Inactivation of murine norovirus on a range of copper alloy surfaces is accompanied by loss of capsid integrity

Norovirus is one of the most common causes of acute viral gastroenteritis. The virus is spread via the fecal-oral route, most commonly from infected food and water, but several outbreaks have originated from contamination of surfaces with infectious virus. In this study, a close surrogate of human norovirus causing gastrointestinal disease in mice, murine norovirus type 1 (MNV-1), retained infectivity for more than 2 weeks following contact with a range of surface materials, including Teflon (polytetrafluoroethylene [PTFE]), polyvinyl chloride (PVC), ceramic tiles, glass, silicone rubber, and stainless steel. Persistence was slightly prolonged on ceramic surfaces. A previous study in our laboratory observed that dry copper and copper alloy surfaces rapidly inactivated MNV-1 and destroyed the viral genome. In this new study, we have observed that a relatively small change in the percentage of copper, between 70 and 80% in copper nickels and 60 and 70% in brasses, had a significant influence on the ability of the alloy to inactivate norovirus. Nickel alone did not affect virus, but zinc did have some antiviral effect, which was synergistic with copper and resulted in an increased efficacy of brasses with lower percentages of copper. Electron microscopy of purified MNV-1 that had been exposed to copper and stainless steel surfaces suggested that a massive breakdown of the viral capsid had occurred on copper. In addition, MNV-1 that had been exposed to copper and treated with RNase demonstrated a reduction in viral gene copy number. This suggests that capsid integrity is compromised upon contact with copper, allowing copper ion access to the viral genome

Adjunctive rifampicin for Staphylococcus aureus bacteraemia (ARREST): a multicentre, randomised, double-blind, placebo-controlled trial.

BACKGROUND: Staphylococcus aureus bacteraemia is a common cause of severe community-acquired and hospital-acquired infection worldwide. We tested the hypothesis that adjunctive rifampicin would reduce bacteriologically confirmed treatment failure or disease recurrence, or death, by enhancing early S aureus killing, sterilising infected foci and blood faster, and reducing risks of dissemination and metastatic infection. METHODS: In this multicentre, randomised, double-blind, placebo-controlled trial, adults (≥18 years) with S aureus bacteraemia who had received ≤96 h of active antibiotic therapy were recruited from 29 UK hospitals. Patients were randomly assigned (1:1) via a computer-generated sequential randomisation list to receive 2 weeks of adjunctive rifampicin (600 mg or 900 mg per day according to weight, oral or intravenous) versus identical placebo, together with standard antibiotic therapy. Randomisation was stratified by centre. Patients, investigators, and those caring for the patients were masked to group allocation. The primary outcome was time to bacteriologically confirmed treatment failure or disease recurrence, or death (all-cause), from randomisation to 12 weeks, adjudicated by an independent review committee masked to the treatment. Analysis was intention to treat. This trial was registered, number ISRCTN37666216, and is closed to new participants. FINDINGS: Between Dec 10, 2012, and Oct 25, 2016, 758 eligible participants were randomly assigned: 370 to rifampicin and 388 to placebo. 485 (64%) participants had community-acquired S aureus infections, and 132 (17%) had nosocomial S aureus infections. 47 (6%) had meticillin-resistant infections. 301 (40%) participants had an initial deep infection focus. Standard antibiotics were given for 29 (IQR 18-45) days; 619 (82%) participants received flucloxacillin. By week 12, 62 (17%) of participants who received rifampicin versus 71 (18%) who received placebo experienced treatment failure or disease recurrence, or died (absolute risk difference -1·4%, 95% CI -7·0 to 4·3; hazard ratio 0·96, 0·68-1·35, p=0·81). From randomisation to 12 weeks, no evidence of differences in serious (p=0·17) or grade 3-4 (p=0·36) adverse events were observed; however, 63 (17%) participants in the rifampicin group versus 39 (10%) in the placebo group had antibiotic or trial drug-modifying adverse events (p=0·004), and 24 (6%) versus six (2%) had drug interactions (p=0·0005). INTERPRETATION: Adjunctive rifampicin provided no overall benefit over standard antibiotic therapy in adults with S aureus bacteraemia. FUNDING: UK National Institute for Health Research Health Technology Assessment

Desk studies on feasibility of horizontal standard rapid methods for detection of E. coli (including E. coli O157) and Salmonella

The emerging methods becoming available for the rapid detection and enumeration of E. coli (including E. coli O157) and Salmonella in sludges, soil and treated biowastes have been evaluated with a view to possible future standardisation. The main methods that are available for the detection and enumeration of E. coli (including E. coli O157) and Salmonella have been developed largely for analysis of food and water and can be broadly divided into four groups. Proprietary Quantitray® technology, equivalent to the 5-tube most probable number (MPN) technique, employing disposable plastic trays for enumeration of E. coli and Salmonella. Immunological, involving a short or overnight pre-enrichment of the target organism followed by specific detection of cellular antigen in either a lateral flow device or following immunomagnetic capture. Molecular, involving PCR amplification of target DNA sequences from low numbers of cells, or preferably following a short pre-enrichment of the organism to amplify numbers and demonstrate viability prior to molecular detection. Physico-chemical, involving techniques such as measurement of impedance changes during enrichment and growth in appropriate media. The merits of each are described, in relation to their suitability for use with sludge, soil and biowastes. Since the majority of agar and MPN broth techniques take between 24-96 hours for identification and enumeration, we define “rapid” as any technique that detects, and if possible, enumerates the target organism in under 24 hours.All of the methods described have strengths and weaknesses, dependent on not only the Regulators’ types of requirements for sludge, soil and biowaste analysis but also their sensitivity, specificity, speed and cost. It is unlikely therefore that there can be only one methodology applicable to both E. coli (and E. coli O157) and Salmonella detection. Nevertheless, it is considered feasible to formulate horizontal standards to cover rapid analysis of E. coli and Salmonella in sludge, soil, soil improvers, growing media, and biowaste. None of the methods have been extensively evaluated for sewage sludge, soils or biowastes. As such, there is an urgent need for their modification and evaluation as part of the next phase of the Project Horizonta

Desk studies on feasibility of horizontal standard rapid methods for detection of Clostridium perfringens and enterococci in sludges, soil, soil improvers, growing media and biowastes

The existing methods currently available for the detection and enumeration of clostridia and enterococci in sludges and treated biowastes have been evaluated with a view to possible standardisation. The main methods used for the detection and enumeration of Clostridium and Enterococcus spp. have been developed largely for analysis of food and water and can be broadly divided into three groups. Quantification of colonies on agar media; most probable number (MPN) quantification in indicator broth using conventional test tube technology; and proprietary Quantitray® technology equivalent to the 5-tube MPN technique employing disposable plastic trays for enumeration of enterococci. The merits of each are described. At least one report has suggested that the use of m-CP agar medium, which is used in the reference method in the European Union, is not suitable for recovering C. perfringens spores from groundwater. This questions its possible use as a method for detecting C. perfringens in sludge, soil, soil improvers, growing media, and biowaste.Indeed, all of the methods described for detection of C. perfringens and enterococci have strengths and weaknesses, dependent on not only the Regulators’ types of requirements for sludge, soil and biowaste analysis but also their sensitivity, specificity, speed and cost. Nevertheless, it is considered feasible to formulate horizontal standards to cover analysis of C. perfringens and enterococci in sludge, soil, soil improvers, growing media, and biowaste. However, none of the methods have been extensively evaluated for these waste types. As such, there is an urgent need for their modification and evaluation as part of the next phase of the Project Horizonta

Inactivation of norovirus on dry copper alloy surfaces

Noroviruses (family Caliciviridae) are the primary cause of viral gastroenteritis worldwide. The virus is highly infectious and touching contaminated surfaces can contribute to infection spread. Although the virus was identified over 40 years ago the lack of methods to assess infectivity has hampered the study of the human pathogen. Recently the murine virus, MNV-1, has successfully been used as a close surrogate. Copper alloys have previously been shown to be effective antimicrobial surfaces against a range of bacteria and fungi. We now report rapid inactivation of murine norovirus on alloys, containing over 60% copper, at room temperature but no reduction of infectivity on stainless steel dry surfaces in simulated wet fomite and dry touch contamination. The rate of inactivation was initially very rapid and proportional to copper content of alloy tested. Viral inactivation was not as rapid on brass as previously observed for bacteria but copper-nickel alloy was very effective. The use of chelators and quenchers of reactive oxygen species (ROS) determined that Cu(II) and especially Cu(I) ions are still the primary effectors of toxicity but quenching superoxide and hydroxyl radicals did not confer protection. This suggests Fenton generation of ROS is not important for the inactivation mechanism. One of the targets of copper toxicity was the viral genome and a reduced copy number of the gene for a viral encoded protein, VPg (viral-protein-genome-linked), which is essential for infectivity, was observed following contact with copper and brass dry surfaces. The use of antimicrobial surfaces containing copper in high risk closed environments such as cruise ships and care facilities could help to reduce the spread of this highly infectious and costly pathogen

Inactivation of MNV on copper surfaces in the presence of quenchers D-mannitol or Tiron (A) or chelators EDTA or BCS (B) and to remove hydroxyl radical or superoxide, copper II or Cu I, respectively.

<p>Approximately 5×10⁴ pfu MNV was inoculated onto metal surfaces in the presence of chelators or quenchers of reactive oxygen species and assessed for infectious virus using plaque assay as described in text. The results were compared to those obtained without chelators or quenchers to ascertain if there was a protective effect. No quenchers or chelators present is represented by white bars; D-mannitol (A) or EDTA (B) represented by diagonal striped bars; Tiron (A) or BCS (B) represented by cross hatched bars. No significant reduction of infectivity occurred in the presence of any quenchers or chelators on stainless steel surfaces (even though D-mannitol has been reported to interfere with HSV replication) (C and D respectively). Error bars represent ± SD and data are from multiple experiments.</p

Destruction of entire MNV genome occurs on copper.

<p>MNV (PEG concentrate) was exposed to copper (lane 1), cartridge brass (lane 2) or stainless steel (lane 3) for 2 hours. Viral RNA was purified using Qiagen mini prep viral RNA kit and fragments separated on non-denaturing 1% agarose gel electrophoresis and visualised in UV light box. Viral RNA has degraded on copper, less on brass and not at all on stainless steel (see control RNA S2 Supplementary Information). Lanes 4, 5 and 6 are PEG precipitation of uninfected cells (mock) applied to stainless steel, brass and copper respectively. Virus added to all surfaces and removed immediately was similar to lane 1 although some reduction in intensity on copper was visible (not shown). DNA ladder is Bioline hyperladder I (HL1 1 Kb)</p

Comparison between inactivation rates of MNV in wet fomite (A) and dry touch (B) contamination on copper surfaces.

<p>Inactivation rates were calculated for various contact times of MNV exposed to test surfaces as described in the text (from the results generated in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0075017#pone-0075017-g001" target="_blank">Figure 1</a>). (copper (white bars), phosphor bronze (95% copper) (forward diagonal striped bars), copper nickel (89% copper) (backward diagonal striped bars), cartridge brass (70% copper) (cross hatch bars), nickel silver (65% copper) (horizontal striped bars) and stainless steel (vertical striped bars)). Error bars represent ± SD and data are from multiple experiments.</p

Composition of metals used in the study.

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