45 S. aureus genomes. Our analyses revealed a distinct lineage of multidrug-resistant ST45 MRSA harbouring qacA, predominantly found in Australia and Singapore. Bayesian inference predicted that the acquisition of qacA occurred in the late 1990s. qacA was integrated into a structurally variable region of the chromosome containing Tn552 (carrying blaZ) and Tn4001 (carrying aac(6’)-aph(2”)) transposable elements. Using mutagenesis and in vitro assays, we provide phenotypic evidence that qacA confers tolerance to chlorhexidine. These findings collectively suggest both antimicrobial resistance and the carriage of qacA may play a role in the successful establishment of ST45 MRSA.

Abstract This study delves into the polarization properties of various hair colors using several techniques, including polarization ray tracing, full Stokes, and Mueller matrix imaging. Our analysis involved studying hair in both indoor and outdoor settings under varying lighting conditions. Our results demonstrate a strong correlation between hair color and the degree of linear polarization. Specifically, light-colored hair, such as white and blond, exhibits high albedo and low DoLP. In contrast, dark hair, like black and brown hair, has low albedo and high DoLP. Our research also revealed that a single hair strand displays high diattenuation near specular reflections but high depolarization in areas with diffuse reflections. Additionally, we investigated the wavelength dependency of the polarization properties by comparing the Mueller matrix under illumination at 450 nm and 589 nm. Our investigation demonstrates the impact of hair shade and color on polarization properties and the Umov effect.

Feline lymphoma, a prevalent cancer in cats, exhibits varied prognoses influenced by anatomical site and cellular characteristics. In this study, we investigated the utility of flow cytometry and clonality analysis via PCR for antigen receptor rearrangement (PARR) with respect to characterizing the disease and predicting prognosis. For this purpose, we received fine needle aspirates and/or blood from 438 feline patients, which were subjected to flow cytometry analysis and PARR. We used a subset of the results from patients with confirmed B- or T-cell lymphomas for comparison to cytological or histological evaluation (n = 53). Using them as a training set, we identified the optimal set of flow cytometry parameters, namely forward scatter thresholds, for cell size categorization by correlating with cytology-defined sizes. Concordance with cytological sizing among this training set was 82%. Furthermore, 90% concordance was observed when the proposed cell sizing was tested on an independent test set (n = 24), underscoring the reliability of the proposed approach. Additionally, lymphoma subtypes defined by flow cytometry and PARR demonstrated significant survival differences, validating the prognostic utility of these methods. The proposed methodology achieves high concordance with cytological evaluations and provides an additional tool for the characterization and management of feline lymphoproliferative diseases.

Dogs with B-cell lymphoma typically respond well to first-line CHOP-based chemotherapy, but there is no standard of care for relapsed patients. To help veterinary oncologists select effective drugs for dogs with lymphoid malignancies such as B-cell lymphoma, we have developed multimodal machine learning models that integrate data from multiple tumor profiling modalities and predict the likelihood of a positive clinical response for 10 commonly used chemotherapy drugs. Here we report on clinical outcomes that occurred after oncologists received a prediction report generated by our models. Remarkably, we found that dogs that received drugs predicted to be effective by the models experienced better clinical outcomes by every metric we analyzed (overall response rate, complete response rate, duration of complete response, patient survival times) relative to other dogs in the study and relative to historical controls.

ABSTRACT Infection with Pasteurella multocida represents a significant economic threat to Australian pig producers, yet our knowledge of its antimicrobial susceptibilities is lagging, and genomic characterization of P. multocida strains associated with porcine lower respiratory disease is internationally scarce. This study utilized high-throughput robotics to phenotypically and genetically characterize an industry-wide collection of 252 clinical P. multocida isolates that were recovered between 2014 and 2019. Overall, antimicrobial resistance was found to be low, with clinical resistance below 1% for all tested antimicrobials except those from the tetracycline class. Five dominant sequence types, representing 64.8% of all isolates, were identified; they were disseminated across farms and had previously been detected in various animal hosts and countries. P. multocida in Australian farms remain controllable via current antimicrobial therapeutic protocols. The identification of highly dominant, interspecies-infecting strains provides insight into the epidemiology of the opportunistic pathogen, and it highlights a biosecurity threat to the Australian livestock industry. IMPORTANCE Pasteurellosis is rated by the World Animal Health Organisation (OIE) as a high-impact disease in livestock. Although it is well understood in many host-disease contexts, our understanding of the organism in porcine respiratory disease is limited. Given its high frequency of involvement in porcine respiratory disease complex (PRDC), it is important that we are aware of its antimicrobial susceptibilities so that we can respond quickly and appropriately with antimicrobial therapy. Genetic insights about the organism can help us to better understand its epidemiology and inform our biosecurity practices and prophylactic management.

Abstract Background The global emergence of community-associated methicillin-resistant Staphylococcus aureus (CA-MRSA) has seen the dominance of specific clones in different regions around the world with the PVL-positive ST93-IV as the predominant CA-MRSA clone in Australia. In this study we applied a genome-wide association study (GWAS) approach on a collection of Australian ST93-IV MRSA genomes to screen for genetic traits that might have assisted the ongoing transmission of ST93-IV in Australia. We also compared the genomes of ST93-IV bacteraemia and non-bacteraemia isolates to search for potential virulence genes associated with bacteraemia. Results Based on single nucleotide polymorphism phylogenetics we revealed two distinct ST93-IV clades circulating concurrently in Australia. One of the clades contained isolates primarily isolated in the northern regions of Australia whilst isolates in the second clade were distributed across the country. Analyses of the ST93-IV genome plasticity over a 15-year period (2002–2017) revealed an observed gain in accessory genes amongst the clone’s population. GWAS analysis on the bacteraemia isolates identified two gene candidates that have previously been associated to this kind of infection. Conclusions Although this hypothesis was not tested here, it is possible that the emergence of a ST93-IV clade containing additional virulence genes might be related to the high prevalence of ST93-IV infections amongst the indigenous population living in the northern regions of Australia. More importantly, our data also demonstrated that GWAS can reveal candidate genes for further investigations on the pathogenesis and evolution of MRSA strains within a same lineage.

IntroductionHuman-snake interactions are common in tropical regions where subsistence-farming and livestock-herding activities predominate alongside proliferation of snakes. Local beliefs and perceptions about snakes and snakebites influence human behaviour. Understanding these beliefs and perceptions can inform the development of resources to drive behaviour change and to minimise the risk of injury to both humans and snakes. This qualitative study, conducted between May and July 2019, sought to explore the beliefs and perceptions regarding snakes and snakebites, and methods of prevention and management among members of the community in Kitui County, Kenya.MethodsSemi-structured interviews were used to collect qualitative data from 23 participants, recruited using a stratified purposeful sampling strategy in four selected sub-counties of Kitui county. Interview data was anonymised and coded and a thematic analysis was conducted using NVivo 12.ResultsPeople from Kitui county mostly had negative perceptions about snakes. There was a generalised awareness of the need to prevent snakebite, predominantly through keeping snakes away from homes/compounds. However, implementation was limited by financial constraints. Participants also identified logistic and financial obstacles to early hospital presentation following a snakebite, and they expressed a strong preference of having their snakebites treated in a hospital over consulting traditional healers. There was a universal recognition of the benefit of early intervention with a specific appreciation of the utility of the black stone. Furthermore, the removal of a snake's "teeth" was an expected treatment outcome for some community members, with the failure to do so perceived as causing poor wound healing or persistence of symptoms. Some religious groups held views which differed from most participants.ConclusionThere is a need to explore and clarify common misconceptions about snakes and first aid treatment of snakebites, encourage learning about the true nature of snakes, and highlight beneficial uses of snakes. A change in the epistemological conception of community education material by enhancing the value and use of local forms of knowledge, and the employment of art techniques to transmit this knowledge, could improve community perception and methods of snakebite prevention. Patient expectations should be appropriately managed by discussing possible outcomes, incorporating follow-up visits and addressing long-term complications of snakebites.

Lomentospora prolificans has caused outbreaks in immunocompromised patients. We performed whole genome sequencing (WGS) on 4 L. prolificans isolates from infections occurring during an 8-month period in the haematology unit at Hospital 1., and 2 isolates from unrelated infections at Hospital 2., showing a high number of mutational differences (>10,000 single nucleotide polymorphisms) between L. prolificans isolates from Hospital 1. Novel typing of isolates by WGS did not demonstrate a single causative strain.

Nonclinical immunotoxicity evaluation is an important component of safety assessment for pharmaceuticals. One in vitro assay that can be applied in a weight of evidence assessment is the human lymphocyte activation (HuLA) assay, an antigen recall assay, similar in many respects to the in vivo T-cell-dependent antibody response (TDAR) in that cooperation of multiple immune cell types are needed to produce responses. This assay uses human cells and is more amenable than the TDAR to compound ranking and mechanistic studies. The HuLA assay requires less time and drug than TDAR assays, uses a relevant antigen (influenza), reflects a human immune response, and applies principles of the 3Rs to non-clinical safety assessment. Peripheral blood mononuclear cells (PBMC) from flu-immunized donors are re-stimulated with flu-vaccine in the presence of test articles, and proliferation is measured. Published data demonstrate the applicability of the HuLA assay, but it has not been evaluated for reproducibility across testing sites. To evaluate assay reproducibility, scientists from a consortium of institutions conducted the assay in parallel, using a common pool of donor PBMC, influenza vaccine, and known immunosuppressant compounds (cyclosporine A and mycophenolic acid). The HuLA assay was highly reproducible in identification of inhibition of antigen-specific responses, and there was significant agreement across testing sites in the half maximal inhibitory concentration (IC50) values. Intra-site variability was the largest contributor to the variability observed within the assay. The HuLA assay was demonstrated to be ideally suited to comparing multiple compounds (i.e. compound ranking or benchmarking) within the same assay. Overall, the data reported herein support the HuLA assay as a useful tool in mechanistic evaluations of antigen-specific immune responses.

Introduction: Enterococcus faecium is an opportunistic pathogen that has become one of the leading causes of hospital acquired infection that are resistant to multiple critically important antimicrobials. Aim: The objective of the study was to describe the molecular characteristics and relationship between major strains of E. faecium harbouring the van operon and to determine if the strains had increasing virulence and antimicrobial resistance determinants over time. Methods: E. faecium harbouring the van operon detected using PCR from surveillance rectal swabs of patients that were admitted to high-risk units at a Perth teaching hospital from 2001 to 2015 were retrospectively analysed using a whole genome sequencing and bioinformatics approach. Results: ST18, ST78, ST80, ST173, ST203 and ST555 were identified as the major STs accounting for 93.7% of E. faecium isolates. Except for ST173, major STs identified at Royal Perth Hospital (RPH) have been reported across Australia and internationally. Isolates from each ST formed independently branched phylogenetic clusters with each harbouring unique virulence and antimicrobial resistance profiles. Depending on the ST, different genes conferring resistance to similar antimicrobial classes were identified. Except for ST80 which harboured the vanA type operon, all major strains harboured the vanB operon conferring only vancomycin resistance. Conclusion: Major strains of E. faecium isolated over 15-years showed unique virulome and resistome profiles with no indication of increasing virulence or antimicrobial resistance determinants. Strains were distantly related and the acquisition of different genes encoding similar antimicrobial resistances suggest the independent evolution of each strain. Data Summary: The whole genome sequences of all isolates from this study are accessible from the NCBI-SRA database under project number PRJNA575940 and PRJNA524213. Published reference sequence Aus0004 was obtained from NCBI-SRA under project number PRJNA86649 DOI:10.1128/JB.00259–12

Key aims of the WHO Strategy to halve snakebite morbidity and mortality include health system strengthening and training of health workers. This requires knowledge of local health system needs and capacity, health worker training needs, and factors influencing health worker decision-making in snakebite management. This study explored health worker experiences and perceptions of snakebite management, both individually and in the context of their local health system.We used a qualitative study design with semi-structured interviews (n = 14) and focus group discussions (n = 4). We employed a combination of sampling strategies aiming to achieve maximum variation among key informants within resource limitations. We recruited health workers (n = 33) of varying roles from purposively selected tier 2, 3 and 4 health facilities (n = 12) and the community (tier 1) in four sub-counties in Kitui County, Kenya. We conducted inductive thematic analysis of all transcripts.The results identified that health workers recognised snake envenoming as a time-critical emergency in which delay in care seeking, sometimes exacerbated by health system referral delays, was a major barrier to effective management of patients. Clinicians strongly voiced a need for training in snakebite management, diagnosis and antivenom administration. Unexpressed needs for training were demonstrated in traditional remedy ineffectiveness, syndromic management, and critical appraisal of treatment effectiveness. Under-resourcing in antivenom, other medication, equipment, infrastructure and staffing also challenged management. Fear of snakebite and fear of antivenom, both linked to past experiences, influenced clinical decision-making.Our findings clearly indicate a need in Kitui County for training programmes that equip health workers for clinical decision-making in snakebite management. We further identify community intervention needs to facilitate prompt presentation to healthcare, including practical affordable transport solutions, and systematic health system resourcing needs. In addition, we recommend supportive supervision and further research in response to the emotional stress resulting from managing difficult cases in under-resourced settings.

Whole-genome sequencing is essential to many facets of infectious disease research. However, technical limitations such as bias in coverage and tagmentation, and difficulties characterising genomic regions with extreme GC content have created significant obstacles in its use. Illumina has claimed that the recently released DNA Prep library preparation kit, formerly known as Nextera Flex, overcomes some of these limitations. This study aimed to assess bias in coverage, tagmentation, GC content, average fragment size distribution, and de novo assembly quality using both the Nextera XT and DNA Prep kits from Illumina. When performing whole-genome sequencing on Escherichia coli and where coverage bias is the main concern, the DNA Prep kit may provide higher quality results; though de novo assembly quality, tagmentation bias and GC content related bias are unlikely to improve. Based on these results, laboratories with existing workflows based on Nextera XT would see minor benefits in transitioning to the DNA Prep kit if they were primarily studying organisms with neutral GC content.

BACKGROUND:Over the last three decades, hospital adapted clonal complex (CC) 17 strains of Enterococcus faecium have acquired and exchanged antimicrobial resistance genes leading to the widespread resistance to clinically important antimicrobials globally. In Australia, a high prevalence of vancomycin resistance has been reported in E. faecium in the last decade. METHODS:In this study, we determined the phylogenetic relationship and genetic characteristics of E. faecium collected from hospitalized patients with blood stream infections throughout Australia from 2015 to 2017 using high throughput molecular techniques. RESULTS:Using single nucleotide polymorphism based phylogenetic inference, three distinct clusters of isolates were observed with additional sub-clustering. One cluster harboured mostly non-CC17 isolates while two clusters were dominant for the vanA and vanB operons. CONCLUSION:The gradual increase in dominance of the respective van operon was observed in both the vanA and vanB dominant clusters suggesting a strain-van operon affinity. The high prevalence of the van operon within isolates of a particular sub-cluster was linked to an increased number of isolates and 30-day all-cause mortality. Different dominant sub-clusters were observed in each region of Australia. Findings from this study can be used to put future surveillance data into a broader perspective including the detection of novel E. faecium strains in Australia as well as the dissemination and evolution of each strain.

Gran parte de las discusiones recientes sobre el positivismo jurídico sostienen que la controversia a propósito de éste se cifra en la distinción entre sus versiones incluyente y excluyente. Podemos obtener una perspectiva todavía más amplia de la discusión saliendo del ámbito del positivismo jurídico incluyente y excluyente trayendo a escena también al no-positivismo. En todo caso, en este ensayo quiero defender que existe una distinción más fundamental en otra parte, aún dentro del propio terreno positivista. La distinción que tengo en mente es la que se da entre el positivismo jurídico naturalizado (J. Austin) y el positivismo jurídico no naturalizado (H. Kelsen). Aún y cuando por razones institucionales el positivismo jurídico ha sido ampliamente discutido en el vacío, a menudo suele darse por hecho que existen vínculos entre el positivismo jurídico y el “positivismo en sentido amplio” como tradición o posición filosófica –o, como se diría en los círculos filosóficos en la actualidad–que existen vínculos entre el positivismo jurídico y el naturalismo. Mi tesis es que debe distinguirse claramente la empresa positivista de Kelsen del positivismo jurídico como naturalismo, donde el principio de separación es simplemente un corolario del naturalismo y en el cual no existe, por supuesto, tesis de normatividad nomológica alguna.

Using whole-genome sequencing, we identified a community-associated methicillin-resistant Staphylococcus aureus (CA-MRSA) sequence type (ST) 398 type V (5C2&5) isolate (typically found in China) in Australia in 2017. This CA-MRSA ST398 variant was highly virulent, similar to other related CA-MRSAs of ST398. This strain should be monitored to prevent more widespread dissemination.

In Australia in 2015, Candida auris sternal osteomyelitis was diagnosed in a 65-year-old man with a history of intensive care treatment in Kenya in 2012 and without a history of cardiac surgery. The isolate was South Africa clade III. Clinicians should note that C. auris can cause low-grade disease years after colonization.

First-line treatments of cancer do not always work, and even when they do, they cure the disease at unequal rates mostly owing to biological and clinical heterogeneity across patients. Accurate prediction of clinical outcome and survival following the treatment can support and expedite the process of comparing alternative treatments. We describe the methodology to dynamically determine remission probabilities for individual patients, as well as their prospects of progression-free survival (PFS). The proposed methodology utilizes the ex vivo drug sensitivity of cancer cells, their immunophenotyping results, and patient information, such as age and breed, in training machine learning (ML) models, as well as the Cox hazards model to predict the probability of clinical remission (CR) or relapse across time for a given patient. We applied the methodology using the three types of data obtained from 242 canine lymphoma patients treated by (L)-CHOP chemotherapy. The results demonstrate substantial enhancement in the predictive accuracy of the ML models by utilizing features from all the three types of data. They also highlight superior performance and utility in predicting survival compared to the conventional stratification method. We believe that the proposed methodology can contribute to improving and personalizing the care of cancer patients.

Prosthetic joint infection is a devastating complication of arthroplasty. Previous epidemiological studies have assessed factors associated with arthroplasty infections but have not assessed the impact of comorbidity on infection at different arthroplasty locations. We used a case-control design to investigate risk factors for prosthetic joint infection with reference to the anatomical site. During an eight-year period at a single hospital, 63 patients developed a prosthetic joint infection (36 hips, 27 knees). Cases of prosthetic hip or knee joint infection were matched 1:2 to controls. The results suggest that factors associated with arthroplasty infections differ with anatomical location. Following knee arthroplasty, wound discharge was associated with an increased risk of prosthetic joint infection whereas the presence of a drain tube reduced the risk. By contrast, increased body mass index, increased drain tube loss and superficial incisional surgical site infections (SSIs) were associated with prosthetic hip infection. When analysed as a combined cohort, systemic steroid use, increased SSI drain tube losses, wound discharge, and superficial incisional SSIs were predictors of infection. [ABSTRACT FROM AUTHOR]

In Western Australia, Neisseria meningitidis serogroup W clonal complex 11 became the predominant cause of invasive meningococcal disease in 2016. We used core-genome analysis to show emergence of a penicillin-resistant clade that had the penA_253 allele. This new penicillin-resistant clade might affect treatment regimens for this disease.

Objective: To undertake the first detailed genomic analysis of methicillin-resistant Staphylococcus aureus (MRSA) isolated in Sri Lanka.Methods: A prospective observational study was performed on 94 MRSA isolates collected over a 4 months period from the Anuradhapura Teaching Hospital, Sri Lanka. Screening for mecA, mecC, and the Panton-Valentine leucocidin (PVL)-associated lukS-PV/lukF-PV genes and molecular characterization by spa typing was undertaken. Whole genome sequencing (WGS) and phylogenetic analysis was performed on selected multilocus sequence type (MLST) clonal complex 5 (CC5) isolates from Sri Lanka, England, Australia, and Argentina.Results: All 94 MRSA harbored the mecA gene. Nineteen spa types belonging to nine MLST clonal complexes were identified. Where origin of the sample was recorded, most isolates were from skin and soft tissue infections (70/91; 76.9%), with fewer causing bacteremia (16/91; 17.6%), empyema (3/91; 3.3%) and osteomyelitis (2/91; 2.2%). Sixty two (65.9%) isolates were PVL positive with the majority (56 isolates; 90.3%) belonging to a dominant CC5 lineage. This lineage, PVL-positive ST5-MRSA-IVc, was associated with both community and hospital-onset infections. Based on WGS, representative PVL-positive ST5-MRSA-IVc isolates from Sri Lanka, England and Australia formed a single phylogenetic clade, suggesting wide geographical circulation.Conclusions: We present the most detailed genomic analysis of MRSA isolated in Sri Lanka to date. The analysis identified a PVL-positive ST5-MRSA-IVc that is prevalent among MRSA causing clinical infections in Sri Lanka. Furthermore, this clone was also found among isolates from the United Kingdom and Australia.

ABSTRACT We examined the oxacillin resistance phenotype and genomic structure of staphylococcal cassette chromosome mec (SCCmec) elements from 77 veterinary methicillin-resistant Staphylococcus pseudintermedius (MRSP) isolates. Isolates were characterized by oxacillin broth microdilution, whole-genome sequencing, and bioformatics analysis. Five previously described SCCmec elements, and a sixth novel element, were identified: SCCmec III (also known as II-III), ΨSCCmec57395, and SCCmecNA45 (a SCCmec VII variant), all previously described in MRSP, and SCCmec IVg and SCCmec VT, previously described in both methicillin-resistant Staphylococcus aureus (MRSA) and MRSP. The sixth element was novel and found among nine geographically clustered isolates. This novel pseudostaphylococcal cassette chromosome (ΨSCCmecKW21) contained a class A mec gene complex but lacked ccr genes. It also harbored heavy metal (cadmium) resistance determinants. The median oxacillin MIC values among ΨSCCmecKW21, SCCmec III, and SCCmec VT isolates were significantly higher than those determined for the SCCmecNA45 VII variant isolates and ΨSCCmec57395 and SCCmec IVg isolates. ΨSCCmecKW21 was found exclusively in sequence type 497 (ST497), an MRSP clone that is locally successful in Victoria, Australia. Future studies are necessary to determine if this clone has disseminated further afield and if ΨSCCmecKW21 has moved into other MRSP lineages or staphylococcal species. IMPORTANCE Staphylococcus pseudintermedius is a significant veterinary pathogen and occasional cause of infections in humans. β-Lactams are an important group of antimicrobials used to treat staphylococcal infections in humans and animals. However, when staphylococci become methicillin resistant via the acquisition of a mobile genetic element called staphylococcal cassette chromosome mec (SCCmec), they become resistant to all β-lactams. This study detected a novel SCCmec element among a cluster of methicillin-resistant S. pseudintermedius isolates from animals in Australia. It also detected SCCmec elements in S. pseudintermedius that had high similarity to those identified in methicillin-resistant Staphylococcus aureus, demonstrating how human and animal pathogens can share the same resistance determinants.

E. coli O157:H7 is a foodborne pathogen that constitutes a global threat to human health. However, the quantification of this pathogen in food and environmental samples may be problematic at the low cell numbers commonly encountered in environmental samples. In this study, we used recombinase polymerase amplification (RPA) for the detection of E. coli O157:H7, real-time quantitative PCR (qPCR) for quantification, and droplet digital PCR (ddPCR) for absolute and accurate quantification of E. coli O157:H7 from spiked and environmental samples. Primer and probe sets were used for the detection of stx1 and stx2 using RPA. Genes encoding for stx1, stx2, eae, and rfbE were used to quantify E. coli O157:H7 in the water samples. Furthermore, duplex ddPCR assays were used to quantify the pathogens in these samples. Duplex assay set 1 used stx1 and rfbE genes, while assay set 2 used stx2 and eae genes. Droplet digital PCR was used for the absolute quantification of E. coli O15:H7 in comparison with qPCR for the spiked and environmental samples. The RPA results were compared to those from qPCR and ddPCR in order to assess the efficiency of the RPA compared with the PCR methods. The assays were further applied to the dairy lagoon effluent (DLE) and the high rate algae pond (HRAP) effluent, which were fed with diluted DLE. The RPA detected was 4 CFU/mL with a high reproducibility. In addition, quantification by qPCR was from 103 to 107 CFU/mL of the wastewater samples. Therefore, the RPA assay has potential as a point of care tool for the detection of E. coli O157:H7 from different environmental sources, followed by quantification of the target concentrations.

Este estudo teve como objetivo desenvolver e verificar as evidências iniciais de validade e a consistência interna de uma Escala de Avaliação de Estressores Psicossociais no Contexto Laboral. Participaram do primeiro estudo 630 trabalhadores (mulheres = 51,5%, média de idade = 32,77; DP = 10,33) e, do segundo, 305 trabalhadores (mulheres = 44,3%; média de idade = 34,62; DP = 12,36). As análises fatoriais exploratórias indicaram uma estrutura fatorial latente de sete fatores, com índices de consistência interna aceitáveis. Tal estrutura foi replicada na análise fatorial confirmatória. As correlações dos fatores com os afetos negativos dirigidos ao trabalho e a satisfação no trabalho foram, em geral, nas direções esperadas. Concluiu-se que a escala apresentou evidências iniciais de validade e consistência interna, o que recomenda seu uso futuro para fins de diagnóstico e pesquisa.

From early 2012, a novel clone of vancomycin resistant Enterococcus faecium (assigned the multi locus sequence type ST796) was simultaneously isolated from geographically separate hospitals in south eastern Australia and New Zealand. Here we describe the complete genome sequence of Ef_aus0233, a representative ST796 E. faecium isolate. We used PacBio single molecule real-time sequencing to establish a high quality, fully assembled genome comprising a circular chromosome of 2,888,087 bp and five plasmids. Comparison of Ef_aus0233 to other E. faecium genomes shows Ef_aus0233 is a member of the epidemic hospital-adapted lineage and has evolved from an ST555-like ancestral progenitor by the accumulation or modification of five mosaic plasmids and five putative prophage, acquisition of two cryptic genomic islands, accrued chromosomal single nucleotide polymorphisms and a 80 kb region of recombination, also gaining Tn1549 and Tn916, transposons conferring resistance to vancomycin and tetracycline respectively. The genomic dissection of this new clone presented here underscores the propensity of the hospital E. faecium lineage to change, presumably in response to the specific conditions of hospital and healthcare environments.

Orientation: This article addresses the issues of executive remuneration and whether it was excessive or not. Research purpose: On 05 August 2015, the US Securities and Exchange Commission (SEC) adopted a rule to operationalise Section 953(b) of the Dodd-Frank Wall Street Reform and Consumer Protection Act 2010 (Dodd-Frank or the Dodd-Frank Act 2010), the ‘pay ratio provision’, as part of a process to ensure sound corporate governance and shed light on assertions that corporate executive remuneration was excessive and detrimental to the economic wellbeing of the USA. This pay ratio rule will be operative starting from 2017 and requires public firms to publish the ratio of chief executive officer (CEO) remuneration to the median remuneration of all its employees. Hence, it is a measure of income distribution. It does not reveal the relationship between executive compensation and the value added to the firm by executives. In anticipation of this rule becoming mandatory and as part of a quest to quantify the value of executives to the firm, Paulo and Le Roux (2014) developed an approach to measure the value executives add to the firm, drawing from audited financial statements and thereby demonstrating that the value added by executive management could be measured according to the requirements of sound research methodology and rigorous epistemology. Motivation for the study: Statutory enactment of the pay ratio provision provided the impetus to create an index, the Paulo–Le Roux Index, that shows how much executives are paid in relation to how much value they add to the firm. Research design, approach and method: Paulo and Le Roux (2014) developed an approach to measure the value executives add to the firm, drawing from audited financial statements and thereby demonstrating that the value added by executive management could be measured according to the requirements of sound research methodology and rigorous epistemology. Statutory enactment of the pay ratio provision provided the impetus to create an index, the Paulo–Le Roux Index, that shows how much executives are paid in relation to how much value they add to the firm. The value added to the firm is a composite of the value drivers, sales, growth, capital requirements (CR), operating profitability (OP), and the discount rate in the form of a weighted average cost of capital (WACC). Main findings: Discussions that hitherto have been normative regarding executive remuneration, and unrelated to the value created by executives, can now be based on rigorous valuations that draw from audited financial statements. Practical/managerial implications: Numerous advantages accrue from the use of this index for all stakeholders, managers, organised labour, investors, as well as for asset allocation and corporate restructuring, the risk incurred in adding value, and the strategies applied. This index can be used for any enterprise, division, functional area, or project, and for any financial period for which audited financial statements are available. Contribution: Using the index ensures sound corporate governance and shed light on assertions that corporate executive remuneration was excessive and detrimental to the economic wellbeing.

This work investigated the molecular epidemiology and antimicrobial resistance of methicillin-resistant Staphylococcus aureus (MRSA) isolated from veterinarians in Australia in 2009. The collection (n = 44) was subjected to extensive molecular typing (MLST, spa, SCCmec, dru, PFGE, virulence and antimicrobial resistance genotyping) and antimicrobial resistance phenotyping by disk diffusion. MRSA was isolated from Australian veterinarians representing various occupational emphases. The isolate collection was dominated by MRSA strains belonging to clonal complex (CC) 8 and multilocus sequence type (ST) 22. CC8 MRSA (ST8-IV [2B], spa t064; and ST612-IV [2B], spa variable,) were strongly associated with equine practice veterinarians (OR = 17.5, 95% CI = 3.3-92.5, P < 0.001) and were often resistant to gentamicin and rifampicin. ST22-IV [2B], spa variable, were strongly associated with companion animal practice veterinarians (OR = 52.5, 95% CI = 5.2-532.7, P < 0.001) and were resistant to ciprofloxacin. A single pig practice veterinarian carried ST398-V [5C2], spa t1451. Equine practice and companion animal practice veterinarians frequently carried multiresistant-CC8 and ST22 MRSA, respectively, whereas only a single swine specialist carried MRSA ST398. The presence of these strains in veterinarians may be associated with specific antimicrobial administration practices in each animal species.

Age-related macular degeneration (AMD) is the leading cause of irreversible blindness in the elderly population worldwide. Although recent studies have demonstrated strong genetic associations between AMD and SNPs in a number of genes, other modes of regulation are also likely to play a role in the etiology of this disease. We identified a significantly decreased level of methylation on the IL17RC promoter in AMD patients. Furthermore, we showed that hypomethylation of the IL17RC promoter in AMD patients led to an elevated expression of its protein and messenger RNA in peripheral blood as well as in the affected retina and choroid, suggesting that the DNA methylation pattern and expression of IL17RC may potentially serve as a biomarker for the diagnosis of AMD and likely plays a role in disease pathogenesis.

O estudo teve como objetivo desenvolver e validar uma escala para avaliação dos afetos dirigidos ao contexto do trabalho. A amostra foi composta por 293 trabalhadores de ambos os sexos, que responderam a uma versão inicial da escala de afetos, com 40 itens, bem como a uma escala de satisfação no trabalho e a uma escala de exaustão emocional. A análise fatorial exploratória resultou na elaboração de uma versão final da escala de afetos composta de 28 itens, distribuídos em duas dimensões - afetos positivos e afetos negativos, e índices de consistência interna de 0,93 e 0,90, respectivamente. As correlações da versão final da escala de afetos com a satisfação no trabalho e a exaustão emocional atestaram sua validade convergente e discriminante. Concluiu-se que as características psicométricas da escala de afetos no trabalho recomendam seu uso em pesquisas brasileiras destinadas a avaliar a freqüência dos afetos dirigidos ao contexto do trabalho.

Age-related macular degeneration (AMD) is a common yet complex retinal degeneration that causes irreversible central blindness in the elderly. Pathology is widely believed to follow loss of retinal pigment epithelium (RPE) and photoreceptor degeneration. Here we report aberrant expression of interleukin-17A (IL17A) and the receptor IL17RC in the macula of AMD patients. In vitro, IL17A induces RPE cell death characterized by the accumulation of cytoplasmic lipids and autophagosomes with subsequent activation of pro-apoptotic Caspase-3 and Caspase-9. This pathology is reduced by siRNA knockdown of IL17RC. IL17-dependent retinal degeneration in a mouse model of focal retinal degeneration can be prevented by gene therapy with adeno-associated virus vector encoding soluble IL17 receptor. This intervention rescues RPE and photoreceptors in a MAPK-dependent process. The IL17 pathway plays a key role in RPE and photoreceptor degeneration and could hold therapeutic potential in AMD.

Abstract Placental hCG and pitutary LH transduce signals in target tissues through a common receptor (LHCGR). We demonstrate that recombinant LHCGR proteins which include the hormone-binding domain are secreted from transfected cells and that natural LHCGR is also secreted from human placental explants. LHCGR recombinant proteins representing varying lengths of the N-terminal extracellular domain were expressed in Chinese Hamster Ovary cells in suspension culture. Secretion was minimal up to 72h but by 96h 24-37% of the LHCGR had been released into the culture medium. The secreted proteins were folded and sensitive to glycosidases suggesting N-linked glycosylation. Secretion was independent of recombinant size and was mediated via structurally defined membrane vesicles (50-150nm). Similarly cultured human early pregnancy placental explants also released LHCGR via microvesicles. These studies provide the first experimental evidence of the possible mechanistic basis of the secretion of LHCGR.

O presente estudo investigou o impacto das atitudes frente às mudanças nas organizações nos comportamentos de cidadania. A pesquisa contou com a participação de 304 funcionários de ambos os sexos, provenientes de duas empresas localizadas no Rio de Janeiro, sendo uma do segmento bancário e outra do segmento educacional, que responderam à Escala de Atitudes frente à Mudança Organizacional e à Escala de Civismo nas Organizações. Os resultados das análises de regressões múltiplas lineares hierárquicas evidenciaram que as atitudes de aceitação às mudanças tiveram um impacto positivo nos comportamentos de cidadania organizacional, que as atitudes de ceticismo frente às mudanças interferiram negativamente nos comportamentos de proteção ao sistema e de criação de clima favorável e que, quanto menor a idade, maior a propensão a apresentar comportamentos de cidadania organizacional voltados para o oferecimento de sugestões criativas para o sistema, de proteção ao sistema e de criação de clima favorável à organização. Tais resultados são discutidos à luz da teoria da troca social, e, à guisa de conclusão, são propostas algumas ações estratégicas destinadas a suscitar atitudes positivas em tempos de mudanças nas organizações, capazes, assim, de estimular os comportamentos de cidadania organizacional de seus membros.

El presente estudio investigó el impacto de las actitudes frente a las mudanzas en las organizaciones en los comportamientos de ciudadanía. La pesquisa contó con la participación de 304 funcionarios de ambos los sexos, provenientes de dos empresas localizadas en Rio de Janeiro, siendo una del segmento bancario y otra del segmento educacional, que respondieron a la Escala de Actitudes frente a la Mudanza Organizacional y a la Escala de Civismo en las Organizaciones. Los resultados de los análisis de regresiones múltiples lineales jerárquicas evidenciaron que las actitudes de aceptación a las mudanzas tuvieron un impacto positivo en los comportamientos de ciudadanía organizacional, que las actitudes de escepticismo frente a las mudanzas interfirieron negativamente en los comportamientos de protección al sistema y de creación de clima favorable, y que, cuanto menor la edad, mayor la propensión a presentar comportamientos de ciudadanía organizacional dirigidos hacia el ofrecimiento de sugestiones creativas para el sistema, de protección al sistema y de creación de clima favorable a la organización. Tales resultados son discutidos a la luz de la teoría del intercambio social, y, a guisa de conclusión, son propuestas algunas acciones estratégicas destinadas a suscitar actitudes positivas en tiempos de mudanzas en las organizaciones, capaces, así, de estimular los comportamientos de ciudadanía organizacional de sus miembros.

The jerantinine family of Aspidosperma indole alkaloids from Tabernaemontana corymbosa are potent microtubule-targeting agents with broad spectrum anticancer activity. The natural supply of these precious metabolites has been significantly disrupted due to the inclusion of T. corymbosa on the endangered list of threatened species by the International Union for Conservation of Nature. This report describes the asymmetric syntheses of (-)-jerantinines A and E from sustainably sourced (-)-tabersonine, using a straight-forward and robust biomimetic approach. Biological investigations of synthetic (-)-jerantinine A, along with molecular modelling and X-ray crystallography studies of the tubulin-(-)-jerantinine B acetate complex, advocate an anticancer mode of action of the jerantinines operating via microtubule disruption resulting from binding at the colchicine site. This work lays the foundation for accessing useful quantities of enantiomerically pure jerantinine alkaloids for future development.

Objective: This article describes how maps were developed from the clinical modifications of the 9th and 10th revisions of the International Classification of Diseases (ICD) to the Abbreviated Injury Scale 2005 Update 2008 (AIS08). The development of the mapping methodology is described, with discussion of the major assumptions used in the process to map ICD codes to AIS severities. There were many intricacies to developing the maps, because the 2 coding systems, ICD and AIS, were developed for different purposes and contain unique classification structures to meet these purposes., Methods: Experts in ICD and AIS analyzed the rules and coding guidelines of both injury coding schemes to develop rules for mapping ICD injury codes to the AIS08. This involved subject-matter expertise, detailed knowledge of anatomy, and an in-depth understanding of injury terms and definitions as applied in both taxonomies. The official ICD-9-CM and ICD-10-CM versions (injury sections) were mapped to the AIS08 codes and severities, following the rules outlined in each coding manual. The panel of experts was composed of coders certified in ICD and/or AIS from around the world. In the process of developing the map from ICD to AIS, the experts created rules to address issues with the differences in coding guidelines between the 2 schemas and assure a consistent approach to all codes., Results: Over 19,000 ICD codes were analyzed and maps were generated for each code to AIS08 chapters, AIS08 severities, and Injury Severity Score (ISS) body regions. After completion of the maps, 14,101 (74%) of the eligible 19,012 injury-related ICD-9-CM and ICD-10-CM codes were assigned valid AIS08 severity scores between 1 and 6. The remaining 4,911 codes were assigned an AIS08 of 9 (unknown) or were determined to be nonmappable because the ICD description lacked sufficient qualifying information for determining severity according to AIS rules. There were also 15,214 (80%) ICD codes mapped to AIS08 chapter and ISS body region, which allow for ISS calculations for patient data sets., Conclusion: This mapping between ICD and AIS provides a comprehensive, expert-designed solution for analysts to bridge the data gap between the injury descriptions provided in hospital codes (ICD-9-CM, ICD-10-CM) and injury severity codes (AIS08). By applying consistent rules from both the ICD and AIS taxonomies, the expert panel created these definitive maps, which are the only ones endorsed by the Association for the Advancement of Automotive Medicine (AAAM). Initial validation upheld the quality of these maps for the estimation of AIS severity, but future work should include verification of these maps for MAIS and ISS estimations with large data sets. These ICD-AIS maps will support data analysis from databases with injury information classified in these 2 different systems and open new doors for the investigation of injury from traumatic events using large injury data sets.

Natural products with anti-cancer activity play a vital role in lead and target discovery. We report here the synthesis and biological evaluation of the plant-derived alkaloid, piperlongumine and analogues. Using a Horner-Wadsworth-Emmons coupling approach, a selection of piperlongumine-like compounds were prepared in good overall yield from a novel phosphonoacetamide reagent. A number of the compounds displayed potent anti-cancer activity against colorectal (HCT 116) and ovarian (IGROV-1) carcinoma cell lines, via a mechanism of action which may involve ROS generation. Contrary to previous reports, no selective action in cancer cell (MRC-5) was observed for piperlongumine analogues.

We present a selection of elegant and diverse biomimetic syntheses of complex natural product dimers. The dimerisation pathways discussed encompass the most prevalent strategies, including: Diels-Alder, Aldol, Mannich, conjugate addition, oxidative, radical and photochemical approaches; each underpinned through rational biosynthetic speculation.

The management of prosthetic joint infections remains a clinical challenge, particularly infections due to methicillin-resistant staphylococci. Previously, this infection was considered a contraindication to debridement and retention strategies. This retrospective cohort study examined the treatment and outcomes of patients with arthroplasty infection by methicillin-resistant staphylococci managed by debridement and retention in conjunction with rifampin-fusidic acid combination therapy. Over an 11-year period, there were 43 patients with infection by methicillin-resistant staphylococci managed with debridement and retention. This consisted of close-interval repeated arthrotomies with pulsatile lavage. Rifampin was combined with fusidic acid for the majority of patients (88%). Patients were monitored for a median of 33.5 months (interquartile range, 20 to 54 months). Overall, 9 patients experienced treatment failure, with 12- and 24-month estimates of infection-free survival of 86% (95% confidence interval [CI], 71 to 93%) and 77% (95% CI, 60 to 87%), respectively. The following factors were associated with treatment failure: methicillin-resistant Staphylococcus aureus (MRSA) arthroplasty infection, a single surgical debridement or ≥4 debridements, and the receipt of less than 90 days of antibiotic therapy. Patients with infection by methicillin-resistant coagulase-negative staphylococci (MR-CNS) were less likely to fail treatment. The overall treatment success rate reported in this study is comparable to those of other treatment modalities for prosthetic joint infections by methicillin-resistant staphylococci. Therefore, the debridement and retention of the prosthesis and rifampin-based antibiotic therapy are a valid treatment option for carefully selected patients.

Information is required about treatment outcomes of Gram-negative prosthetic joint infections treated with prosthesis retention and surgical debridement, especially where biofilm-active antibiotics such as fluoroquinolones are used. The outcome of 17 consecutive patients with an early Gram-negative prosthetic joint infection who had been treated with prosthesis retention and surgical debridement was analysed. Enterobacteriaceae were isolated in 16 patients and infections were mixed with other organisms in 13 (76%) patients. The median joint age was 17 days and the median duration of symptoms before debridement was 7 days. All patients initially received intravenous β-lactam antibiotic therapy and 14 patients were then treated with oral ciprofloxacin. Treatment failure occurred in two patients over a median period of follow-up of 28 months. In only one patient was a relapsed Gram-negative infection responsible for the failure and this patient had not been treated with ciprofloxacin. The 2-year survival rate free of treatment failure was 94% (95% CI, 63-99%). Prosthesis retention with surgical debridement, in combination with antibiotic regimens including ciprofloxacin, was effective and should be considered for patients with early Gram-negative prosthetic joint infection., (2010 The Authors. Clinical Microbiology and Infection; 2010 European Society of Clinical Microbiology and Infectious Diseases.)

An Eritrean-born man observed over an extended period had upper gastrointestinal symptoms, fever, hepatosplenomegaly and pancytopenia in the setting of advanced HIV infection and poor adherence to antiretroviral therapy. Despite thorough investigation, it was not until a repeat gastroscopic examination and gastric biopsy were performed 18 months after initial presentation that Leishmania infection was diagnosed. The species was identified by polymerase chain reaction assay as L. donovani. Physicians managing HIV-infected patients from regions where Leishmania is endemic should consider visceral leishmaniasis, even in patients who have not lived in a Leishmania-endemic region for many years.

There is growing evidence of the efficacy of treating early staphylococcal infections of prosthetic joints with surgical debridement and prosthesis retention, combined with oral antibiotic regimens that include rifampicin in combination with a fluoroquinolone. With rising rates of fluoroquinolone-resistant staphylococci, evidence concerning the efficacy of alternative combinations of antibiotics is required. Twenty patients with staphylococcal prosthetic joint infections who had been treated with surgical debridement and prosthesis retention, and a combination of rifampicin and fusidic acid were analysed. The mean duration of symptoms before initial debridement was 16 (range 2-75) days. The median time of follow-up was 32 (range 6-76) months. Treatment failure occurred in two patients. The cumulative risk of treatment failure after 1 year was 11.76% (95% CI 3.08-39.40%). Two patients had their treatment changed because of nausea. Ten of 11 patients with infections involving methicillin-resistant Staphylococcus aureus had successful outcomes. Debridement without prosthesis removal, in combination with rifampicin and fusidic acid treatment, was effective and should be considered for patients with early staphylococcal prosthetic joint infections, including those with infections involving fluoroquinolone-resistant organisms.

Impaired lymph drainage is an inevitable consequence of any form of surgery that disrupts lymphatics, resulting in a degree of lymphoedema that may vary from subtle to dramatic and although classically involving an entire limb, may be more localised, confined to only a small area such as a skin flap. Infection is a well-recognised complication of lymphoedema. However, not all inflammatory episodes occurring in the setting of lymphatic dysfunction can be clearly attributed to infection as this article demonstrates. Five patients presented over a 5-year period with distinctive erysipelas-like inflammation affecting the breast which occurred several weeks following reduction mammaplasty in four patients and breast reconstruction in one patient. No clinical response was obtained with standard antibiotics. This inflammatory problem may represent a previously unreported complication of breast surgery with an incidence of 4% following reduction mammaplasty. Recent research supports the notion that this type of episode is most likely to be due to a non-infective inflammatory process related to lymphatic dysfunction induced by surgery.

Disseminated tuberculosis is notoriously difficult to diagnose and, with the decrease in tuberculosis incidence in Australia, familiarity with its manifestations has dwindled. We describe four bacteriologically proven cases which illustrate the range of presentations and diagnostic difficulties. Surprisingly, immunosuppressive therapy need not cause rapid deterioration. Disseminated tuberculosis should be considered in any patient with multisystem illness who is at risk of tuberculosis, particularly if born overseas. In the absence of confirmatory results, a prompt therapeutic trial may be life-saving.