WITHDRAWN: Evaluation of Fourier Transform Infrared spectroscopy (IR Biotyper) as a complement to Whole genome sequencing (WGS) to characterise Enterobacter cloacae , Citrobacter freundii and Klebsiella pneumoniae isolates recovered from hospital sinks.

The authors have withdrawn their manuscript due to becoming aware of methodology issues related to the curation of the training set used to determine cut-off values for Biotyper cluster assignation and lack of replicate measurements on different days for the isolates analysed. It is therefore unclear whether the conclusions of the manuscript are founded and no further work is possible to correct these issues as the instrument is no longer available to the authors. If you have any questions, please contact the corresponding author.

A workflow for the detection of antibiotic residues, measurement of water chemistry and preservation of hospital sink drain samples for metagenomic sequencing.

BACKGROUND: Hospital sinks are environmental reservoirs that harbour healthcare-associated (HCA) pathogens. Selective pressures in sink environments, such as antibiotic residues, nutrient waste and hardness ions, may promote antibiotic resistance gene (ARG) exchange between bacteria. However, cheap and accurate sampling methods to characterize these factors are lacking. AIMS: To validate a workflow to detect antibiotic residues and evaluate water chemistry using dipsticks. Secondarily, to validate boric acid to preserve the taxonomic and ARG ('resistome') composition of sink trap samples for metagenomic sequencing. METHODS: Antibiotic residue dipsticks were validated against serial dilutions of ampicillin, doxycycline, sulfamethoxazole and ciprofloxacin, and water chemistry dipsticks against serial dilutions of chemical calibration standards. Sink trap aspirates were used for a 'real-world' pilot evaluation of dipsticks. To assess boric acid as a preservative of microbial diversity, the impact of incubation with and without boric acid at ∼22 °C on metagenomic sequencing outputs was evaluated at Day 2 and Day 5 compared with baseline (Day 0). FINDINGS: The limits of detection for each antibiotic were: 3 µg/L (ampicillin), 10 µg/L (doxycycline), 20 µg/L (sulfamethoxazole) and 8 µg/L (ciprofloxacin). The best performing water chemistry dipstick correctly characterized 34/40 (85%) standards in a concentration-dependent manner. One trap sample tested positive for the presence of tetracyclines and sulphonamides. Taxonomic and resistome composition were largely maintained after storage with boric acid at ∼22 °C for up to five days. CONCLUSIONS: Dipsticks can be used to detect antibiotic residues and characterize water chemistry in sink trap samples. Boric acid was an effective preservative of trap sample composition, representing a low-cost alternative to cold-chain transport.

Quality Improvement Within a Mental Health Setting: Alcohol Detoxification.

Aims We describe a clinical audit on alcohol detoxification, using NICE guidelines as a comparable standard. NICE guidelines recommend completing a thorough alcohol history, documentation of a physical examination including screening for Wernicke's encephalopathy, monitoring of vital signs and liver investigations. Breath alcohol level and standardised assessment of withdrawal should be completed in addition to documentation of chlordiazepoxide and thiamine prescriptions. The reported mental health service completed the first cycle of the audit as part of a large-scale, international audit on alcohol detoxification by the Prescribing Observatory for Mental Health, UK (POMH-UK). Two additional audit cycles were completed within the service to ensure continuous quality improvement and clinical effectiveness. Methods Retrospective chart reviews were performed for admissions within pre-defined 6-month periods. Inclusion criteria: ICD-10 F10 diagnosis; prescription of alcohol detoxification schedule. Results This mental health service demonstrated greater compliance with the NICE standards in comparison to other services in the POMH-UK audit. The second-cycle audit showed increased compliance in most areas compared to the initial results. The third-cycle audit focused on two specific areas that required improvement to optimise quality improvement - Breath Alcohol Level and Clinical Institute of Withdrawal Assessment, documentation of which improved from 79% to 85% and 39% to 91% respectively in the final audit cycle. Conclusion The results of this audit indicate that adherence to defined clinical standards within this mental health service exceeds that of the benchmark POMH-UK data. The effectiveness of electronic patient records in improving adherence to set clinical standards, specifically in relation to documentation of clinical parameters is evident. The report also confirms continued improved results with each audit cycle within the service.

External Beam Radiotherapy in Western Africa: 1969-2019.

AIMS: We describe the absolute and per capita numbers of megavoltage radiotherapy machines (MVMs) in Western Africa from 1969 to 2019. MATERIALS AND METHODS: Western Africa was defined in accordance with the United Nations' delineation and inclusive of 16 countries. A literature search for publications detailing the number of cobalt-60 machines (COs) and linear accelerators (LINACs) in radiotherapy centres was carried out. Population data from the World Bank Group and crude cancer rates from the International Agency for Research on Cancer were used to calculate ratios of million persons per MVM and MVMs per 1000 cancer cases. RESULTS: The numbers of MVMs in Western Africa in 1969, 1979, 1989, 1999 and 2009 were zero, two, three, six and nine, respectively. In 2019 there were 22 MVMs distributed across Ghana (five), Côte d'Ivoire (two), Mali (one), Mauritania (two), Nigeria (nine) and Senegal (three). Nine countries (56.3%) had no history of external beam radiotherapy (EBRT). The largest increase in absolute EBRT capacity occurred from 2017 to 2019, during which 13 MVMs were commissioned. The largest decrease in EBRT capacity occurred from 2015 to 2017, during which four LINACs and three COs were rendered non-operational. The ratio of million persons per MVM improved from 67.0 in 1979 to 17.8 in 2019. As of 2019, there was 0.09 MVM per 1000 cancer cases. CONCLUSIONS: Western African nations have experienced an increase in the absolute number of MVMs and per capita radiotherapy capacity during the last 50 years, especially in the last decade. As non-functional LINACs contributed to a temporary decline in the EBRT infrastructure, dual use of CO/LINAC technologies may act to promote the availability of EBRT treatment in centres with capacity for multiple MVMs.

The Practice of Paediatric Radiation Oncology in Low- and Middle-income Countries: Outcomes of an International Atomic Energy Agency Study.

AIMS: Childhood cancer survival is suboptimal in most low- and middle-income countries (LMICs). Radiotherapy plays a significant role in the standard care of many patients. To assess the current status of paediatric radiotherapy, the International Atomic Energy Agency (IAEA) undertook a global survey and a review of practice in eight leading treatment centres in middle-income countries (MICs) under Coordinated Research Project E3.30.31; 'Paediatric radiation oncology practice in low and middle income countries: a patterns-of-care study by the International Atomic Energy Agency.' MATERIALS AND METHODS: A survey of paediatric radiotherapy practices was distributed to 189 centres worldwide. Eight leading radiotherapy centres in MICs treating a significant number of children were selected and developed a database of individual patients treated in their centres comprising 46 variables related to radiotherapy technique. RESULTS: Data were received from 134 radiotherapy centres in 42 countries. The percentage of children treated with curative intent fell sequentially from high-income countries (HICs; 82%) to low-income countries (53%). Increasing deficiencies were identified in diagnostic imaging, radiation staff numbers, radiotherapy technology and supportive care. More than 92.3% of centres in HICs practice multidisciplinary tumour board decision making, whereas only 65.5% of centres in LMICs use this process. Clinical guidelines were used in most centres. Practice in the eight specialist centres in MICs approximated more closely to that in HICs, but only 52% of patients were treated according to national/international protocols whereas institution-based protocols were used in 41%. CONCLUSIONS: Quality levels in paediatric radiotherapy differ among countries but also between centres within countries. In many LMICs, resources are scarce, coordination with paediatric oncology is poor or non-existent and access to supportive care is limited. Multidisciplinary treatment planning enhances care and development may represent an area where external partners can help. Commitment to the use of protocols is evident, but current international guidelines may lack relevance; the development of resources that reflect the capacity and needs of LMICs is required. In some LMICs, there are already leading centres experienced in paediatric radiotherapy where patient care approximates to that in HICs. These centres have the potential to drive improvements in service, training, mentorship and research in their regions and ultimately to improve the care and outcomes for paediatric cancer patients.

An evaluation of the electronic reporting system for the enhanced surveillance of carbapenemase-producing Gram-negative bacteria in England.

BACKGROUND: An electronic reporting system (ERS) for the enhanced surveillance of carbapenemase-producing Gram-negative bacteria (CPGNB) was launched by Public Health England in May 2015. AIM: This evaluation aimed to assess uptake, timeliness and completeness of data provided and explore potential barriers and facilitators to adopting the system. METHODS: The evaluation comprised a retrospective analysis of surveillance data and semi-structured interviews with ERS users. FINDINGS: The proportion of organisms referred for investigation of carbapenem resistance via ERS increased over the first 12 months post-implementation from 35% to 73%; uptake varied widely across regions of England. Completeness of enhanced data fields was poor in 78% of submitted isolates. The median number of days to report confirmatory test results via ERS was 1 day for the regional service and nine days for the national reference laboratory, which additionally conducts phenotypic testing to confirm carbapenemase negativity. Hindrances to ERS utility included: a lack of designated, ongoing resource for system maintenance, technical support and development; uncertainty about how and when to use ERS and workload. Incomplete data prevented gaining a better understanding of important risk factors and transmission routes of CPGNB in England. CONCLUSION: The ERS is the only surveillance system in England with the potential to gather intelligence on important risk factors for CPGNB to inform public health measures to control their spread. Although the ERS captures more information on CPGNB than other surveillance systems, timeliness and completeness of the enhanced data require substantial improvements in order to deliver the desired health benefits.

Herpesvirus skin disease in free-living common frogs Rana temporaria in Great Britain.

Infectious disease is a significant driver of global amphibian declines, yet despite this, relatively little is known about the range of pathogens that affect free-living amphibians. Recent detection of the tentatively named Ranid herpesvirus 3 (RHV3), associated with skin disease in free-living common frogs Rana temporaria in Switzerland, helps to address this paucity in knowledge, but the geographic distribution and epidemiology of the pathogen remains unclear. Syndromic surveillance for ranid herpesvirus skin disease was undertaken throughout Great Britain (GB), January 2014 to December 2016. Reports of common frogs with macroscopic skin lesions with a characteristic grey appearance were solicited from members of the public. Post-mortem examination was conducted on one affected frog found dead in 2015 at a site in England. In addition, archived samples from an incident involving common frogs in England in 1997 with similar macroscopic lesions were further investigated. Transmission electron microscopy identified herpes-like virions in skin lesions from both the 1997 and 2015 incidents. RHV3, or RHV3-like virus, was detected in skin lesions from the 2015 case by PCR and sequencing. Our findings indicate that herpesvirus skin disease is endemic in common frogs in GB, with widespread distribution at apparently low prevalence. Further research into the role of host immunity, virus latency and the significance of infection to host survival is required to better understand the epidemiology and impact of cutaneous herpesvirus infections in amphibian populations.

Author Correction: Rapid susceptibility profiling of carbapenem-resistant Klebsiella pneumoniae.

A correction to this article has been published and is linked from the HTML and PDF versions of this paper. The error has not been fixed in the paper.

First outbreak of colonization by linezolid- and glycopeptide-resistant Enterococcus faecium harbouring the cfr gene in a UK nephrology unit.

AIM: To describe an outbreak of colonization by linezolid- and glycopeptide-resistant Enterococcus faecium harbouring the cfr gene in a UK nephrology unit. METHODS: Isolates of linezolid-resistant E. faecium were typed by pulsed-field gel electrophoresis (PFGE), and examined by polymerase chain reaction (PCR) and sequencing for the transmissible cfr gene that confers resistance to linezolid. Enhanced environmental cleaning, initial and weekly screening of all patients, and monitoring of adherence to standard infection control precautions were implemented. FINDINGS: Five patients with pre-existing renal disease were found to have rectal colonization with linezolid-resistant E. faecium over a two-week period. The index case was a 57-year-old male from India who had travelled to the UK. One patient also had a linezolid-resistant E. faecium of a different PFGE profile isolated from a heel wound. All isolates were confirmed to harbour the cfr gene by PCR and Sanger sequencing, and all were resistant to glycopeptides (VanA phenotype). CONCLUSIONS: This article describes the first UK outbreak with a single strain of linezolid- and glycopeptide-resistant E. faecium harbouring the cfr gene, affecting five patients in a nephrology unit. Following the implementation of aggressive infection control measures, no further cases were detected beyond a two-week period.

Rapid susceptibility profiling of carbapenem-resistant Klebsiella pneumoniae.

The expanding global distribution of multi-resistant Klebsiella pneumoniae demands faster antimicrobial susceptibility testing (AST) to guide antibiotic treatment. Current ASTs rely on time-consuming differentiation of resistance and susceptibility after initial isolation of bacteria from a clinical specimen. Here we describe a flow cytometry workflow to determine carbapenem susceptibility from bacterial cell characteristics in an international K. pneumoniae isolate collection (n = 48), with a range of carbapenemases. Our flow cytometry-assisted susceptibility test (FAST) method combines rapid qualitative susceptible/non-susceptible classification and quantitative MIC measurement in a single process completed shortly after receipt of a primary isolate (54 and 158 minutes respectively). The qualitative FAST results and FAST-derived MIC (MICFAST) correspond closely with broth microdilution MIC (MICBMD, Matthew's correlation coefficient 0.887), align with the international AST standard (ISO 200776-1; 2006) and could be used for rapid determination of antimicrobial susceptibility in a wider range of Gram negative and Gram positive bacteria.

Major role of pKpQIL-like plasmids in the early dissemination of KPC-type carbapenemases in the UK.

Objectives: Klebsiella pneumoniae carbapenemase (KPC)-producing Enterobacteriaceae were first seen in the UK in 2003 and have been increasingly reported since 2010, largely owing to an ongoing outbreak in North-West England. We examined the role of clonal spread and plasmid transmission in their emergence. Methods: Isolates comprised KPC-positive K. pneumoniae ( n = 33), Escherichia coli ( n = 7) and Enterobacter spp. ( n = 4) referred to the national reference laboratory between 2008 and 2010 from 17 UK centres, including three in North-West England. Isolates were typed by MLST. Plasmids were transferred by electroporation and characterized by PCR or sequencing. PCR screening assays were developed to distinguish plasmid pKpQIL variants. Results: The K. pneumoniae isolates included 10 STs, of which three belonged to clonal group (CG) 258. CG258 ( n = 19) isolates were detected in 13 centres but accounted for only 7/19 (36.8%) of those from North-West England. Most KPC-producers (37/44, 84.1%), including 16/19 CG258 K. pneumoniae , carried bla KPC on IncFII K2 plasmids. Sequencing of a subset of these plasmids ( n = 11) revealed similarities with published pKpQIL. One variant, pKpQIL-UK [identified in K. pneumoniae CG258 ( n = 5) and ST468 ( n = 1) isolates from distinct centres] had only a few nucleotide changes from classical pKpQIL, whereas pKpQIL-D1 ( n = 1) and pKpQIL-D2 ( n = 4), from isolates of various species in the North-West, harboured large variations, reflecting replacement of the partitioning and replication functions and potentially thereby facilitating spread. PCR revealed that 36/37 (97.3%) IncFII K2 -type plasmids in KPC-positive isolates had pKpQIL markers. Conclusions: pKpQIL-like plasmids played a major role in the early dissemination of KPC enzymes in the UK.

Curious bivalves: Systematic utility and unusual properties of anomalodesmatan mitochondrial genomes.

Mitogenomic trees for Bivalvia have proved problematic in the past, but several highly divergent lineages were missing from these analyses and increased representation of these groups may yet improve resolution. Here, we add seven new sequences from the Anomalodesmata and one unidentified semelid species (Bryopa lata, Euciroa cf. queenslandica, Laternula elliptica, Laternula truncata, Lyonsia norwegica, Myadora brevis, Tropidomya abbreviata, "Abra" sp.). We show that relationships in a mitogenomic tree for the Class are improved by the addition of seven anomalodesmatans from this highly divergent clade, but are still not completely consistent with relationships recovered in studies of nuclear genes. We suggest that some anomalous relationships (for instance the non-monophyly of Bivalvia) may be partially explained by compositional heterogeneity in the mitogenome and suggest that the addition of more taxa may help resolve both this effect and possible instances of long branch attraction. We also identify several curious features about anomalodesmatan mitogenomes. For example, many protein-coding gene boundaries are poorly defined in marine bivalves, but particularly so in anomalodesmatans, primarily due to non-conserved boundary sequences. The use of transcriptomic and genomic data together enabled better definition of gene boundaries, the identification of possible pseudogenes and suggests that most genes are translated monocistronically, which contrasts with many other studies. We also identified a possible case of gene duplication of ND5 in Myadora brevis (Myochamidae). Mitogenome size in the Anomalodesmata ranges from very small compact molecules, with the smallest for Laternula elliptica (Laternulidae) only 14,622bp, to Bryopa lata (Clavagellidae) which is at least 31,969bp long and may be >40,000bp. Finally, sampled species show a high degree of sequence divergence and variable gene order, although intraspecific variation in Laternula elliptica is very low.

Evaluation of meat, fruit and vegetables from retail stores in five United Kingdom regions as sources of extended-spectrum beta-lactamase (ESBL)-producing and carbapenem-resistant Escherichia coli.

We determined the prevalence and types of extended-spectrum ß-lactamase (ESBL)-producing and carbapenem-resistant Escherichia coli in raw retail beef, chicken, pork, fruit and vegetables in five UK regions in 2013-14. Raw meat (n=397), and fruit and vegetable samples (n=400) were purchased from retail stores in London, East Anglia, North West England, Scotland and Wales. Samples were tested for the presence of ESBL-producing E. coli by plating enriched samples on CHROMagar CTX and CHROMagar ESBL, for AmpC-type E. coli by plating on "CHROMagar FOX" (CHROMagar ECC+16mg/L cefoxitin), and for carbapenem-resistant E. coli by plating on CHROMagar KPC. Additionally, pre-enrichment counts were performed on the above agars, and on CHROMagar ECC. Isolates of interest were characterised by MALDI-ToF to confirm identification, by PCR for blaCIT,blaCTX-M,blaOXA, blaSHV and blaTEM genes; ESBL or blaCIT genes were sequenced. Only 1.9% and 2.5% of beef and pork samples, respectively were positive for ESBL-producing E. coli after enrichment compared with 65.4% of chicken samples. 85.6% positive samples from chicken meat carried blaCTX-M-1; blaCTX-M-15 was not detected. None of the fruits or vegetables yielded ESBL-producing E. coli and none of the meat, fruit or vegetable samples yielded carbapenem-resistant E. coli. Retail chicken was more frequently a source of ESBL-producing E. coli than were beef, pork, fruit or vegetables. None of the foodstuffs yielded E. coli with CTX-M-15 ESBL, which dominates in human clinical isolates in the UK, and none yielded carbapenem-resistant E. coli.

The role of whole genome sequencing in antimicrobial susceptibility testing of bacteria: report from the EUCAST Subcommittee.

Whole genome sequencing (WGS) offers the potential to predict antimicrobial susceptibility from a single assay. The European Committee on Antimicrobial Susceptibility Testing established a subcommittee to review the current development status of WGS for bacterial antimicrobial susceptibility testing (AST). The published evidence for using WGS as a tool to infer antimicrobial susceptibility accurately is currently either poor or non-existent and the evidence / knowledge base requires significant expansion. The primary comparators for assessing genotypic-phenotypic concordance from WGS data should be changed to epidemiological cut-off values in order to improve differentiation of wild-type from non-wild-type isolates (harbouring an acquired resistance). Clinical breakpoints should be a secondary comparator. This assessment will reveal whether genetic predictions could also be used to guide clinical decision making. Internationally agreed principles and quality control (QC) metrics will facilitate early harmonization of analytical approaches and interpretive criteria for WGS-based predictive AST. Only data sets that pass agreed QC metrics should be used in AST predictions. Minimum performance standards should exist and comparative accuracies across different WGS laboratories and processes should be measured. To facilitate comparisons, a single public database of all known resistance loci should be established, regularly updated and strictly curated using minimum standards for the inclusion of resistance loci. For most bacterial species the major limitations to widespread adoption for WGS-based AST in clinical laboratories remain the current high-cost and limited speed of inferring antimicrobial susceptibility from WGS data as well as the dependency on previous culture because analysis directly on specimens remains challenging. For most bacterial species there is currently insufficient evidence to support the use of WGS-inferred AST to guide clinical decision making. WGS-AST should be a funding priority if it is to become a rival to phenotypic AST. This report will be updated as the available evidence increases.

Carbapenemase-producing Enterobacteriaceae in hospital wastewater: a reservoir that may be unrelated to clinical isolates.

BACKGROUND: Carbapenemase-producing Enterobacteriaceae (CPE) are an emerging infection control problem in hospitals worldwide. Identifying carriers may help reduce potential spread and infections. AIM: To assess whether testing hospital wastewater for CPE can supplement patient-based screening for infection prevention purposes in a hospital without a recognized endemic CPE problem. METHODS: Wastewater collected from hospital pipework on 16 occasions during February to March 2014 was screened for CPE using chromID(®) CARBA agar and chromID(®) CPS agar with a 10µg ertapenem disc and combination disc testing. Minimum inhibitory concentrations were determined using British Society for Antimicrobial Chemotherapy methodology and carbapenemase genes detected by polymerase chain reaction or whole-genome sequencing. Selected isolates were typed by pulsed-field gel electrophoresis. FINDINGS: Suspected CPE were recovered from all 16 wastewater samples. Of 17 isolates sent to the Antimicrobial Resistance and Healthcare Associated Infections Reference Unit, six (four Citrobacter freundii and two Enterobacter cloacae complex) were New Delhi metallo-ß-lactamase (NDM) producers and the remaining 11 (six Klebsiella oxytoca and five Enterobacter cloacae complex) were Guiana-Extended-Spectrum-5 (GES-5) producers, the first to be described among Enterobacteriaceae in the UK. The four NDM-producing C. freundii, two NDM-producing E. cloacae complex, and four out of five GES-5-producing E. cloacae complex were each indistinguishable isolates of the same three strains, whereas the six GES-5-producing K. oxytoca overall shared 79% similarity. CONCLUSION: CPE are readily isolated from hospital wastewater using simple culture methods. There are either undetected carriers of CPE excreting into the wastewater, or these CPE represent colonization of the pipework from other sources. Surveillance of hospital wastewater for CPE does not appear helpful for infection control purposes within acute hospitals.

Microevolution of Monophasic Salmonella Typhimurium during Epidemic, United Kingdom, 2005-2010.

Microevolution associated with emergence and expansion of new epidemic clones of bacterial pathogens holds the key to epidemiologic success. To determine microevolution associated with monophasic Salmonella Typhimurium during an epidemic, we performed comparative whole-genome sequencing and phylogenomic analysis of isolates from the United Kingdom and Italy during 2005-2012. These isolates formed a single clade distinct from recent monophasic epidemic clones previously described from North America and Spain. The UK monophasic epidemic clones showed a novel genomic island encoding resistance to heavy metals and a composite transposon encoding antimicrobial drug resistance genes not present in other Salmonella Typhimurium isolates, which may have contributed to epidemiologic success. A remarkable amount of genotypic variation accumulated during clonal expansion that occurred during the epidemic, including multiple independent acquisitions of a novel prophage carrying the sopE gene and multiple deletion events affecting the phase II flagellin locus. This high level of microevolution may affect antigenicity, pathogenicity, and transmission.

Dynamics of piscine francisellosis differs amongst tilapia species (Oreochromis spp.) in a controlled challenge with Francisella noatunensis subsp. orientalis.

A 25-week immersion challenge was conducted exposing Oreochromis mossambicus, Oreochromis aureus and Oreochromis urolepis hornorum to Francisella noatunensis subsp. orientalis (Fno). Two populations were compared for each fish species; 'resident fish' were defined as fish maintained in tanks since week 0 of challenge, whereas 'naïve fish' were defined as fish added to tanks once temperature in water reached <26 °C at 21 weeks post-challenge. Fno genome equivalents (GEs) in water were similar in all treatments 1 h post-challenge; however, significantly lower Fno GEs were detected 2 weeks post-challenge in all tanks, and the only treatment with detectable Fno GE after 4 weeks of challenge were the O. mossambicus tanks. Twenty-one weeks post-challenge, naïve fish were stocked with 'resident' cohorts. Over a 4-week period, mortalities occurred consistently only in O. mossambicus naïve cohorts. Overall presence of granulomas in spleen of survivors was similar (>55%) in all resident populations; however, in naïve populations, only O. mossambicus presented granulomas. Similarly, only O. mossambicus presented viable Fno in the spleen of survivors, and Fno GEs were only detected in O. mossambicus, and in resident O. aureus. In conclusion, the results of this study suggest different susceptibility of tilapia species to piscine francisellosis.