Epidemiology and outcomes of vancomycin-resistant enterococcus infections: a systematic review and meta-analysis.

Vancomycin-resistant enterococci (VRE) cause many infections in the healthcare context. Knowledge regarding the epidemiology and burden of VRE infections, however, remains fragmented. We aimed to summarize recent studies on VRE epidemiology and outcomes in hospitals, long-term-care facilities (LTCFs) and nursing homes worldwide based on current epidemiological reports. We searched MEDLINE/PubMed, the Cochrane Library, and Web of Science for observational studies, which reported on VRE faecium and faecalis infections in in-patients published between January 2014 and December 2020. Outcomes were incidence, infection rate, mortality, length of stay (LOS), and healthcare costs. We conducted a meta-analysis on mortality (PROSPERO registration number: CRD42020146389). Of 681 identified publications, 57 studies were included in the analysis. Overall quality of evidence was moderate to low. VRE incidence was rarely and heterogeneously reported. VRE infection rate differed highly (1-55%). The meta-analysis showed a higher mortality for VRE faecium bloodstream infections (BSIs) compared with VSE faecium BSIs (risk ratio, RR 1.46; 95% confidence interval (CI) 1.17-1.82). No difference was observed when comparing VRE faecium vs VRE faecalis BSI (RR 1.00, 95% CI 0.52-1.93). LOS was higher in BSIs caused by E. faecium vs E. faecalis. Only three studies reported healthcare costs. In contrast to previous findings, our meta-analysis of included studies indicates that vancomycin resistance independent of VRE species may be associated with a higher mortality. We identified a lack of standardization in reporting outcomes, information regarding healthcare costs, and state-of-the-art microbiological species identification methodology, which may inform the set-up and reporting of future studies.

Machine-assisted interpretation of auramine stains substantially increases through-put and sensitivity of microscopic tuberculosis diagnosis.

Of all bacterial infectious diseases, infection by Mycobacterium tuberculosis poses one of the highest morbidity and mortality burdens on humans throughout the world. Due to its speed and cost-efficiency, manual microscopy of auramine-stained sputum smears remains a crucial first-line detection method. However, it puts considerable workload on laboratory staff and suffers from a limited sensitivity. Here we validate a scanning and analysis system that combines fully-automated microscopy with deep-learning based image analysis. After automated scanning, the system summarizes diagnosis-relevant image information and presents it to the microbiologist in order to assist diagnosis. We tested the benefit of the automated scanning and analysis system using 531 slides from routine workflow, of which 56 were from culture positive specimen. Assistance by the scanning and analysis system allowed for a higher sensitivity (40/56 positive slides detected) than manual microscopy (34/56 positive slides detected), while greatly reducing manual slide-analysis time from a recommended 5-15 min to around 10 s per slide on average.

A randomised phase II trial and feasibility study of palliative chemotherapy in frail or elderly patients with advanced gastroesophageal cancer (321GO).

BACKGROUND: Elderly patients are commonly under-represented in cancer clinical trials. The 321GO was undertaken in preparation for a definitive phase three trial assessing different chemotherapy regimens in a frail and/or elderly population with advanced gastroesophageal (GO) cancer. METHODS: Patients with advanced GO cancer considered unfit for conventional dose chemotherapy were randomly assigned in a 1 : 1 : 1 ratio to: epirubicin, oxaliplatin and capecitabine (EOX); oxaliplatin and capecitabine (OX); and capecitabine alone (X) (all 80% of full dose and unblinded). The primary end point was patient recruitment over an 18-month period. A registration study recorded treatment choice for all patients with advanced GO cancer at trial centres. RESULTS: A total of 313 patients were considered for palliative chemotherapy for GO cancer over the 18-month period: 115 received full dose treatment, 89 less than standard treatment or entered 321GO and 111 no treatment. Within 321GO, 55 patients were randomly assigned (19 to OX and X; 17 to EOX). Progression-free survival (PFS) for all patients was 4.4 months and by arm 5.4, 5.6 and 3.0 months for EOX, OX and X, respectively. The number of patients with a good overall treatment utility (OTU), a novel patient-centred endpoint, at 12 weeks was 3 (18%), 6 (32%) and 1 (6%) for EOX, OX and X, respectively. At 6 weeks, 22 patients (41%) had experienced a non-haematologic toxicity ⩾grade 3, most commonly lethargy or diarrhoea. The OTU was prognostic for overall survival in patients alive at week 12 (logrank test P=0.0001). CONCLUSIONS: It is feasible to recruit elderly and/or frail patients with advanced GO cancer to a randomised clinical trial. The OX is the preferred regimen for further study. Overall treatment utility shows promise as a comparator between treatment regimens for feasibility and randomised trials in the elderly and/or frail GO cancer population.

Lethal and sub-lethal responses of the biogenic reef forming polychaete Sabellaria alveolata to aqueous chlorine and temperature.

Sabellaria alveolata, a reef-forming marine polychaete, was exposed to aqueous chlorine which is routinely used as an anti-fouling agent in power station cooling water. Worms were treated to a range of chlorination levels (0, 0.02, 0.1 and 0.5 mg l(-1) Total Residual Oxidant referred to as control, low, intermediate and high TRO) at mean and maximum summer temperatures (18 and 23 °C respectively). Overall mortality was relatively low, however a combination of high temperature and intermediate and high TRO resulted in a significant increase in mortality compared to the control and low TRO treatments. In contrast the extension of dwelling tubes was reduced at high TRO, but increased at low and intermediate TRO levels relative to the controls independent of temperature. Finally, tube strength was found to decrease with increasing TRO, again independent of temperature. On the basis of these findings, S. alveolata can be considered tolerant of one month exposures to low TRO at water temperatures up to and including the summer maxima for southern UK waters. However, at higher TRO levels and during warm weather, high mortality would be predicted.

Keratan sulphate in the interglobular domain has a microstructure that is distinct from keratan sulphate elsewhere on pig aggrecan.

The microstructure of keratan sulphate purified from the interglobular domain, the keratan sulphate-rich region and total aggrecan was compared using fluorophore-assisted-carbohydrate-electrophoresis. Keratan sulphate in the interglobular domain was substantially less sulphated than keratan sulphate elsewhere on aggrecan, based on the ratio of unsulphated: monosulphated disaccharides generated by endo-beta-galactosidase digestion, and the ratio of monosulphated: disulphated disaccharides generated by keratanase II digestion. The ratio of unsulphated: monosulphated: disulphated disaccharides was 1:4:5 for keratan sulphate from total aggrecan and the keratan sulphate-rich region, but only 1:0.9:0.8 for the interglobular domain. These results show that keratan sulphate in the interglobular domain of pig aggrecan has a microstructure that is distinct from keratan sulphate in the keratan sulphate-rich region.

Mortality within 30 days of chemotherapy: a clinical governance benchmarking issue for oncology patients.

No national benchmark figures exist for early mortality due to chemotherapy unlike for surgical interventions. Deaths within 30 days of chemotherapy during a 6-month period were identified from the Royal Marsden Hospital electronic patient records. Treatment intention--curative or palliative, cause of death and number of previous treatments--were documented. Between April 2005 and September 2005, 1976 patients received chemotherapy with 161 deaths within 30 days of chemotherapy (8.1%). Of these, 124 deaths (77.0%) were due to disease progression. Of the other 37 deaths, 12 (7.5%) were related to chemotherapy, six each for solid tumours and haematological malignancies, of which seven (4.3%) were due to neutropenic sepsis. For the remaining 25 deaths (15.5%) there was insufficient information. There were more deaths after third and subsequent lines of therapy than with first and secondlines of therapy. Only 12 of the 161 deaths occurred in patients who were receiving potentially curative chemotherapy to give a mortality rate in breast and gastrointestinal malignancy of 0.5 and 1.5%, respectively. It is possible to audit mortality within 30 days of chemotherapy and this should become a benchmark for standard practice nationally. Most deaths were due to disease progression in the palliative setting. We practice this form of audit each quarter and feed back to the treating teams so that deaths are discussed and practice monitored.

The activity of methylated and non-methylated selenium species in lymphoma cell lines and primary tumours.

BACKGROUND: Diffuse large B-cell lymphoma patients with low serum selenium concentration at presentation have a lower response rate and overall survival than patients with higher serum selenium. The co-administration of selenium with conventional chemotherapy may be useful in these patients. PATIENTS AND METHODS: We investigated the activity of two selenium species, methylseleninic acid (MSA) and selenodiglutathione (SDG) in a panel of human lymphoma cell lines and in a primary lymphoma culture system. RESULTS: Both compounds demonstrated cytostatic and cytotoxic activity with EC(50) values in the range 1.0-10.2 microM. Cell death was associated with an increase in the sub-G1 (apoptotic) fraction by flow cytometry and was not preceded by any obvious cell cycle arrest. SDG, but not MSA, resulted in marked increases in intracellular ROS, particularly in CRL2261 and SUD4 cells in which the cytotoxic activity of SDG was partly, or completely, inhibited by n-acetyl cysteine, suggesting a dependence on ROS for activity in some cells. Both MSA and SDG showed a concentration dependent reduction in percentage viability after a 2-day exposure in primary lymphoma cultures, with EC(50) values in the range 39-300 microM and 9-28 microM, respectively. CONCLUSION: The selenium compounds MSA and SDG induce cell death in lymphoma cell lines and primary lymphoma cultures, which with SDG may be partly attributable to the generation of ROS.

Perlecan from human epithelial cells is a hybrid heparan/chondroitin/keratan sulfate proteoglycan.

Perlecan is a multidomain proteoglycan, usually substituted with heparan sulphate (HS), and sometimes substituted with both HS and chondroitin sulphate (CS). In this paper, we describe perlecan purified from HEK-293 cells substituted with HS, CS and keratan sulphate (KS). KS substitution was confirmed by immunoreactivity with antibody 5D4, sensitivity to keratanase treatment, and fluorophore-assisted carbohydrate electrophoresis. HEK-293 perlecan failed to promote FGF-dependent cell growth in an in vitro assay. This study is the first to report perlecan containing KS, and makes perlecan one of only a very few proteoglycans substituted with three distinct types of glycosaminoglycan chains.

Basic science knowledge of dental students on conventional and problem-based learning (PBL) courses at Liverpool.

This cross-sectional study investigated performances of two consecutive cohorts of second year dental students on completion of a conventional didactic course and two succeeding cohorts of second year dental students on a recently introduced problem-based learning (PBL) course at The University of Liverpool School of Dentistry. A 40 part true/false questionnaire tested recall of factual knowledge in anatomy, biochemistry, oral biology and physiology. The results showed no significant difference in the total scores when negatively marked between the conventional and PBL course groups but higher total scores in the PBL groups when positively marked. Performances in anatomy, oral biology and physiology did not differ between the groups when negatively marked but the scores of the conventional course groups in biochemistry were significantly lower than the others. With positive marking biochemistry scores were not significantly different but all other subjects were significantly higher in the PBL course groups. The PBL course groups offered fewer blank responses than the conventional course groups but the ratio of correct to incorrect responses, for both definite and intelligent guess responses, were similar in these groups. The compositional profile of the study groups was similar with respect to educational background but the PBL course groups included more females and more older students. The results of this study show that the overall knowledge recall in the basic sciences by dental students on PBL or conventional didactic courses does not differ and may be helpful to those considering the introduction of PBL into the curriculum.

Mutations in the interglobular domain of aggrecan alter matrix metalloproteinase and aggrecanase cleavage patterns. Evidence that matrix metalloproteinase cleavage interferes with aggrecanase activity.

We have expressed G1-G2 mutants with amino acid changes at the DIPEN(341) downward arrow(342)FFGVG and ITEGE(373) downward arrow(374)ARGSV cleavage sites, in order to investigate the relationship between matrix metalloproteinase (MMP) and aggrecanase activities in the interglobular domain (IGD) of aggrecan. The mutation DIPEN(341) to DIGSA(341) partially blocked cleavage by MMP-13 and MMP-8 at the MMP site, while the mutation (342)FFGVG to (342)GTRVG completely blocked cleavage at this site by MMP-1, -2, -3, -7, -8, -9, -13, -14. Each of the MMP cleavage site mutants, including a four-amino acid deletion mutant lacking residues ENFF(343), were efficiently cleaved by aggrecanase, suggesting that the primary sequence at the MMP site had no effect on aggrecanase activity in the IGD. The mutation (374)ARGSV to (374)NVYSV completely blocked cleavage at the aggrecanase site by aggrecanase, MMP-8 and atrolysin C but had no effect on the ability of MMP-8 and MMP-13 to cleave at the Asn(341) downward arrowPhe bond. Susceptibility to atrolysin C cleavage at the MMP site was conferred in the DIGSA(341) mutant but absent in the wild-type, (342)GTRVG, (374)NVYSV, and deletion mutants. To further explore the relationship between MMP and aggrecanase activities, sequential digest experiments were done in which MMP degradation products were subsequently digested with aggrecanase and vice versa. Aggrecanase-derived G1 domains with ITEGE(373) C termini were viable substrates for MMPs; however, MMP-derived G2 fragments were resistant to cleavage by aggrecanase. A 10-mer peptide FVDIPENFFG, which is a substrate analogue for the MMP cleavage site, inhibited aggrecanase cleavage at the Glu(373) downward arrowAla bond. This study demonstrates that MMPs and aggrecanase have unique substrate recognition in the IGD of aggrecan and suggests that sequences at the C terminus of the DIPEN(341) G1 domain may be important for regulating aggrecanase cleavage.

Matrix metalloproteinases 19 and 20 cleave aggrecan and cartilage oligomeric matrix protein (COMP).

Matrix metalloproteinase (MMP)-19 and MMP-20 (enamelysin) are two recently discovered members of the MMP family. These enzymes are involved in the degradation of the various components of the extracellular matrix (ECM) during development, haemostasis and pathological conditions. Whereas MMP-19 mRNA is found widely expressed in body tissues, including the synovium of normal and rheumatoid arthritic patients, MMP-20 expression is restricted to the enamel organ. In this study we investigated the ability of MMP-19 and MMP-20 to cleave two of the macromolecules characterising the cartilage ECM, namely aggrecan and the cartilage oligomeric matrix protein (COMP). Both MMPs hydrolysed aggrecan efficiently at the well-described MMP cleavage site between residues Asn(341) and Phe(342), as shown by Western blotting using neo-epitope antibodies. Furthermore, the two enzymes cleaved COMP in a distinctive manner, generating a major proteolytic product of 60 kDa. Our results suggest that MMP-19 may participate in the degradation of aggrecan and COMP in arthritic disease, whereas MMP-20, due to its unique expression pattern, may primarily be involved in the turnover of these molecules during tooth development.

Generation and novel distribution of matrix metalloproteinase-derived aggrecan fragments in porcine cartilage explants.

We have studied aggrecan catabolism mediated by matrix metalloproteinases (MMPs) in a porcine cartilage culture system. Using antibodies specific for DIPEN(341) and (342)FFGVG neoepitopes, we have detected MMP-derived fragments in conditioned medium and cultured cartilage, by radioimmunoassay, Western blotting, and immunolocalization. Radioimmunoassay revealed that the amount (pmol of epitope/mg of total glycosaminoglycan) of (342)FFGVG epitope released from cartilage remained constant over a 5-day culture period and was not increased by IL-1alpha or retinoate. However, the proportion (pmol of epitope/mg of released glycosaminoglycan) of (342)FFGVG epitope released was decreased upon stimulation, consistent with the involvement of a non-MMP proteinase, such as aggrecanase. The data suggest that in vitro MMPs may be involved in the base-line catabolism of aggrecan. Immunolocalization experiments showed that DIPEN(341) and ITEGE(373) epitopes were increased by treatment with IL-1alpha and retinoate. Confocal microscopy revealed that ITEGE(373) epitope was largely intracellular but with matrix staining in the superficial zone, whereas DIPEN(341) epitope was cell-associated and widely distributed in the matrix. Surprisingly, the majority of (342)FFGVG epitope, determined by radioimmunoassay and Western blotting, was retained in the tissue despite the absence of a G1 domain anchor. Interleukin-1alpha stimulation caused a marked increase in tissue DIPEN(341) and (342)FFGVG epitope, and the (342)FFGVG fragments retained in the tissue were larger than those released into the medium. Active porcine aggrecanase was unable to cleave (342)FFGVG fragments at the downward arrowGlu(373) downward arrowAla(374) bond but cleaved intact aggrecan at this site, suggesting that (342)FFGVG fragments are not substrates for aggrecanase. The apparent retention of large (342)FFGVG fragments within cartilage, and their resistance to N-terminal cleavage by aggrecanase suggests that (342)FF6V6 fragments may have a role in cartilage homeostasis.

The value of a questionnaire in assessing the acquisition and retention of basic science knowledge by dental students.

This cross-sectional study aimed to assess and compare, by performances in a questionnaire, the level of knowledge of basic medical sciences in 6th-form school pupils studying science subjects as entrance requirements to University and in 2nd, 3rd and 4th-year undergraduate dental students. A 40-part multiple response, true/false questionnaire, testing recall of factual knowledge in anatomy, biochemistry, physiology and oral biology, was used as the method of assessment. The results suggested that this simple format was an acceptable and useful method of assessment of the knowledge level of the study groups. The difference in scores of knowledge, expected to be higher in 2nd-year students compared to 6th form groups, was greatest in anatomy and oral biology, less in biochemistry and, unexpectedly, was not apparent in physiology. A difference in performance on the knowledge questionnaire was observed between 4th and 2nd year dental students, attributable primarily to decreased scores for 4th year dental students in biochemistry and, to a lesser extent, anatomy. The results obtained with this standardised test of factual knowledge recall may be of value to teachers compiling medical sciences courses for dental undergraduates and to those planning and evaluating new curricula with a less didactic approach.

Effects on tooth movement of force delivery from nickel-titanium archwires.

The aim of this project was to determine the in vivo effects of tooth movement with nickel-titanium archwires on the periodontium during the early stages of orthodontic treatment. The extent of tooth movement, severity of gingival inflammation, pocket probing depth, gingival crevicular fluid (GCF) flow, and the amount of the chondroitin sulphate (CS) glycosaminoglycan (GAG) component of the GCF of one maxillary canine in each of 33 patients treated with a pre-adjusted appliance were measured before and at four stages during the first 22 weeks of treatment. The methods involved the use of a reflex metrograph to determine the type of tooth movement and electrophoresis to quantitate the CS in the GCF. It was found that GCF flow increased after 4 weeks of tooth movement whereas the increase in the amount of CS in the GCF, which is taken to be indicative of periodontal tissue turnover, occurred at the later stage of 10 weeks. Teeth which showed the greatest amount of tooth movement continued to express large amounts of CS in large volumes of GCF until 22 weeks, whilst the CS levels in those teeth moving to a smaller extent declined. These data suggest that nickel-titanium archwires may produce a super-elastic plateau effect in vivo on canine teeth, which are initially displaced from the arch such that large amounts of tooth movement occur in the first 22 weeks of treatment.

Recombinant human aggrecan G1-G2 exhibits native binding properties and substrate specificity for matrix metalloproteinases and aggrecanase.

A recombinant human aggrecan G1-G2 fragment comprising amino acids Val(1)-Arg(656) has been expressed in Sf21 cells using a baculovirus expression system. The recombinant G1-G2 (rG1-G2) was purified to homogeneity by hyaluronan-Sepharose affinity chromatography followed by high performance liquid chromatography gel filtration, and gave a single band of M(r) 90,000-95,000 by silver stain or immunoblotting with monoclonal antibody 1-C-6. The expressed G1-G2 bound to both hyaluronan and link protein indicating that the immunoglobulin-fold motif and proteoglycan tandem repeat loops of the G1 domain were correctly folded. Further analysis of secondary structure by rotary shadowing electron microscopy confirmed a double globe appearance, but revealed that the rG1-G2 was more compact than its native counterpart. The size of rG1-G2 by SDS-polyacrylamide gel electorphoresis was unchanged following digestion with keratanase and keratanase II and reduced by only 2-5 kDa following digestion with either O-glycosidase or N-glycosidase F. Recombinant G1-G2 was digested with purified matrix metalloproteinases (MMP), isolated aggrecanase, purified atrolysin C, or proteinases present in conditioned medium from cartilage explant cultures, and the products analyzed on SDS gels by silver stain and immunoblotting. Neoepitope antibodies recognizing the N-terminal F(342)FGVG or C-terminal DIPEN(341) sequences were used to confirm MMP cleavage at the Asn(341) downward arrow Phe bond, while neoepitope antibodies recognizing the N-terminal A(374)RGSV or C-terminal ITEGE(373) sequences were used to confirm aggrecanase cleavage at the Glu(373) downward arrow Ala bond. Cleavage at the authentic MMP and aggrecanase sites revealed that these proteinases have the same specificity for rG1-G2 as for native aggrecan. Incubation of rG1-G2 with conditioned medium from porcine cartilage cultures revealed that active soluble aggrecanase but no active MMPs, was released following stimulation with interleukin-1alpha or retinoic acid. Atrolysin C, which cleaves native bovine aggrecan at both the aggrecanase and MMP sites, efficiently cleaved rG1-G2 at the aggrecanase site but failed to cleave at the MMP site. In contrast, native glycosylated G1-G2 with or without keratanase treatment was cleaved by atrolysin C at both the aggrecanase and MMP sites. The results suggest that the presence or absence per se of keratan sulfate on native G1-G2 does not affect the activity of atrolysin C toward the two sites.

Membrane-type 1 MMP (MMP-14) cleaves at three sites in the aggrecan interglobular domain.

An aggrecan G1-G2 substrate was used to determine sites within the interglobular domain that were susceptible to cleavage by MT1-MMP. Degradation products were identified by Western blotting with neo-epitope antibodies specific for MMP-derived N- and C-terminal sequences. The results showed that MT1-MMP cleaved at the N341-F342 and D441-L442 bonds, as shown for other MMPs, and also at a site 13 amino acids C-terminal to the N341-F342 site. The G2 product of this additional cleavage was identified by sequence analysis and revealed an N-terminus commencing T355VxxPDVELPLP. The data are consistent with MT1-MMP cleavage at three sites in the aggrecan interglobular domain; one at N342-F342, a second at D441-L442 and a third at Q354-T355.