Description and validation of a new automated surveillance system for Clostridium difficile in Denmark.

The surveillance of Clostridium difficile (CD) in Denmark consists of laboratory based data from Departments of Clinical Microbiology (DCMs) sent to the National Registry of Enteric Pathogens (NREP). We validated a new surveillance system for CD based on the Danish Microbiology Database (MiBa). MiBa automatically collects microbiological test results from all Danish DCMs. We built an algorithm to identify positive test results for CD recorded in MiBa. A CD case was defined as a person with a positive culture for CD or PCR detection of toxin A and/or B and/or binary toxin. We compared CD cases identified through the MiBa-based surveillance with those reported to NREP and locally in five DCMs representing different Danish regions. During 2010-2014, NREP reported 13 896 CD cases, and the MiBa-based surveillance 21 252 CD cases. There was a 99·9% concordance between the local datasets and the MiBa-based surveillance. Surveillance based on MiBa was superior to the current surveillance system, and the findings show that the number of CD cases in Denmark hitherto has been under-reported. There were only minor differences between local data and the MiBa-based surveillance, showing the completeness and validity of CD data in MiBa. This nationwide electronic system can greatly strengthen surveillance and research in various applications.

Monitoring meticillin resistant Staphylococcus aureus and its spread in Copenhagen, Denmark, 2013, through routine whole genome sequencing.

Typing of meticillin resistant Staphylococcus aureus (MRSA) by whole genome sequencing (WGS) is performed routinely in Copenhagen since January 2013. We describe the relatedness, based on WGS data and epidemiological data, of 341 MRSA isolates. These comprised all MRSA (n = 300) identified in Copenhagen in the first five months of 2013. Moreover, because MRSA of staphylococcal protein A (spa)-type 304 (t304), sequence type (ST) 6 had been associated with a continuous neonatal ward outbreak in Copenhagen starting in 2011, 41 t304 isolates collected in the city between 2010 and 2012 were also included. Isolates from 2013 found to be of t304, ST6 (n=14) were compared to the 41 earlier isolates. In the study, isolates of clonal complex (CC) 22 were examined in detail, as this CC has been shown to include the hospital-acquired epidemic MRSA (EMRSA-15) clone. Finally, all MRSA ST80 were also further analysed, as representatives of an important community-acquired MRSA in Europe. Overall the analysis identified 85 spa-types and 35 STs from 17 CCs. WGS confirmed the relatedness of epidemiologically linked t304 neonatal outbreak isolates. Several non-outbreak related patients had isolates closely related to the neonatal isolates suggesting unrecognised community chains of transmission and insufficient epidemiological data. Only four CC22 isolates were related to EMRSA-15. No community spread was observed among the 13 ST80 isolates. WGS successfully replaced conventional typing and added information to epidemiological surveillance. Creation of a MRSA database allows clustering of isolates based on single nucleotide polymorphism (SNP) calling and has improved our understanding of MRSA transmission.

Validation of dipslides as a tool for environmental sampling in a real-life hospital setting.

Environmental sampling in hospitals is becoming increasingly important because of the rise in nosocomial infections. In order to monitor and track these infections and optimize cleaning and disinfection, we need to be able to locate the fomites with the highest amount of microorganisms, but the optimal method for this is not clear. The aim of this study was to evaluate which of four different dipslides or a standard TSA contact plate were best at recovering human bacteria from the environment. We tested four different dipslides with selective and non-selective agars versus a standard TSA contact plate in order to find the best sampling media. Two hundred sites in a children's medical ward in Copenhagen University hospital were sampled in autumn 2012. There was no difference in total bacteria count between the TSA contact plate and the dipslides. Faecal indicator bacteria recovery was the same for the dipslides and the TSA contact plate. Dipslides may be equally well suited for environmental sampling and hygiene assessment as TSA contact plates. Dipslides have some advantages, such as better sample security, easier sampling in confined spaces and longer shelf life that may speak in favour of choosing these for bacteria environmental sampling in hospitals, depending on the task.

A systematic review of peri-operative melatonin.

We systematically reviewed randomised controlled trials of peri-operative melatonin. We included 24 studies of 1794 participants that reported eight peri-operative outcomes: anxiety; analgesia; sleep quality; oxidative stress; emergence behaviour; anaesthetic requirements; steal induction; and safety. Compared with placebo, melatonin reduced the standardised mean difference (95% CI) pre-operative anxiety score by 0.88 (0.44-1.33) and postoperative pain score by 1.06 (0.23-1.88). The magnitude of effect was unreliable due to substantial statistical heterogeneity, with I(2) 87% and 94%, respectively. Qualitative reviews suggested the melatonin improved sleep quality and emergence behaviour, and might be capable of reducing oxidative stress and anaesthetic requirements.

Repeated introduction and spread of the MRSA clone t304/ST6 in northern Europe.

OBJECTIVES: During the last decades several methicillin-resistant Staphylococcus aureus (MRSA) clones with the capability of global spread have emerged in the community. Here, we have investigated a large collection of clinical isolates belonging to MRSA clone t304/ST6, which has emerged in many European countries over the last years, in order to retrace its phylogeny and its spread. METHODS: We characterized 466 ST6 isolates from Denmark (n = 354), France (n = 10), Norway (n = 24), Sweden (n = 27) and the UK (n = 51). All had spa-type t304 (n = 454) or t304-related spa-types (n = 12) and whole genome sequencing (WGS) was carried out on Illumina Miseq or Hiseq with 100-300 bp reads. cgMLST was performed using Ridom SeqSphere. RESULTS: A minimum spanning tree (MST) of all 466 isolates showed one large cluster including 182 isolates collected only from Denmark and related to a long-term neonatal outbreak in Copenhagen. This cluster contrasted with numerous small clusters, including the remaining Danish isolates and isolates from the other countries that interspersed throughout the tree. Most isolates were Panton-Valentine leukocidin (PVL) negative (95%) and harboured SCCmec IVa. One genome was closed using Oxford Nanopore technology and Illumina MiSeq. It contained a plasmid of 19.769 bp including the blaZ gene. A similar plasmid was found in 78% of all isolates. DISCUSSION: t304/ST6 is a successful emerging clone and the fact that isolates from five countries are interspersed throughout the MST indicates a common origin. This clone is commonly described in the Middle East and its emergence in Europe coincides with influx of refugees from the Syrian Civil War.

Genetic relationship between bacteria isolated from intraoperative air samples and surgical site infections at a major teaching hospital in Ghana.

BACKGROUND: In low- and middle-income countries (LMICs) the rate of surgical site infections (SSI) is high, leading to negative patient outcomes and excess healthcare costs. A causal relationship between airborne bacteria in the operating room and SSI has not been established, at a molecular or genetic level. We studied the relationship between intraoperative airborne bacteria and bacteria causing SSI in an LMIC. METHODS: Active air sampling using a portable impactor was performed during clean or clean-contaminated elective surgical procedures. Active patient follow-up consisting of phone calls and clinical examinations was performed 3, 14 and 30 days after surgery. Bacterial isolates recovered from SSI and air samples were compared by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF) identification, ribotyping, whole genome sequencing (WGS), and metagenomic analysis. RESULTS: Of 128 included patients, 116 (91%) completed follow-up and 11 (9%) developed SSI. Known pathogenic bacteria were isolated from intraoperative air samples in all cases with SSI. A match between air and SSI isolates was found by MALDI-TOF in eight cases. Matching ribotypes were found in six cases and in one case both WGS and metagenomic analysis showed identity between air- and SSI-isolates. CONCLUSION: The study showed high levels of intraoperative airborne bacteria, an SSI-rate of 9% and a genetic link between intraoperative airborne bacteria and bacteria isolated from SSIs. This indicates the need for awareness of intraoperative air quality in LMICs.

Spread of bacteria on surfaces when cleaning with microfibre cloths.

The impact of environmental contamination on nosocomial cross-transmission is mostly unresolved and in Danish hospitals assessment of cleaning is based on visible criteria only. The use of premoistened microfibre cloths and the 16-side method have been introduced into Danish hospitals because of economic and ergonomic advantages but they have not been evaluated for applicability in hospital cleaning. Our hypothesis was that this method may spread bacteria. A surface was contaminated with bacteria (4 cfu/bacteria/cm(2)), and cleaned with a premoistened microfibre cloth folded to 16-side use. Each of 15 sterile surfaces was cleaned with a new side of the microfibre cloth; imprints were made and the experiment repeated 12 times. After cleaning, the contaminated surface imprints of microfibre cloths showed a median of 45.5 cfu/plate for E. faecalis and 2.5 cfu/plate for B. cereus. Median values from imprints from cloth sides 2-16 were between 1 and 12 cfu/plate for E. faecalis and 0 cfu/plate for B. cereus. Imprints of the contaminated surfaces were a median of 45.5 cfu/plate for E. faecalis, giving a reduction of 5.6-fold. For B. cereus the median value was 0 cfu/plate. The surface numbers 2-16 had median values between 0.5 and 7.5 for E. faecalis, which was spread to 11-15 of the 15 sterile surfaces (P<0.01). B. cereus was found in six out of 180 imprints on surfaces 2-16, all with 1 cfu/plate (non-significant). The implication is that although there was an overall reduction in bacterial counts on the contaminated surface, bacteria were spread to subsequently cleaned surfaces.

MR colonography with fecal tagging: barium vs. barium ferumoxsil.

RATIONALE AND OBJECTIVES: Both magnetic resonance (MR) and computed tomographic (CT) colonography are useful for colon examination. With sensitivities close to those for conventional colonoscopy (CC) for polyps, colonography has been proposed as an alternative to diagnostic CC. MR colonography (MRC) with fecal tagging may be a method of gaining further patient acceptance and widespread use, but the method has to be optimized. The aim of our study was to evaluate the quality of a new contrast agent mixture and to validate a new method for evaluating the tagging efficiency of contrast agents. MATERIALS AND METHODS: Twenty patients referred to CC underwent dark lumen MRC prior to the colonoscopy. Two groups of patients received two different oral contrast agents (barium sulfate and barium sulfate/ferumoxsil) as a laxative-free fecal tagging prior to the MRC. After MRC, the contrast agent was rated qualitatively (with the standard method using contrast-to-wall ratio) and subjectively (using a visual analog scale [VAS]) by three different blinded observers. RESULTS: Evaluated both qualitatively and subjectively, the tagging efficiency of barium sulfate/ferumoxsil was significantly better (P < .05) than barium sulfate alone. The VAS method for evaluating the tagging efficiency of contrast agents showed a high correlation (observer II, r = 0.91) to the standard method using contrast-to-wall ratio and also a high interclass correlation (observer II and III = 0.89/0.85). MRC found 1 of 22 (5%) polyps <6 mm, 2 of 3 (67%) polyps 6-10 mm, and 2 of 2 (100%) polyps >10 mm. CONCLUSION: MRC with fecal tagging using barium sulfate/ferumoxsil as contrast agent will give better overall assessment of the colon wall compared to barium sulfate alone. Furthermore, the VAS method of evaluating fecal tagging efficiency correlated with the standard method of calculating the contrast-to-wall ratio.

Traffic flow and microbial air contamination in operating rooms at a major teaching hospital in Ghana.

BACKGROUND: Current literature examining the relationship between door-opening rate, number of people present, and microbial air contamination in the operating room is limited. Studies are especially needed from low- and middle-income countries, where the risk of surgical site infections is high. AIM: To assess microbial air contamination in operating rooms at a Ghanaian teaching hospital and the association with door-openings and number of people present. Moreover, we aimed to document reasons for door-opening. METHODS: We conducted active air-sampling using an MAS 100® portable impactor during 124 clean or clean-contaminated elective surgical procedures. The number of people present, door-opening rate and the reasons for each door-opening were recorded by direct observation using pretested structured observation forms. FINDINGS: During surgery, the mean number of colony-forming units (cfu) was 328 cfu/m3 air, and 429 (84%) of 510 samples exceeded a recommended level of 180 cfu/m3. Of 6717 door-openings recorded, 77% were considered unnecessary. Levels of cfu/m3 were strongly correlated with the number of people present (P = 0.001) and with the number of door-openings/h (P = 0.02). In empty operating rooms, the mean cfu count was 39 cfu/m3 after 1 h of uninterrupted ventilation and 52 (51%) of 102 samples exceeded a recommended level of 35 cfu/m3. CONCLUSION: The study revealed high values of intraoperative airborne cfu exceeding recommended levels. Minimizing the number of door-openings and people present during surgery could be an effective strategy to reduce microbial air contamination in low- and middle-income settings.

Control of a methicillin-resistant Staphylococcus aureus (MRSA) outbreak in a day-care institution.

This article describes an outbreak of methicillin-resistant Staphylococcus aureus (MRSA) in two institutions for multi-handicapped children in Copenhagen. The aim of the study was to determine whether it was possible to eradicate MRSA in a setting with multi-handicapped children and staff where there was a high degree of physical interaction. This was a prospective interventional uncontrolled cohort study that took place from January 2003 to March 2005. All individuals in close contact with the two institutions and/or in close contact with an MRSA-colonized subject from the outbreak were included in the study: 38 children, 60 staff members and 12 close relatives of colonized subjects. Infection control measures included screening all individuals. When MRSA infection or colonization was found, an attempt was made to eradicate MRSA, staff education was undertaken and attempts were made to determine the route of transmission. Eleven individuals were found to be positive for MRSA (10.0%). All isolates were identical by pulsed-field gel electrophoresis and harboured the staphylococcal cassette chromosome mec (SCCmec) type IV. All colonized and infected individuals were associated with a single room in one of the institutions. MRSA was eradicated from all the colonized and infected subjects. This study shows that it is possible to control an MRSA outbreak in institutions for multi-handicapped children where there is a high degree of physical contact.

Pharmacokinetics of Alternative Administration Routes of Melatonin: A Systematic Review.

BACKGROUND: Melatonin is traditionally administered orally but has a poor and variable bioavailability. This study aims to present an overview of studies investigating the pharmacokinetics of alternative administration routes of melatonin. METHODS: A systematic literature search was performed and included experimental or clinical studies, investigating pharmacokinetics of alternative administration routes of melatonin in vivo. Alternative administration routes were defined as all administration routes except oral and intravenous. RESULTS: 10 studies were included in the review. Intranasal administration exhibited a quick absorption rate and high bioavailability. Transdermal administration displayed a variable absorption rate and possible deposition of melatonin in the skin. Oral transmucosal administration of melatonin exhibited a high plasma concentration compared to oral administration. Subcutaneous injection of melatonin displayed a rapid absorption rate compared to oral administration. CONCLUSION: Intranasal administration of melatonin has a large potential, and more research in humans is warranted. Transdermal application of melatonin has a possible use in a local application, due to slow absorption and deposition in the skin. Oral transmucosal administration may potentially be a clinically relevant due to avoiding first-pass metabolism. Subcutaneous injection of melatonin did not document any advantages compared to other administration routes.

Melatonin as Protection Against Radiation Injury: A Systematic Review.

INTRODUCTION: Radiation is widely used in the treatment of various cancers and in radiological imaging procedures. Ionizing radiation causes adverse effects, leading to decreased quality of life in patients, by releasing free radicals that cause oxidative stress and tissue damage. The sleep-hormone melatonin is a free radical scavenger, and induces several anti-oxidative enzymes. This review investigates the scientific literature on the protective effects of melatonin against exposure to ionizing radiation, and discusses the clinical potential of melatonin as prophylactic treatment against ionizing radiation damage. METHODS: A systematic literature search was performed and included experimental or clinical studies written in English that investigated the protective effects of melatonin against gamma or X-ray irradiation in vivo. Studies were excluded if patients were treated with chemotherapy concomitantly. RESULTS: 37 studies were included in the review. All were of experimental case-control design and employed animals. The studies demonstrated that exogenous melatonin reduced oxidative stress and inflammation in all investigated tissues. Furthermore, melatonin increased 30-day survival and protected against radiation enteritis. These protective effects were only documented when melatonin was administered prior to exposure to ionizing radiation. DISCUSSION: This review documents that melatonin effectively protects animals against injury to healthy tissues from ionizing radiation. However, no studies have been performed in humans. If clinical studies can document similar protective effects, melatonin could have a great potential to prevent side effects of radiotherapy for cancer, to protect against increased long-term cancer risk in radiological imaging procedures, and to protect from radiation due to nuclear incidents.

Test and validation of methods to sample and detect human virus from environmental surfaces using norovirus as a model virus.

BACKGROUND: Viruses cause a major proportion of human infections, especially gastroenteritis and respiratory infections in children and adults. Indirect transmission between humans via environmental surfaces may play a role in infections, but methods to investigate this have been sparse. AIM: To validate and test efficient and reliable procedures to detect multiple human pathogenic viruses on surfaces. METHODS: The study was divided into two parts. In Part A, six combinations of three different swabs (consisting of cotton, foamed cotton, or polyester head) and two different elution methods (direct lysis or immersion in alkaline glycine buffer before lysis) were tested for efficient recovery of human norovirus GII.7 and mengovirus from artificially contaminated surfaces. In Part B we determined the detection limit for norovirus GI.1 and GII.3 using the best procedure found in Part A linked with a commercial multiplex real-time quantitative polymerase chain reaction detection assay. FINDINGS: Combining the polyester swab with direct lysis allowed recovery down to 100 and 10 genome copies/cm(2) of norovirus GI.1 and GII.3, respectively. This procedure resulted in the significant highest recovery of both norovirus and mengovirus, whereas no differences in amplification efficiencies were observed between the different procedures. CONCLUSION: The results indicate that it is possible to detect low concentrations of virus on environmental surfaces. We therefore suggest that a polyester swab, followed by direct lysis, combined with a multiplex qPCR detection assay is an efficient screening tool that merits study of different respiratory and gastrointestinal viruses on environment surfaces.

Effect of cleaning and disinfection of toys on infectious diseases and micro-organisms in daycare nurseries.

BACKGROUND: The rising number of children in daycare nurseries increases opportunities for the transmission of infectious diseases. Pathogens may be transmitted directly from child to child via sneezing, coughing and touching, or indirectly via the environment. Toys are among the fomites with the highest pathogen load, but their role in disease transmission is unknown. AIM: To determine if washing and disinfection of toys can reduce sickness absence and microbial pathogen load in the nursery environment. METHODS: Twelve nurseries (caring for 587 children) were randomized to intervention and control groups. The intervention consisted of washing and disinfection of toys and linen every two weeks for three months by a commercial cleaning company. The extent and causes of sickness absence among the children were recorded in both groups before and after introduction of the intervention. Ten sampling points in each nursery were examined for bacteria and respiratory viruses. RESULTS: The presence of respiratory virus DNA/RNA was widespread, but very few pathogenic bacteria were found in the environment. The intervention reduced the presence of adenovirus [odds ratio (OR) 2.4, 95% confidence interval (CI) 1.1-5.0], rhinovirus (OR 5.3, 95% CI 2.3-12.4) and respiratory syncytial virus (OR 4.1, 95% CI 1.5-11.2) compared with the control group, but the intervention had no effect on sickness absence or disease patterns in the nurseries. CONCLUSION: Although cleaning and disinfection of toys every two weeks can decrease the microbial load in nurseries, it does not appear to reduce sickness absence among nursery children.

IgM anti-GM1 antibodies in the Guillain-Barré syndrome: a serological predictor of the clinical course.

It has been suggested that antibodies against GM1 are involved in the pathogenesis of the Guillain-Barré syndrome (GBS). Recently, we have developed a standardized ELISA assay for anti-GM1 antibodies of IgM type well-suited for longitudinal patient studies. The relationship between serum antibodies against GM1 and Campylobacter jejuni was investigated in patients with GBS and in patients with C. jejuni infection. Patients with a short-lasting anti-GM1 elevation had a fast recovery, whereas patients with slow recovery had a long-lasting anti-GM1 elevation. A linear relationship was found between significant clinical recovery and the time until the anti-GM1 peak was halved (R = 0.9, p < 0.01). The absolute level of anti-GM1 did not predict the length of the recovery nor was the level of anti-GM1 related to the clinical disability at its nadir. Our data indicate that monitoring of the IgM anti-GM1 level can predict clinical recovery in GBS patients.

Treatment of H. pylori infected mice with antioxidant astaxanthin reduces gastric inflammation, bacterial load and modulates cytokine release by splenocytes.

Helicobacter pylori is a gram-negative bacterium affecting about half of the world population, causing chronic gastritis type B dominated by activated phagocytes. In some patients the disease evolves into gastric ulcer, duodenal ulcer, gastric cancer or MALT lymphoma. The pathogenesis is in part caused by the immunological response. In mouse models and in human disease, the mucosal immune response is characterized by activated phagocytes. Mucosal T-lymphocytes are producing IFN-gamma thus increasing mucosal inflammation and mucosal damage. A low dietary intake of antioxidants such as carotenoids and vitamin C may be an important factor for acquisition of H. pylori by humans. Dietary antioxidants may also affect both acquisition of the infection and the bacterial load of H. pylori infected mice. Antioxidants, including carotenoids, have anti-inflammatory effects. The aim of the present study was to investigate whether dietary antoxidant induced modulation of H. pylori in mice affected the cytokines produced by H. pylori specific T-cells. We found that treatment of H. pylori infected mice with an algal cell extract containing the antioxidant astaxanthin reduces bacterial load and gastric inflammation. These changes are associated with a shift of the T-lymphocyte response from a predominant Th1-response dominated by IFN-gamma to a Th1/Th2-response with IFN-gamma and IL-4. To our knowledge, a switch from a Th1-response to a mixed Th1/Th2-response during an ongoing infection has not been reported previously.