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.

Automated surveillance system for hospital-acquired urinary tract infections in Denmark.

BACKGROUND: The Danish Hospital-Acquired Infections Database (HAIBA) is an automated surveillance system using hospital administrative, microbiological, and antibiotic medication data. AIM: To define and evaluate the case definition for hospital-acquired urinary tract infection (HA-UTI) and to describe surveillance data from 2010 to 2014. METHODS: The HA-UTI algorithm defined a laboratory-diagnosed UTI as a urine culture positive for no more than two micro-organisms with at least one at ≥10(4)cfu/mL, and a probable UTI as a negative urine culture and a relevant diagnosis code or antibiotic treatment. UTI was considered hospital-acquired if a urine sample was collected ≥48h after admission and <48h post discharge. Incidence of HA-UTI was calculated per 10,000 risk-days. For validation, prevalence was calculated for each day and compared to point prevalence survey (PPS) data. FINDINGS: HAIBA detected a national incidence rate of 42.2 laboratory-diagnosed HA-UTI per 10,000 risk-days with an increasing trend. Compared to PPS the laboratory-diagnosed HA-UTI algorithm had a sensitivity of 50.0% (26/52) and a specificity of 94.2% (1842/1955). There were several reasons for discrepancies between HAIBA and PPS, including laboratory results being unavailable at the time of the survey, the results considered clinically irrelevant by the surveyor due to an indwelling urinary catheter or lack of clinical signs of infection, and UTIs being considered HA-UTI in PPS even though the first sample was taken within 48h of admission. CONCLUSION: The HAIBA algorithm was found to give valid and valuable information and has, among others, the advantages of covering the whole population and allowing continuous standardized monitoring of HA-UTI.

A validation of the Danish microbiology database (MiBa) and incidence rate of Actinotignum schaalii (Actinobaculum schaalii) bacteraemia in Denmark.

Actinotignum schaalii (former named Actinobaculum schaalii) can cause urinary tract infections (UTIs) and bacteraemia, mainly in the elderly. A. schaalii is difficult to identify with conventional biochemical tests, and it is often overlooked if the urine is only cultured in ambient air. The aim of this study was to validate data from the nationwide Danish microbiology database (MiBa) with data from the laboratory information system (LIS) at the local department of microbiology in Viborg-Herning, and to evaluate the incidence rate of bacteraemia caused by A. schaalii in Denmark by using data from the MiBa. All departments of microbiology in Denmark report data to the MiBa. All microbiological samples with A. schaalii in Denmark were extracted for a period of 5 years from the MiBa and from the local LISs. All data obtained from our local LIS were also found in the MiBa, except for data on real-time PCR, which were not registered, owing to missing ID codes in the MiBa. From 2010 to 2014, there was a significant increase in the incidence rate of blood cultures with A. schaalii, from 1.8 to 6.8 cases per million, which was probably due to coincident implementation of matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF MS) in routine diagnostics. We found that A. schaalii caused bacteraemia and UTIs mainly in the elderly. In conclusion, the MiBa can be a useful source of nationwide microbiological data in Denmark. Our results suggest that the incidence rate of A. schaalii as a cause of bacteraemia has been underestimated, and that culture of urine in CO2 can improve the detection of A. schaalii.

Improving national surveillance of Lyme neuroborreliosis in Denmark through electronic reporting of specific antibody index testing from 2010 to 2012.

Our aim was to evaluate the results of automated surveillance of Lyme neuroborreliosis (LNB) in Denmark using the national microbiology database (MiBa), and to describe the epidemiology of laboratory-confirmed LNB at a national level. MiBa-based surveillance includes electronic transfer of laboratory results, in contrast to the statutory surveillance based on manually processed notifications. Antibody index (AI) testing is the recommend laboratory test to support the diagnosis of LNB in Denmark. In the period from 2010 to 2012, 217 clinical cases of LNB were notified to the statutory surveillance system, while 533 cases were reported AI positive by the MiBa system. Thirty-five unconfirmed cases (29 AI-negative and 6 not tested) were notified, but not captured by MiBa. Using MiBa, the number of reported cases was increased almost 2.5 times. Furthermore, the reporting was timelier (median lag time: 6 vs 58 days). Average annual incidence of AI-confirmed LNB in Denmark was 3.2/100,000 population and incidences stratified by municipality ranged from none to above 10/100,000. This is the first study reporting nationwide incidence of LNB using objective laboratory criteria. Laboratory-based surveillance with electronic data-transfer was more accurate, complete and timely compared to the surveillance based on manually processed notifications. We propose using AI test results for LNB surveillance instead of clinical reporting.

Utilization of blood cultures in Danish hospitals: a population-based descriptive analysis.

This national population-based study was conducted as part of the development of a national automated surveillance system for hospital-acquired bacteraemia and ascertains the utilization of blood cultures (BCs). A primary objective was to understand how local differences may affect interpretation of nationwide surveillance for bacteraemia. From the Danish Microbiology Database, we retrieved all BCs taken between 2010 and 2013 and linked these to admission data from the National Patient Registry. In total, 4 587 295 admissions were registered, and in 11%, at least one BC was taken. Almost 50% of BCs were taken at admission. The chance of having a BC taken declined over the next days but increased after 4 days of admission. Data linkage identified 876 290 days on which at least one BC was taken; 6.4% yielded positive results. Ten species, Escherichia coli, Staphylococcus aureus, Klebsiella pneumoniae, Streptococcus pneumoniae, Enterococcus faecium, Enterococcus faecalis, Pseudomonas aeruginosa, Candida albicans, Enterobacter cloacae and Klebsiella oxytoca, accounted for 74.7% of agents for this purpose classified as pathogenic. An increase in BCs and positive BCs was observed over time, particularly among older patients. BCs showed a seasonal pattern overall and for S. pneumoniae particularly. A predominance of male patients was seen for bacteraemias due to S. aureus, E. faecium and K. pneumoniae. Minor differences in BCs and positive BCs between departments of clinical microbiology underpin the rationale of a future automated surveillance for bacteraemia. The study also provides important knowledge for interpretation of surveillance of invasive infections more generally.

The Danish Microbiology Database (MiBa) 2010 to 2013.

The Danish Microbiology Database (MiBa) is a national database that receives copies of reports from all Danish departments of clinical microbiology. The database was launched in order to provide healthcare personnel with nationwide access to microbiology reports and to enable real-time surveillance of communicable diseases and microorganisms. The establishment and management of MiBa has been a collaborative process among stakeholders, and the present paper summarises lessons learned from this nationwide endeavour which may be relevant to similar projects in the rapidly changing landscape of health informatics.

Low vaccine effectiveness against influenza A(H3N2) virus among elderly people in Denmark in 2012/13--a rapid epidemiological and virological assessment.

In Denmark, the 2012/13 influenza season has been dominated by influenza A(H3N2). We estimated the vaccine effectiveness (VE) of the trivalent influenza vaccine by linking national registers in a test-negative case-control study of patients tested for influenza aged ≥65 years. The adjusted VE against laboratory-confirmed influenza A and B was -11% (95% CI: -41 to 14) and 69% (95% CI: 26 to 87), respectively. Genetic characterisation of the influenza A(H3N2) viruses indicated genetic drift, with seven substitutions at key antigenic sites.

Increased incidence of Mycoplasma pneumoniae infections detected by laboratory-based surveillance in Denmark in 2010.

In Denmark recurrent epidemics of Mycoplasma pneumoniae infections have been described since the 1950s at intervals of approximately four to six years. The latest epidemic occurred in 2004/05 followed by two years of high incidence and more than three years of low incidence. Due to a recent increase in diagnosed cases since late summer 2010, we conducted a survey of positive M. pneumoniae PCR tests performed by clinical microbiology departments in Denmark, which indicated that a new epidemic may be underway.

Caveolae, caveolin and cav-p60 in smooth muscle and renin-producing cells in the rat kidney.

AIMS: In vascular smooth muscle cells caveolae are important for signalling mechanisms regulating vascular contraction. In smooth muscle layer of the renal afferent arteriole juxtaglomerular cells (JG cells) are non-contractile renin producing cells that have the capacity to change their phenotype into smooth muscle cells and back again by metaplastic transformation. Signalling mechanisms in JG cells are not fully understood and we therefore investigated if caveolae were present, and thereby could be involved as integrators of cellular signalling in both of these phenotypes of smooth muscle cells. METHODS: Using electron microscopy we compared the number of caveolae in JG cells and smooth muscle cells in the afferent arteriole of the rat kidney. The expression of caveolin and cav-p60 was examined using a combination of immunogold electron microscopy and immunofluorescence microscopy. RESULTS: We found that JG cells have sixfold less caveolae per cell surface sectional length than smooth muscle cells. The expression of cavolin-1 and cav-p60 correlated with the number of caveolae. An examination of the general distribution of caveolae, cav-p60 and caveolins in the rat kidney showed that cav-p60, like caveolin-1, is a specific maker of caveolae. CONCLUSION: The number of caveolae in JG cells is very low, and this makes it unlikely that caveolae are of major importance for the renin secretion specific for JG cells.

cav-p60 expression in rat muscle tissues. Distribution of caveolar proteins.

Caveolae are plasmalemmal invaginations of uncertain function. In view of the large number of hypotheses on caveolar functions, it is important to identify which components of caveolae are tissue specific and which are general. The only well-characterized major protein of caveolae is caveolin, which exists in three tissue-specific isoforms: caveolin-1, -2, and -3. Recently cav-p60 was characterized as a 60-kDa caveola-specific protein in adipocytes. The distributions of cav-p60 and caveolin isoforms in different rat muscle tissues were examined by immunofluorescence and immunoelectron microscopy. Cav-p60 was present in caveolae of skeletal and heart muscle, in vascular and intestinal smooth muscle, and in adipocyte caveolae. Furthermore cav-p60 was present in endothelial cells and cells of perineural sheaths. Caveolin-1 and -2 were present in adipocytes, endothelial cells, and cells of perineural sheaths. In all kinds of vascular and intestinal smooth muscle, caveolin-1 and -2 were present at high levels, whereas caveolin-3 expression was low or undetectable, depending on the specific smooth muscle subtype. High levels of caveolin-3 were found only in caveolae and T tubules of skeletal and heart muscle. We conclude that cav-p60 is a highly specific marker of caveolae in many if not all cell types having caveolae.

A 60-kDa protein abundant in adipocyte caveolae.

To search for caveolar proteins, mice were immunised with rat adipocyte membranes. Hybridoma supernatants were screened for antibodies to proteins on the cytosolic face of caveolae by indirect immunoelectron microscopy of immunogold-labelled adipocyte plasma membrane sheets adsorbed on electron-microscope (EM) grids. One of the hybridoma supernatants (2F11) produced a specific labelling of caveolae which was much more intense than that obtained with caveolin-1 antibodies. In Western blots of sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE) separated proteins in crude membrane fractions from different rat tissues, 2F11 labelled a band corresponding to 60 kDa. The intensity of 2F11 labelling was high in adipose tissue and in other tissues varied in parallel to caveolin- labelling. In blots of plasma membrane (PM) and light-microsomal (LM) fractions from a homogenate of adipocytes, prior insulin stimulation of the adipocytes translocated GLUT-4 from the LM to the PM fraction, but was without effect on the distribution of the 60-kDa protein labelled by 2F11. Digestion with endoproteinase lys-C produced the same pattern of immunoreactive fragments of the protein in the vesicular PM and LM fractions, indicating similar membrane topology of the 2F11-reactive, 60-kDa protein in vesicles of PM and LM fractions.

Human neutrophils are devoid of the integral membrane protein caveolin.

The secretory vesicles of human neutrophils are rapidly mobilizable vesicles that contain several GPI-linked proteins, a characteristic feature of caveolae in other cells. To investigate whether secretory vesicles are structurally related to caveolae, we examined human neutrophils for the presence of caveolin, a major constituent of caveolae, by immunoblotting using monoclonal and polyclonal antibodies. Caveolin was not detected in lysates of human neutrophils nor in isolated plasma membrane/light membrane fractions in which secretory vesicles localize. In contrast, caveolin was readily detected in isolated membranes of adipose cells. We conclude that human neutrophils are devoid of caveolin and that secretory vesicles are not related to caveolae nor dependent on caveolae for mobilization.

Expression of GLUT1 in stratified squamous epithelia and oral carcinoma from humans and rats.

Most cells express facilitative glucose transporters. Four isoforms (GLUT1-4) transporting D-glucose across the plasma membrane show a specific tissue distribution, which is the basis for tissue-specific patterns in glucose metabolism. GLUT1 is expressed at high levels in tissue barriers such as the blood-brain barrier, and this isoform has been suggested as an indicator of such barriers. GLUT1 has been found in basal layers of human epidermis where no such tissue barrier is present. To further clarify these issues, we examined the distribution of GLUT1 and GLUT4 in skin, different types of oral mucosa from rat and man, and a human oral carcinoma by indirect immunofluorescence microscopy. The results showed that GLUT1 was expressed in the basal and parabasal layers of the different stratified squamous epithelia, with some variations between keratinized and non-keratinized subtypes. GLUT1 was also expressed in ductal- and myoepithelial cells of minor salivary glands and perineural sheath located in the lamina propra, and furthermore in the cells of an oral carcinoma. GLUT4 was not expressed in any of the tissues examined. This distribution of GLUT1 does not fit with the idea of GLUT1 as a general indicator of tissue barriers. In contrast, our results support the prevailing, but limited knowledge of glucose metabolism in squamous stratified epithelia, a metabolism believed to depend mostly on glycolysis, especially in the basal layers. High-level expression seemed to be confined to keratinocytes without glycogen stores.

Quantity of Na/K-ATPase and glucose transporters in the plasma membrane of rat adipocytes is reduced by in vivo triiodothyronine.

The expression of sodium-potassium pumps and glucose transporters in pure adipocyte plasma membranes from a hyperthyroid animal model was studied. Hyperthyroidism was induced by enteral administration of five doses of 90 micrograms of triiodothyronine every second day to 8-week-old rats. Following isolation of epididymal adipocytes, 3-O-methylglucose transport was measured and the number of Na/K-ATPase-(alpha 1- and alpha 2-isoforms) and glucose transporter (GLUT1 and GLUT4) molecules in sheets of adipocyte plasma membrane were determined by quantitative immunoelectron microscopy, using gold labelling. Maximal in vitro insulin stimulation of adipocytes increased the glucose transport rate and the amount of GLUT4 in the plasma membrane 15-fold, whereas the amount of alpha 2 was unaffected. In adipocytes from hyperthyroid rats, mean adipocyte volume was decreased by 18% and the quantities of GLUT4 per unit area of plasma membrane (maximal insulin stimulation) and of alpha 2 were decreased by 19% and 15%, respectively. Thus, hypotrophia of fat tissue in the hyperthyroid state is associated with a decreased expression in the plasma membrane of the glucose transporter GLUT4 and the alpha 2-isoform of Na/K-ATPase.

Quantitation of Na+/K(+)-ATPase and glucose transporter isoforms in rat adipocyte plasma membrane by immunogold labeling.

We have quantitated and studied the topology of isoforms of the Na+/K(+)-ATPase and of the glucose transporter in rat adipocyte plasma membranes. Adipocytes were incubated with or without insulin for 15 min. Sheets of native plasma membrane, with the cytoplasmic face exposed, were prepared by adsorption to EM grids. Grids were incubated in parallel with monoclonal antibodies against the Na+/K(+)-ATPase isoforms alpha 1 and alpha 2, and the glucose transporter isoforms GLUT1 and GLUT4, followed by immunogold labeling, negative staining and quantitation by counting of the gold particles in electron micrographs. In addition, the distribution of glucose transporters and Na+/K(+)-ATPase isoforms in subcellular membrane fractions prepared by an established fractionation procedure was monitored by Western blotting. We found that the Na+/K(+)-ATPases and the glucose transporters were confined to the planar part of the plasma membrane, without association to caveolar invaginations. The vast majority of the Na+/K(+)-ATPase molecules in the adipocyte plasma membrane were of the alpha 2 isoform; GLUT4 was the dominating glucose transporter isoform. The total number of Na+/K(+)-ATPase molecules labeled in the plasma membrane was 3.5 x 10(5) per cell, independent of insulin stimulation. Concomitantly, insulin increased GLUT4 labeling sevenfold to a value of 3.5 x 10(5) per cell.

Metformin ameliorates diabetes but does not normalize the decreased GLUT 4 content in skeletal muscle of obese (fa/fa) Zucker rats.

We studied the expression of the glucose transporter GLUT 4 in the soleus and red gastrocnemius muscles from obese, diabetic (fa/fa) Zucker rats compared to their lean littermates (Fa/-), with and without treatment with the antidiabetic drug metformin. In the untreated groups of rats, the GLUT 4 content in a crude membrane fraction of both the soleus and the red gastrocnemius muscles were significantly lower in the obese (fa/fa) rats (3.46 +/- 0.28 vs. 6.04 +/- 0.41, p < 0.001 and 6.0 +/- 0.24 vs. 9.1 +/- 0.48, p < 0.0001, respectively). Differences in GLUT 4 expression in soleus muscle from the same rats were confirmed by quantitative immunofluorescence microscopy, and the results were significantly correlated with the results obtained from quantitative immunoblotting (rho = 0.70, p < 0.0005). The decreased expression of GLUT 4 in fa/fa rats could contribute to the well-established insulin resistance in skeletal muscle of these animals. After 4 weeks of treatment with metformin, weight gain was not affected in either the diabetic (fa/fa) rats or the lean (Fa/-) rats. Improvement of glucose homeostasis by metformin was not associated with normalization of the GLUT 4 expression in the skeletal muscles studied, indicating (1) that the decreased GLUT 4 expression is not directly related to hyperinsulinaemia and diabetes mellitus and (2) that metformin does not normalize the expression of GLUT 4 in skeletal muscle of the diabetic (fa/fa) Zucker rats.