An evaluation of a pilot of daily testing of SARS-CoV-2 contacts in acute hospital and ambulance trusts in England.

OBJECTIVES: Healthcare worker (HCW) SARS-CoV-2 contacts in England have been required to quarantine, creating staff shortages. We piloted daily contact testing (DCT) to assess its feasibility as an alternative. STUDY DESIGN: Observational service evaluation. METHODS: We conducted an observational service evaluation of 7-day DCT using antigen lateral flow devices (LFDs) at four acute hospital trusts and one ambulance trust in England. Mixed methods were used, using aggregate and individual-level test monitoring data, semi-structured interviews, and a survey of eligible contacts. RESULTS: In total, 138 HCWs were identified as contacts of a confirmed SARS-CoV-2 case. Of these, 111 (80%) consented to daily LFD testing, of whom 82 (74%) completed the required programme without interruption and 12 (11%) completed with interruption. Fifty-eight participants (52%) and two non-participants (7.4%) completed the survey. In total, 28 interviews were conducted with participants, site and infection control leads, and union representatives. One participant tested positive on LFD and polymerase chain reaction (PCR) test. Three participants tested positive on PCR but not LFD. DCT was well-accepted by trusts and staff. Participants reported no relaxation of their infection prevention and control behaviours. No incidents of transmission were detected. An estimated 729 potential days of work absence were averted. CONCLUSIONS: DCT can be acceptably operated in a healthcare setting, averting quarantine-related work absences in HCW SARS-CoV-2 contacts.

Detection of Bacillus anthracis spores from environmental water using bioluminescent reporter phage.

AIMS: We investigated the ability of a temperate Bacillus anthracis reporter phage (Wß::luxAB-2), which transduces bioluminescence to infected cells, to detect viable spores from deliberately contaminated environmental water samples. METHODS AND RESULTS: Environmental water was inoculated with spores and assayed with Wß::luxAB-2. Bioluminescent signals directly correlated with input phage and spore concentrations. A limit of detection of 101 and 102 CFU per ml within 8 h was achieved from pond and lake water, respectively. Detection was greatly simplified by minimizing sample processing steps without spore extraction. The complex endogenous microbial flora and salt content of brackish water challenged the assay, extending the detection time to 12 h for a sensitivity of 102 CFU per ml. Phage-mediated bioluminescence was strictly dependent on bacterial physiology, being significantly reduced in mid/late log phase cells. This was shown to be due to an inability of the phage to adsorb. CONCLUSIONS: The reporter phage Wß::luxAB-2 displays potential for simplified detection of viable spores from contaminated water samples within 12 h. SIGNIFICANCE AND IMPACT OF THE STUDY: A deliberate aerosol release of spores could lead to widespread contamination, leaving large areas uninhabitable until remediation. An essential requirement of this restoration process is the development of simplified detection assays in different environmental matrices.

The decline in fitness with inbreeding: evidence for negative dominance-by-dominance epistasis in Drosophila melanogaster.

Genetic interactions can play an important role in the evolution of reproductive strategies. In particular, negative dominance-by-dominance epistasis for fitness can theoretically favour sex and recombination. This form of epistasis can be detected statistically because it generates nonlinearity in the relationship between fitness and inbreeding coefficient. Measures of fitness in progressively inbred lines tend to show limited evidence for epistasis. However, tests of this kind can be biased against detecting an accelerating decline due to line losses at higher inbreeding levels. We tested for dominance-by-dominance epistasis in Drosophila melanogaster by examining viability at five inbreeding levels that were generated simultaneously, avoiding the bias against detecting nonlinearity that has affected previous studies. We find an accelerating rate of fitness decline with inbreeding, indicating that dominance-by-dominance epistasis is negative on average, which should favour sex and recombination.

The effect of parasites on sex differences in selection.

The life history strategies of males and females are often divergent, creating the potential for sex differences in selection. Deleterious mutations may be subject to stronger selection in males, owing to sexual selection, which can improve the mean fitness of females and reduce mutation load in sexual populations. However, sex differences in selection might also maintain sexually antagonistic genetic variation, creating a sexual conflict load. The overall impact of separate sexes on fitness is unclear, but the net effect is likely to be positive when there is a large sex difference in selection against deleterious mutations. Parasites can also have sex-specific effects on fitness, and there is evidence that parasites can intensify the fitness consequences of deleterious mutations. Using lines that accumulated mutations for over 60 generations, we studied the effect of the pathogenic bacterium Pseudomonas aeruginosa on sex differences in selection in the fruit fly Drosophila melanogaster. Pseudomonas infection increased the sex difference in selection, but may also have weakened the intersexual correlation for fitness. Our results suggest that parasites may increase the benefits of sexual selection.

Rapid detection and simultaneous antibiotic susceptibility analysis of Yersinia pestis directly from clinical specimens by use of reporter phage.

Yersinia pestis is a tier 1 agent due to its contagious pneumopathogenicity, extremely rapid progression, and high mortality rate. As the disease is usually fatal without appropriate therapy, rapid detection from clinical matrices is critical to patient outcomes. We previously engineered the diagnostic phage ΦA1122 with luxAB to create a "light-tagged" reporter phage. ΦA1122::luxAB rapidly detects Y. pestis in pure culture and human serum by transducing a bioluminescent signal response. In this report, we assessed the analytical specificity of the reporter phage and investigated diagnostic utility (detection and antibiotic susceptibility analysis) directly from spiked whole blood. The bioreporter displayed 100% (n = 59) inclusivity for Y. pestis and consistent intraspecific signal transduction levels. False positives were not obtained from species typically associated with bacteremia or those relevant to plague diagnosis. However, some non-pestis Yersinia strains and Enterobacteriaceae did elicit signals, albeit at highly attenuated transduction levels. Diagnostic performance was assayed in simple broth-enriched blood samples and standard aerobic culture bottles. In blood, <10(2) CFU/ml was detected within 5 h. In addition, Y. pestis was identified directly from positive blood cultures within 20 to 45 min without further processing. Importantly, coincubation of blood samples with antibiotics facilitated simultaneous antimicrobial susceptibility profiling. Consequently, the reporter phage demonstrated rapid detection and antibiotic susceptibility profiling directly from clinical samples, features that may improve patient prognosis during plague outbreaks.

Cervical cancer screening and abnormalities among women in a residential drug-rehabilitation program.

Women in a residential drug-rehabilitation program had lower rates of cervical screening attendance and higher rates ofdetected abnormalities than women attending a local Well Women's Clinic. As a result ofthis study we plan to include a more comprehensive sexual health history into routine women's health consultations.

Magnetic resonance imaging characteristics of necrotizing meningoencephalitis in Pug dogs.

BACKGROUND: The magnetic resonance imaging (MRI) characteristics of necrotizing meningoencephalitis (NME) are not well documented. OBJECTIVES: To describe common MRI features of NME, to compare the MRI features to histopathologic findings, and to determine whether or not MRI lesions are predictive of survival time. ANIMALS: Eighteen Pugs with NME. METHODS: Retrospective MRI case study of Pugs identified by a search of medical records at 6 veterinary institutions. Eighteen dogs met inclusion criteria of histopathologically confirmed NME and antemortem MRI exam. MRI lesions were characterized and compared with histopathology with the kappa statistic. Survival times were compared with MRI findings by use of Mann-Whitney U-tests and Spearman's rho. RESULTS: Twelve of 18 lesions were indistinctly marginated with mild parenchymal contrast enhancement. Prosencephalic (17/18) lesion distribution included the parietal (16/18), temporal (16/18), and occipital (16/18) lobes. There were cerebellar (4/18) and brainstem (3/18) lesions. Asymmetric lesions were present in both gray and white matter in all dogs. Falx cerebri shift was common (11/18), and 6 dogs had brain herniation. Leptomeningeal enhancement was present in 9/18 dogs. A moderate positive association was found between parenchymal contrast enhancement and both necrosis (kappa= 0.45; P= .045) and monocytic inflammation (kappa= 0.48; P= .025). Higher MRI lesion burden was correlated with longer time from disease onset to MRI (P= .045). MRI lesion burden did not correlate to survival time. CONCLUSIONS AND CLINICAL IMPORTANCE: Asymmetric prosencephalic grey and white matter lesions with variable contrast enhancement were consistent MRI changes in Pugs with confirmed NME. While not pathognomonic for NME, these MRI characteristics should increase confidence in a presumptive diagnosis of NME in young Pugs with acute signs of neurologic disease.

Cervical vertebral malformation-malarticulation syndrome in the Bernese mountain dog: clinical and magnetic resonance imaging features.

OBJECTIVES: To describe the clinical and magnetic resonance imaging features of cervical vertebral malformation-malarticulation in Bernese mountain dogs. METHODS: Seven Bernese mountain dogs (four males and three females) were diagnosed with cervical vertebral malformation-malarticulation by magnetic resonance imaging. The following data were evaluated retrospectively: (1) abnormalities of the cervical vertebral column and spinal cord, (2) spinal cord compression, (3) intervertebral disc degeneration and herniation, (4) severity of clinical signs pretreatment and after treatment, (5) type of treatment and (6) outcome. RESULTS: Spin echo T1-weighted and T2-weighted images disclosed multi-level, extradural compressive spinal cord lesions (ventral, dorsolateral or both) spanning from intervertebral disc spaces C3-4 to C6-7. In all seven dogs, T2-weighted images disclosed one or more intramedullary hyperintensities associated with extradural spinal cord compression. Surgery was performed in five dogs. Two dogs were managed medically. The prognosis for surgical or conservative management in Bernese mountain dogs was similar to cervical vertebral malformation-malarticulation in other breeds. CLINICAL SIGNIFICANCE: Cervical vertebral malformation-malarticulation is an important differential diagnosis for young to middle-aged Bernese mountain dogs with a C1-5 or C6-T2 neuroanatomic localisation. Dorsolateral spinal cord compression associated with articular process hypertrophy was the most common feature of cervical vertebral malformation-malarticulation in the seven Bernese mountain dogs evaluated.

Suspected Phenobarbital-Induced Pseudolymphoma in a Dog.

Pseudolymphoma is a drug reaction to anti-epileptics that is well recognized in humans; it has been reported in one cat but not dogs. In this report, lymphoma-like clinical signs are suspected to be secondary to phenobarbital administration in a dog. A 2.5-year-old male, neutered Shepherd mix presented for a 3-day history of progressive ataxia, dazed mentation, pyrexia, and lethargy. While hospitalized, the dog developed generalized lymphadenopathy and sustained pyrexia. The dog was receiving levetiracetam and phenobarbital for epilepsy, and serum concentrations of both were within standard therapeutic ranges. Abdominal ultrasound revealed hepatomegaly, splenomegaly, and generalized lymphadenopathy. Cytology of the peripheral lymph nodes was consistent with reactive lymph nodes, and aspirates of the liver and spleen revealed histiocytic-neutrophilic inflammation. Phenobarbital was discontinued and replaced with zonisamide. Within 24 hours, the dog was normothermic, and other clinical signs resolved within a week. This case highlights a potentially serious yet reversible adverse reaction to phenobarbital in a dog. This idiosyncratic reaction could be mistaken for neoplasia and is an important differential for lymphoma-like signs in any dog administered phenobarbital.

A dominant negative mutant of the insulin-like growth factor-I receptor inhibits the adhesion, invasion, and metastasis of breast cancer.

The 5-year survival rate for women with metastatic breast cancer is only 25-30%; thus, the need to improve treatment is apparent. Overexpression of insulin-like growth factor-I receptor (IGF-IR) correlates with poor prognosis and local recurrence. In this study, we addressed whether functional impairment of IGF-IR affects adhesion, invasion, and metastasis of breast cancer. Impairment of IGF-IR function was achieved by transfecting a dominant negative form of the receptor, termed 486stop, into MDA-MB-435 metastatic breast cancer cells. The protein product of 486stop is secreted extracellularly, resulting in a bystander effect. Cellular adhesion to laminin and collagen was inhibited 94 and 88%, respectively. Furthermore, 486stop inhibited insulin-like growth factor-I-stimulated invasion through collagen IV by 75%. The dominant negative receptor was secreted, as evidenced by the observation that MDA-MB-435 and MDA-MB-231 cells were prevented from binding to laminin by 90% when treated with conditioned medium (CM) from 486stop-transfected cells. CM also inhibited the invasion of MDA-MB-231 cells across collagen IV by 80%. Finally, CM made MDA-MB-231 cells 30% more sensitive to Taxol-induced cell death. Growth in soft agar was suppressed by 486stop, but growth in monolayer was unaffected. When injected into the mammary fat pad, 486stop did not significantly suppress growth of the primary tumor, but metastasis to the lungs, livers, lymph nodes, and lymph vessels was significantly decreased compared to the vector control. In conclusion, inhibition of IGF-IR resulted in suppression of adhesion, invasion, and metastasis, providing a mechanistic rationale for targeting IGF-IR in the treatment of metastatic breast cancer.

Regulation of c-fgr proto-oncogene expression in Burkitt's lymphoma cells: effect of interferon treatment and relationship to EBV status and c-myc mRNA levels.

The proto-oncogene c-fgr is expressed in transformed human B lymphoid cell lines and has been reported to be induced in cells infected with the Epstein-Barr virus (EBV). We have compared the levels of c-fgr mRNA in several B cell lines and have examined the effects of interferon-induced changes in growth rate of Daudi cells on the concentration of this mRNA. High levels were found in exponentially growing EBV-positive Raji and Daudi cells but the amounts in B95-8 and P3HR-1 cells (also EBV-positive) were lower than in the EBV-negative cell line Ramos. Growth inhibition of Daudi cells by interferon-alpha is preceded by a reduction in c-fgr expression, with a 56% decrease observed within 6 h. The differences in the amounts of c-fgr mRNA in the various cell lines and in control versus interferon-treated cells are similar to the differences in the c-myc mRNA contents of these cells. These results indicate that c-fgr expression bears little relationship to the EBV status of B lymphoid cell lines but may play a role, in conjunction with c-myc expression, in growth regulation by interferons. Other conditions which influence Daudi cell proliferation, such as treatment with a phorbol ester or growth to high cell density, also inhibit c-fgr expression but to a lesser extent than interferon treatment.

Epstein-Barr virus gene expression in interferon-treated cells. Implications for the regulation of protein synthesis and the antiviral state.

This paper presents data on the effects of interferon treatment on Epstein-Barr virus (EBV) gene expression in latently infected Daudi Burkitt's lymphoma cells, and reviews the possible role of viral gene products in the regulation of translation. In Daudi cells the main virally coded RNAs are the small untranslated RNAs EBER-1 and EBER-2, two mRNAs for the DNA binding protein EBNA-1, and a number of small RNAs containing sequences from the BamHI W repeat region of the viral genome. Interferon treatment does not change the qualitative pattern of EBV gene expression but decreases the levels of the EBNA-1 mRNAs. The chromatographic behaviour of EBV-encoded RNAs on CF11-cellulose indicates that many contain double-stranded regions; these RNAs co-purify with RNA that activates the interferon-induced, dsRNA-sensitive protein kinase DAI. Computer analysis indicates that the exons transcribed from the BamHI W repeats have the potential for formation of very stable secondary structures. Many viruses can counteract the inhibition of protein synthesis mediated by the DAI-catalysed phosphorylation of initiation factor eIF-2 and our data suggest that the small RNA EBER-1 may fulfil this function in the EBV system. During the infection and immortalization of B lymphocytes by EBV the synthesis of large amounts of EBER-1 RNA might thus allow the virus to circumvent one of the interferon-mediated mechanisms of host cell defence.

Pulmonary hypertension predicts mortality in infants with omphalocele.

OBJECTIVE: The objective of this study was to identify predictors of mortality in infants with omphalocele. METHODS: Medical records of infants with omphalocele born between January 1992 and June 2012, with follow-up toDecember 2012, were retrospectively reviewed. Survivors and non-survivors were compared. Evidence for pulmonary hypertension was sought between the second and seventh day after birth. All included infants had increased right ventricular pressures (RVP >40 mmhg) on echocardiogram on the second day of life with increased oxygen requirements, therefore, the finding of increased pressure was not considered a result of the transitional circulation. Logistic regression was used to evaluate the importance and independence of various factors. RESULTS: Of 51 infants whose records were reviewed, 13 died (25%) and 38 survived (75%). The median time to death was 34 days (range: 4 -408 days). The median follow-up time for those who died was 1.5 years (range: 0.01-15 years) and for survivors was 2.6 years (range: 0.08-15 years). Logistic regression revealed that respiratory insufficiency at birth (OR: 14.8; 95% CI: 2.5-85.0) and pulmonary hypertension (OR: 6.4; 95% CI: 1.1-39.0) were independently associated with mortality. CONCLUSION: Respiratory insufficiency after birth and pulmonary hypertension are independent predictors of mortality in infants with omphalocele.

Alpha-dystroglycan deficiency correlates with elevated serum creatine kinase and decreased muscle contraction tension in golden retriever muscular dystrophy.

The dystrophin-glycoprotein complex was examined in dystrophin-deficient dogs with golden retriever muscular dystrophy (GRMD) using immunoblot and immunofluorescence analysis. The dystrophin-associated proteins were substantially reduced in muscle from dogs with GRMD. Interestingly, regression analysis revealed a strong correlation between the amount of alpha-dystroglycan and serum creatine kinase levels and the contraction tension measured for a given peroneus longus muscle.

Relative transcriptional inducibility of the human interferon-alpha subtypes conferred by the virus-responsive enhancer sequence.

This paper addresses the role of transcriptional regulation in the determination of the levels of expression of different interferon-alpha subtypes secreted from Namalwa cells following infection with Sendai virus. Using RT-PCR to determine the relative abundance of mRNA species coding for the various subtypes, we found a general correlation with corresponding protein levels, indicative of a role for transcriptional control in the determination of levels of individual subtypes. We have used reporter gene constructs to compare the inducibility of the virus-response elements from the IFNA1, A2, A4, and A14 subtype genes cloned upstream of a secreted alkaline phosphatase gene. The inducibility of these reporter gene constructs broadly correlated with the relative mRNA abundances in both transiently and stably transfected Namalwa cells. During work with stable cell lines, we found that G418, the drug used for the selection of transfected cells, inhibited the induction of interferon by both Sendai virus and double-stranded RNA. This inhibition was reversible when G418 was removed from the medium 24 h before the addition of virus.

Detection of rare allelic variants of the interferon-alpha 2 gene in human genomic DNA.

We have analyzed human donor DNA for the presence of sequences corresponding to allelic variants of the IFN-alpha 2 locus. Using both restriction enzyme digestion of PCR-amplified fragments and sequence analysis of these fragments, we have identified the three reported allelic variants, IFN-alpha 2a, IFN-alpha 2b, and IFN-alpha 2c, in genomic DNA derived from donors of African or Afro-Caribbean origin. This is the first report of the IFN-alpha 2a and IFN-alpha 2c alleles occurring in human donor DNA and supports the view that these are variants of the predominant IFN-alpha 2b allele rather than arising from mutations occurring in cultured cells.

Evaluation of the dystrophin-glycoprotein complex, alpha-actinin, dysferlin and calpain 3 in an autosomal recessive muscular dystrophy in Labrador retrievers.

Labrador retrievers suffer from an autosomal recessive muscular dystrophy of unknown aetiology. Dogs affected with this disease develop generalized weakness associated with severe, generalized skeletal muscle atrophy and mild elevations in creatine kinase in the first few months of life. The severity of signs tends to progress over the first year of life but can vary from mild exercise intolerance to non-ambulatory tetraparesis. Beyond 1 year of age, the signs usually stabilize and although muscle mass does not increase, affected dogs' strength may improve slightly. The pathological changes present on muscle biopsy include marked variation in muscle fibre size with hypertrophied and round atrophied fibres present. There is an increased number of fibres with central nuclei and split fibres can be seen. It has been suggested that the disorder is a model for limb-girdle muscular dystrophy. In recent years, mutations in genes encoding the proteolytic enzyme, calpain 3, a novel protein named dysferlin, and components of the dystrophin-glycoprotein complex have been identified as causes of autosomal recessive limb-girdle muscular dystrophy. We have evaluated these proteins in normal dogs and in three Labrador retrievers with autosomal recessive muscular dystrophy using immunohistochemistry and Western blot analysis on frozen skeletal muscle. The results demonstrate that dystrophin, the sarcoglycans, alpha-actinin, dysferlin and calpain 3 are present in the normal and affected dogs. We conclude that this autosomal recessive muscular dystrophy is not due to a deficiency of alpha-actinin, or any of the known autosomal recessive limb-girdle muscular dystrophy proteins, although we cannot rule out a malfunction of any of these proteins.

Molecular analysis of a spontaneous dystrophin 'knockout' dog.

We have determined the molecular basis for skeletal myopathy and dilated cardiomyopathy in two male German short-haired pointer (GSHP) littermates. Analysis of skeletal muscle demonstrated a complete absence of dystrophin on Western blot analysis. PCR analysis of genomic DNA revealed a deletion encompassing the entire dystrophin gene. Molecular cytogenetic analysis of lymphocytes from the dam and both dystrophic pups confirmed a visible deletion in the p21 region of the affected canine X chromosome. Utrophin is up-regulated in the skeletal muscle, but does not appear to ameliorate the dystrophic canine phenotype. This new canine model should further our understanding of the physiological and biochemical processes in Duchenne muscular dystrophy.

Frequency of serum low-density lipoprotein cholesterol measurement and frequency of results < or=100 mg/dl among patients who had coronary events (Northwest VA Network Study).

This population-based, cross-sectional analysis targeted all veterans with coronary heart disease (CHD) who were active patients in primary care or cardiology clinics in the Veterans Health Administration Northwest Network from July 1998 to June 1999. We report guideline compliance rates, including whether low-density lipoprotein (LDL) was measured, and if measured, whether the LDL was < or=100 mg/dl. In addition, we utilized multivariate logistic regression to determine patient characteristics associated with LDL measurements and levels. Of 13,891 active patients with CHD, 5,552 (40.0%) did not have a current LDL measurement. Of those with LDL measurements, 39.1% were at the LDL goal of < or =100 mg/dl, whereas 26.5% had LDL > or =130 mg/dl. Male gender, younger age, history of angioplasty or coronary artery bypass grafting, current hypertension, diabetes mellitus, and angina pectoris were associated with increased likelihood of LDL measurement. Older age and current diabetes and angina were associated with increased likelihood of LDL being < or =100 mg/dl, if measured. Although these rates of guideline adherence in the CHD population compare well to previously published results, they continue to be unacceptably low for optimal clinical outcomes. Attention to both LDL measurement and treatment (if elevated) is warranted.