Prevalence of antimicrobial resistance at two private veterinary referral hospitals.

Antimicrobial resistance (AMR) is an emerging human and animal issue. The frequency of resistance to high importance antimicrobials, isolation of microbes of One Health importance and the nature and frequency of multi-drug resistant (MDR) profiles in Australian small animal referral practice have not been described previously. Medical databases of two private small animal referral hospitals in Queensland, Australia were reviewed for culture and susceptibility (C&S) results from 1 January to 31 December 2020. Hospital site (H1 and H2), culture sample, C&S results and MDR were documented for samples from services operating at both locations. There were 631 microbial isolates and 386 susceptibility profiles from 438 samples. The predominant organism was Staphylococcus pseudintermedius at H1 (n = 95) and Escherichia coli at H2 (n = 23). The majority of samples were integumentary (H1 n = 216, H2 n = 75) or urogenital (H1 n = 74, H2 n = 70). MDR isolates were reported at both hospitals, and were significantly more likely at H1 (69/262, 26.3% vs. 12/121, 9.9%; P < 0.001). High levels of AMR including MDR profiles were reported at the two hospitals evaluated, but they had significantly different resistance patterns and microbial profiles. These results highlight the need to practice appropriate antimicrobial stewardship in veterinary medicine, and are supportive for individual hospital surveillance with antibiograms.

Estimated glomerular filtration rate progression in UK primary care patients with type 2 diabetes and diabetic kidney disease: a retrospective cohort study.

AIMS: To examine the rates of diabetic kidney disease (DKD) progression and associated factors, we undertook a study of estimated glomerular filtration rate (eGFR) in a historical cohort of UK primary care patients with type 2 diabetes mellitus (T2DM) and associated DKD from the Clinical Practice Research Datalink. METHODS: Our eligible population were patients with definitive T2DM from a recorded diagnostic code with either a diagnosis of chronic kidney disease (CKD) or renal function test values and renal abnormalities consistent with a CKD diagnosis, identified between 1 October 2006 and 31 December 2011. Only patients with albuminuria results reported in mg/l were used for the longitudinal statistical analyses of the eGFR rate of change using multilevel models. RESULTS: We identified 111,030 patients with T2DM. Among them 58.6% (95% confidence interval (CI): 58.3-58.9) had CKD and 37.2% (95% CI: 36.9-37.5%) had presumed DKD at baseline. Only 19.4% of patients had urinary albumin test results expressed as mg/l in the year prior to index date. Almost two-thirds (63.8%) of patients with T2DM and presumed DKD received prescriptions for angiotensin-converting enzyme (ACE) inhibitors or angiotensin type 1 receptor blockers (ARB) or both. Time-dependent variables that predict subsequent eGFR decline include increased albuminuria, time from index date and older age. CONCLUSION: Only a minority of diabetic patients with DKD had quantitative albuminuria assessments. The relatively low proportion of DKD patients with ACEi or ARB prescriptions suggests a gap between healthcare practice and available scientific evidence during the study period. Increased albuminuria and older age were the most consistent predictors of subsequent eGFR decline.

Patient characteristics and treatment patterns for patients with benign prostatic hyperplasia, erectile dysfunction or co-occurring benign prostatic hyperplasia and erectile dysfunction in general practices in the UK: a retrospective observational study.

AIMS: The aim of this study was to assess patient characteristics, medication treatment patterns and healthcare resource utilization among men with existing erectile dysfunction (ED) or benign prostatic hyperplasia (BPH), who are newly diagnosed with the second condition (BPH or ED) compared with those with only one condition. METHODS: This retrospective cohort study utilized the Clinical Practice Research Datalink. Males, aged 40 years or older, newly diagnosed with ED or symptomatic BPH between 1 June 2010 and 31 May 2011, were selected. Patient demographics, existing comorbidities and baseline medication use were analysed. Treatments initiated for the incident condition and treatment patterns were reported at 6, 12, 18 and 24-months postdiagnosis. Referrals to urologists and visits to general practitioners were reported around diagnosis and during follow-up. RESULTS: This study included 11,501 incident patients with BPH, of which 23% had a prior ED diagnosis and 9,734 incident patients with ED, of which 17% had a prior BPH diagnosis. The average age at diagnosis of BPH was similar across both cohorts. Among incident patients with ED, those with prior diagnosis of BPH were diagnosed at an older average age (65 ± 9.2 years) compared to those without BPH (57 ± 9.1 years). The majority of patients in both incident BPH cohorts (62.9-65.5%) were prescribed alpha-blockers as initial treatment. The majority of patients in both incident ED cohorts (49.6-51.6%) were prescribed sildenafil as initial treatment followed by tadalafil (24.3-26.0%). At 12 months, 50% of incident patients with BPH and 80% of patients with ED had discontinued the therapy initiated. CONCLUSION: This study found that in the UK, patients with co-occurring BPH and ED when newly diagnosed with the second condition initiated the same treatments as those without prior ED or BPH. During the first year, treatment patterns including discontinuation were comparable in the groups with one of the conditions and co-occurring BPH and ED.

Use of serology and urine antigen detection to estimate the proportion of adult community-acquired pneumonia attributable to Streptococcus pneumoniae.

Streptococcus pneumoniae is a common cause of community-acquired pneumonia (CAP) but existing diagnostic tools have limited sensitivity and specificity. We enrolled adults undergoing chest radiography at three Indian Health Service clinics in the Southwestern United States and collected acute and convalescent serum for measurement of PsaA and PspA titres and urine for pneumococcal antigen detection. Blood and sputum cultures were obtained at the discretion of treating physicians. We compared findings in clinical and radiographic CAP patients to those in controls without CAP. Urine antigen testing showed the largest differential between CAP patients and controls (clinical CAP 13%, radiographic CAP 17%, control groups 2%). Serological results were mixed, with significant differences between CAP patients and controls for some, but not all changes in titre. Based on urine antigen and blood culture results, we estimated that 11% of clinical and 15% of radiographic CAP cases were due to pneumococcus in this population.

Measuring the incidence of adult community-acquired pneumonia in a Native American community.

Few population-based studies have investigated the epidemiology of adult community-acquired pneumonia (CAP). We aimed to determine the incidence of CAP in a population at high-risk for pneumococcal disease and to evaluate a standardized method for interpreting chest radiographs adapted from the World Health Organization paediatric chest radiograph interpretation guidelines. We reviewed radiology records at the two healthcare facilities serving the White Mountain Apache tribe to identify possible pneumonia cases > or =40 years of age. We categorized patients with clinical criteria and a physician diagnosis of pneumonia as clinical CAP and those with clinical criteria and an acute infiltrate as radiographic CAP. We identified 100 (27/1000 person-years) and 60 (16/1000 person-years) episodes of clinical and radiographic CAP, respectively. The incidence of CAP increased with age. Both radiographic and clinical CAP were serious illnesses with more than half of patients hospitalized. Our case definitions and methods may be useful for comparing data across studies and conducting vaccine trials.

Metal-catalyzed oxidation induces carbonylation of peroxisomal proteins and loss of enzymatic activities.

Peroxisomes are involved in oxidative metabolic reactions and have the capacity to generate large amounts of reactive oxygen species that could damage biomolecules including their own resident proteins. The purpose of this study was to determine whether peroxisomal proteins are susceptible to oxidation and whether oxidative damage affects their enzymatic activity. Peroxisomal proteins were subjected to metal-catalyzed oxidation (MCO) with CuCl(2)/ascorbate and derivatized with 2,4-dinitrophenylhydrazine which allowed for spectrophotometric quantification of carbonylation. Immunochemical detection of carbonylated peroxisomal proteins, resolved by gel electrophoresis and detected with anti-DNP antibodies, revealed five oxidatively modified proteins with the following molecular weights: 80, 66, 62, 55, and 50 kDa. The proteins at 66, 62, and 55 kDa were identified as malate synthase (MS), isocitrate lyase, and catalase (CAT), respectively. MS and CAT were estimated to contain 2-3 mol of carbonyl/mol of protein as a result of MCO. Enzymatic assays revealed varying degrees of activity loss. Isocitrate lyase and malate synthase showed significant loss of activity while catalase and malate dehydrogenase were less inhibited by carbonylation. Our findings show that peroxisomal proteins are vulnerable to MCO damage and thus may also be affected by in vivo exposure to reactive oxygen species.

Linkage disequilibrium maps constructed with common SNPs are useful for first-pass disease association screens.

To develop an efficient strategy for mapping genetic factors associated with common diseases, we constructed linkage disequilibrium (LD) maps of human chromosomes 5, 7, 17, and X. These maps consist of common single nucleotide polymorphisms at an average intermarker distance of 100 kb. The genotype data from these markers in a panel of American samples of European descent were analyzed to produce blocks of markers in strong pair-wise LD. Power calculations were used to guide block definitions and predicted that high-level LD maps would be useful in initial genome scans for susceptibility alleles in case-control association studies of complex diseases. As anticipated, LD blocks on the X chromosome were larger and covered more of the chromosome than those found on the autosomes.

A method for parallel, automated, thermal cycling of submicroliter samples.

A large fraction of the cost of DNA sequencing and other DNA-analysis processes results from the reagent costs incurred during cycle sequencing or PCR. In particular, the high cost of the enzymes and dyes used in these processes often results in thermal cycling costs exceeding $0.50 per sample. In the case of high-throughput DNA sequencing, this is a significant and unnecessary expense. Improved detection efficiency of new sequencing instrumentation allows the reaction volumes for cycle sequencing to be scaled down to one-tenth of presently used volumes, resulting in at least a 10-fold decrease in the cost of this process. However, commercially available thermal cyclers and automated reaction setup devices have inherent design limitations which make handling volumes of <1 microL extremely difficult. In this paper, we describe a method for thermal cycling aimed at reliable, automated cycling of submicroliter reaction volumes.

Single-nucleotide polymorphisms in the public domain: how useful are they?

There is a concerted effort by a number of public and private groups to identify a large set of human single-nucleotide polymorphisms (SNPs). As of March 2001, 2.84 million SNPs have been deposited in the public database, dbSNP, at the National Center for Biotechnology Information (http://www.ncbi.nlm.nih.gov/SNP/). The 2.84 million SNPs can be grouped into 1.65 million non-redundant SNPs. As part of the International SNP Map Working Group, we recently published a high-density SNP map of the human genome consisting of 1.42 million SNPs (ref. 3). In addition, numerous SNPs are maintained in proprietary databases. Our survey of more than 1,200 SNPs indicates that more than 80% of TSC and Washington University candidate SNPs are polymorphic and that approximately 50% of the candidate SNPs from these two sources are common SNPs (with minor allele frequency of > or =20%) in any given population.

Differences in the susceptibility of some penaeid prawn species to gill-associated virus (GAV) infection.

Four species of penaeid prawn cultured in Australia (Penaeus monodon, Penaeus esculentus, Marsupenaeus japonicus and Fenneropenaeus merguiensis) were injected with a virulent preparation of gill-associated virus (GAV). P. monodon (average weight = 8.9, 13.9 and 19.2 g), P. esculentus (average weight = 19.5 g), F. merguiensis (average weight = 10.5 g), and small (average weight = 5.8 g) M. japonicus displayed overt signs of disease and mortalities which reached 82 to 100% within 23 d post-injection. Cumulative mortalities in P. esculentus and F. merguiensis were significantly lower than for P. monodon of the same size class. Medium (average weight = 13.0 g) M. japonicus also developed overt signs of disease but cumulative mortalities were not significantly higher than uninfected controls. Large (average weight = 20.3 g) M. japoncius did not display symptoms of disease and there were no significant mortalities up to 23 d post-injection.

Nutritional requirements of Xylella fastidiosa, which causes Pierce's disease in grapes.

A defined medium (XF-26) containing 3 inorganic salts, 2 tricarboxylic acids, 17 amino acids, potato starch, phenol red, and agar was used as the starting point for the study. Deletions of one or more ingredients were performed to prepare various media. A medium was considered able to support growth of Xylella fastidiosa strains responsible for Pierce's disease in grapes, only after 10 serial passages had been completed. Of 3 inorganic salts, K2HPO4 and MgSO4 x 7H2O were essential, and (NH4)2HPO4 was nonessential for growth. Of the Krebs cycle intermediates, all (citrate, alpha-ketoglutarate, succinate, fumarate, malate, and oxaloacetate) but isocitrate supported growth of cultivated strains, whereas only citrate alone or citrate plus succinate supported the primary isolation of PD bacterium. Of 17 amino acids, 6 uncharged polar R groups (asparagine, cysteine, glutamine, glycine, serine, and threonine) supported growth, whereas 8 nonpolar R groups (alanine, isoleucine, leucine, methionine, phenylalanine, proline, tryptophan, and valine) or 3 positively charged polar groups (arginine, histidine, and lysine) did not. Starch proved to be nonessential.

A kinetic enzyme immunoassay for the quantitation of antibodies to a humanized monoclonal antibody in human serum.

A kinetic enzyme immunoassay was developed and validated to quantitate human antibodies to the humanized monoclonal antibody CAMPATH1-1H (C1H) in human serum. The assay was configured using C1H-coated 96-well plates which were blocked with bovine serum albumin, and incubated with dilutions of human serum containing anti-C1H antibody. Antibody was detected using biotinylated C1H followed by streptavidin-conjugated alkaline phosphatase and p-nitrophenyl phosphate. Absorbance data were collected for 10 min, and mOD min-1 data were exported to MultiCalc data analysis software. A 4-parameter logistic-log algorithm was shown to model the data through the range of the standard curve within 15% of nominal values. The overall assay performance coefficient of variation by ANOVA was 9.2%. The lower limit of detection was defined at 160 Units ml-1. The anti-idiotype antibody standard stock solution is stable at 4 degrees C and at -80 degrees C for at least 11 months in buffer. The anti-idiotype antibody controls are stable for at least seven freeze-thaw cycles and at least 6 months in human serum stored at -20 degrees C. A strategy was devised by which to establish the specific antibody potency for any given batch of anti-C1H antibody standard relative to the Reference Standard. This EIA has been used to quantify and characterize anti-C1H antibody in human serum in support of clinical safety and efficacy studies.

Estrogen receptor alpha is essential for induction of oxytocin receptor by estrogen.

The induction of oxytocin receptor (OTR) synthesis in the periphery and in the brain by estrogen is critical for reproductive success. Oxytocin receptors are involved in the control of parturition, milk ejection, and sexual and maternal behaviors. The discovery of a second estrogen receptor (ERbeta) in the brain and the failure of in vitro transcription studies using OTR promoter constructs to replicate the in vivo transcriptional regulation have raised questions regarding the molecular mechanisms involved in the regulation of the OTR gene by estrogen. Using mice genetically deficient in estrogen receptor alpha (ERalpha), we demonstrate that ERalpha is not necessary for basal OTR synthesis, but is absolutely necessary for the induction of OTR binding in the brain by estrogen.

Increased frequency of P-glycoprotein gene amplification in colchicine-resistant Rat-1 clones transformed by v-src.

A rat fibroblast cell line (Rat-1) carrying a temperature-sensitive mutation of v-src was used to determine whether inducible cellular transformation altered the ability of cells to amplify the p-glycoprotein gene in response to colchicine selection. Transformed and nontransformed Rat-1 fibroblasts selected under 4 times the LD50 generated the same number of colchicine-resistant colonies. We next examined colchicine-resistant colonies derived from transformed cells and compared them to colchicine-resistant colonies derived from nontransformed cells. When Rat-1 cells were selected at 35 degrees C (transforming temperature), 7 out of 7 clones exhibited a 3- to 5-fold p-glycoprotein gene amplification. These results contrasted to those found at the nontransforming temperature (40 degrees C); none of the 8 colchicine-resistant clones examined had amplified the p-glycoprotein gene. Thus in Rat-1 cells carrying a temperature-sensitive v-src gene, p-glycoprotein gene amplification was observed at a high frequency only in transformed clones selected at the temperature permissive for v-src activity.

Binding of the peroxisomal targeting sequence SKL is specified by a low-affinity site in castor bean glyoxysomal membranes. A domain next to the SKL binds to a high-affinity site.

The carboxyl-terminal amino acid sequence serine-lysine-leucine (SKL) is the consensus peroxisomal targeting sequence 1 (PTS1) and is sufficient to direct a polypeptide to peroxisomes in vivo in plants, animals, and yeasts. However, there are also two sites on alkali-stripped glyoxysomal membranes from castor bean (Ricinus communis) endosperm that bind the peptide YHKHLKPLQSKL (SKLp), the sequence of the last 12 amino acids of acyl-coenzyme A oxidase (N.E. Wollins, R.P. Donaldson [1994] J Biol Chem 289: 1149-1153). It was hypothesized that one of these sites interacts with information other than the PTS1. To explore the sequence requirements for each SKLp binding site, we tested the peptides YHKHLKPQSKG and YHKHLKPLQS and found that they bound to the high-affinity site, but not to the low-affinity site. When the high-affinity site was blocked with YHKHLKPQSKG, SKLp bound to the low-affinity site with a dissociation constant (Kd) of 8.5 microM. In an attempt to disrupt high-affinity binding, two the upstream, positively charged residues were replaced with negatively charged residues to make the peptide YHKETEPLQSKL. YHKETEPLQSKL did not bind to either site on the glyoxysomal membranes. These results indicate that the PTS1 binds to the low-affinity site and that the adjacent, positively charged domain binds to the high-affinity site.

Essential food hygiene certificate examination: examination papers reviewed.

One thousand papers for the Essential Food Hygiene examination, chosen consecutively from those being processed in RSH House on 6-7 April 1994, were reviewed. The purpose of the review was to assess the impact of the redesigned course and the new examination papers which were introduced from 1 January 1994. The pass rate for the sample was 96.7% which was somewhat lower than the pass rate of 99.2% for the whole of 1993 when the previous examination papers were in use. Out of 1000 papers 33 were failures. Eighteen candidates had obtained the 70% pass mark yet still failed because one of the two questions on temperature control had been answered incorrectly. The requirement to answer both those questions correctly in order to obtain a certificate was introduced with the new examination papers and marked a major change. The conclusion from this snapshot study was that the majority of Tutors and the candidates from their courses had achieved a high degree of examination success. In particular, Tutors had secured a major objective by ensuring that trainees had a clear understanding of the concepts of temperature control. Only a minority were having difficulty. Of the 33 individuals who failed, 18 came from just four of the 108 courses.