Device-associated infection rates,mortality, length of stay and bacterial resistance in intensive care units in Ecuador: International Nosocomial Infection Control Consortium's findings

AIM To report the results of the International Nosocomial Infection Control Consortium(INICC) study conducted in Quito, Ecuador.METHODS A device-associated healthcare-acquired infection(DAHAI) prospective surveillance study conducted from October 2013 to January 2015 in 2 adult intensive care units(ICUs) from 2 hospitals using the United States Centers for Disease Control/National Healthcare Safety Network(CDC/NHSN) definitions and INICC methods. RESULTS We followed 776 ICU patients for 4818 bed-days. The central line-associated bloodstream infection(CLABSI) rate was 6.5 per 1000 central line(CL)-days, the ventilator-associated pneumonia(VAP) rate was 44.3 per 1000 mechanical ventilator(MV)-days, and the catheterassociated urinary tract infection(CAUTI) rate was 5.7 per 1000 urinary catheter(UC)-days. CLABSI and CAUTI rates in our ICUs were similar to INICC rates [4.9(CLABSI) and 5.3(CAUTI)] and higher than NHSN rates [0.8(CLABSI) and 1.3(CAUTI)]- although device use ratios for CL and UC were higher than INICC and CDC/NSHN's ratios. By contrast, despite the VAP rate was higher than INICC(16.5) and NHSN's rates(1.1), MV DUR was lower in our ICUs. Resistance of A. baumannii to imipenem and meropenem was 75.0%, and of Pseudomonas aeruginosa to ciprofloxacin and piperacillin-tazobactam was higher than 72.7%, all them higher than CDC/NHSN rates. Excess length of stay was 7.4 d for patients with CLABSI, 4.8 for patients with VAP and 9.2 for patients CAUTI. Excess crude mortality in ICUs was 30.9% for CLABSI, 14.5% for VAP and 17.6% for CAUTI. CONCLUSION DA-HAI rates in our ICUs from Ecuador are higher than United States CDC/NSHN rates and similar to INICC international rates.     

A novel human artificial chromosome gene expression system using herpes simplex virus type 1 vectors

Human artificial chromosome (HAC) vectors are an important gene transfer system for expression and complementation studies. We describe a significant advance in HAC technology using infectious herpes simplex virus type 1 (HSV-1) amplicon vectors for delivery. This highly efficient method has allowed gene-expressing HACs to be established in glioma-, kidney- and lung-derived cells. We also developed an HSV-1 hypoxanthine phosphoribosyltransferase (HPRT) HAC vector, which generated functional HPRT-expressing HACs that complemented the genetic deficiency in human cells. The transduction efficiency of the HSV-1 HAC amplicons is several orders of magnitude higher than lipofection-mediated delivery. Studies on HAC stability between cell types showed important differences that have implications for HAC development and gene expression in human cells. This is the first report of establishing gene-expressing HACs in human cells by using an efficient, high-capacity viral vector and by identifying factors that are involved in cell-type-specific HAC instability. The work is a significant advance for HAC technology and the development of HAC gene expression systems in human cells.     

Efficiency of de novo centromere formation in human artificial chromosomes

In a comparative study, we show that human artificial chromosome (HAC) vectors based on alpha-satellite (alphoid) DNA from chromosome 17 but not the Y chromosome regularly form HACs in HT1080 human cells. We constructed four structurally similar HAC vectors, two with chromosome 17 or Y alphoid DNA (17alpha, Yalpha) and two with 17alpha or Yalpha and the hypoxanthine guanine phosphoribosyltransferase locus (HPRT1). The 17alpha HAC vectors generated artificial minichromosomes in 32-79% of the HT1080 clones screened, compared with only approximately 4% for the Yalpha HAC vectors, indicating that Yalpha is inefficient at forming a de novo centromere. The 17alpha HAC vectors produced megabase-sized, circular HACs containing multiple copies of alphoid fragments (60-250 kb) interspersed with either vector or HPRT1 DNA.The 17alpha-HPRT1 HACs were less stable than those with 17alpha only, and these results may influence the design of new HAC gene transfer vectors.     

An artificially constructed de novo human chromosome behaves almost identically to its natural counterpart during metaphase and anaphase in living cells

Human artificial chromosomes (HACs) are promising reagents for the analysis of chromosome function. While HACs are maintained stably, the segregation mechanisms of HACs have not been investigated in detail. To analyze HACs in living cells, we integrated 256 copies of the Lac operator into a precursor yeast artificial chromosome (YAC) containing alpha-satellite DNA and generated green fluorescent protein (GFP)-tagged HACs in HT1080 cells expressing a GFP-Lac repressor fusion protein. Time-lapse analyses of GFP-HACs and host centromeres in living mitotic cells indicated that the HAC was properly aligned at the spindle midzone and that sister chromatids of the HAC separated with the same timing as host chromosomes and moved to the spindle poles with mobility similar to that of the host centromeres. These results indicate that a HAC composed of a multimer of input alpha-satellite YACs retains most of the functions of the centromeres on natural chromosomes. The only difference between the HAC and the host chromosome was that the HAC oscillated more frequently, at higher velocity, across the spindle midzone during metaphase. However, this provides important evidence that an individual HAC has the capacity to maintain tensional balance in the pole-to-pole direction, thereby stabilizing its position around the spindle midzone.     

Construction of a novel human artificial chromosome vector for gene delivery

Potential problems of conventional transgenes include insertional disruption of the host genome and unpredictable, irreproducible expression of the transgene by random integration. Alternatively, human artificial chromosomes (HACs) can circumvent some of the problems. Although several HACs were generated and their mitotic stability was assessed, a practical way for introducing exogenous genes by the HACs has yet to be explored. In this study, we developed a novel HAC from sequence-ready human chromosome 21 by telomere-directed chromosome truncation and added a loxP sequence for site-specific insertion of circular DNA by the Cre/loxP system. This 21HAC vector, delivered to a human cell line HT1080 by microcell fusion, bound centromere proteins A, B, and C and was mitotically stable during long-term culture without selection. The EGFP gene inserted in the HAC vector expressed persistently. These results suggest that the HAC vector provides useful system for functional studies of genes in isogenic cell lines.     

Human artificial chromosomes constructed using the bottom-up strategy are stably maintained in mitosis and efficiently transmissible to progeny mice

Human artificial chromosomes (HACs) are alternative vectors that promise to overcome problematic transgene expression often occurring with conventional vectors in mammalian cells and bodies. We have successfully generated HACs by multimerization of a cloned long alphoid stretch in a human cell line, HT1080. Furthermore, we developed technologies for cloning large genomic regions into HACs by means of co-transfection of clones with the alphoid array and clones encoding the genomic region of interest. The purpose of this study was to investigate the mitotic and meiotic stability of such HACs in mouse cells and bodies. We transferred a circular HAC containing the guanosine triphosphate cyclohydrolase I gene (GCH1-HAC) and a linear HAC containing the human globin gene cluster (globin-HAC) from HT1080 cells into mouse embryonic stem (ES) cells by microcell-mediated chromosome transfer. The HACs were stably maintained in mouse ES cells for 3 months. GCH1-HACs in every ES cell line and globin-HACs in most ES cell lines maintained their structures without detectable rearrangement or acquisition of mouse genomic DNA except one globin-HAC in an ES cell line rearranged and acquired mouse-type centromeric sequences and long telomeres. Creation of chimeric mice using ES cells containing HAC and subsequent crossing showed that both the globin-HAC that had rearranged and acquired mouse type centromeric sequences/long telomeres and GCH1-HACs were retained in tissues of mice and transmitted to progeny. These results indicate that human artificial chromosomes constructed using the bottom-up strategy based on alphoid DNA are stable in mouse bodies and are transmissible.     

Integration-free iPS cells engineered using human artificial chromosome vectors

Human artificial chromosomes (HACs) have unique characteristics as gene-delivery vectors, including episomal transmission and transfer of multiple, large transgenes. Here, we demonstrate the advantages of HAC vectors for reprogramming mouse embryonic fibroblasts (MEFs) into induced pluripotent stem (iPS) cells. Two HAC vectors (iHAC1 and iHAC2) were constructed. Both carried four reprogramming factors, and iHAC2 also encoded a p53-knockdown cassette. iHAC1 partially reprogrammed MEFs, and iHAC2 efficiently reprogrammed MEFs. Global gene expression patterns showed that the iHACs, unlike other vectors, generated relatively uniform iPS cells. Under non-selecting conditions, we established iHAC-free iPS cells by isolating cells that spontaneously lost iHAC2. Analyses of pluripotent markers, teratomas and chimeras confirmed that these iHAC-free iPS cells were pluripotent. Moreover, iHAC-free iPS cells with a re-introduced HAC encoding Herpes Simplex virus thymidine kinase were eliminated by ganciclovir treatment, indicating that the HAC safeguard system functioned in iPS cells. Thus, the HAC vector could generate uniform, integration-free iPS cells with a built-in safeguard system.     

Human artificial chromosomes containing chromosome 17 alphoid DNA maintain an active centromere in murine cells but are not stable

Human artificial chromosomes (HACs) are autonomous molecules that can function and segregate as normal chromosomes in human cells. De novo HACs have successfully been used as gene expression vectors to complement genetic deficiencies in human cultured cells. HACs now offer the possibility of studying the regulation and expression of large genes in a variety of cell types from different tissues and correcting gene deficiencies caused by human inherited diseases. Complementary gene expression studies in mice, especially in mouse models of human genetic diseases, are also important in determining if large human transgenes can be expressed appropriately from artificial chromosomes. Toward this aim we are establishing artificial chromosomes in murine cells as novel gene expression vectors. Initially we transferred HAC vectors into murine cells, but were unable to generate de novo HACs at a reasonable frequency. We then transferred HACs previously established in human HT1080 cells to three different murine cell types by microcell fusion, followed by positive selection. We observed that the HACs in murine cells bound centromere protein C (CENP-C), a marker of active centromeres, and were detected under selection but rapidly lost when selection was removed. These results suggest that the HACs maintain at least a partially functional centromere complex in murine cells, but other factors are required for stability and segregation. Artificial chromosomes containing mouse centromeric sequences may be required for better stability and maintenance in murine cells.     

A Novel System for Simultaneous or Sequential Integration of Multiple Gene-Loading Vectors into a Defined Site of a Human Artificial Chromosome

Human artificial chromosomes (HACs) are gene-delivery vectors suitable for introducing large DNA fragments into mammalian cells. Although a HAC theoretically incorporates multiple gene expression cassettes of unlimited DNA size, its application has been limited because the conventional gene-loading system accepts only one gene-loading vector (GLV) into a HAC. We report a novel method for the simultaneous or sequential integration of multiple GLVs into a HAC vector (designated as the SIM system) via combined usage of Cre, FLP, Bxb1, and φC31 recombinase/integrase. As a proof of principle, we first attempted simultaneous integration of three GLVs encoding EGFP, Venus, and TdTomato into a gene-loading site of a HAC in CHO cells. These cells successfully expressed all three fluorescent proteins. Furthermore, microcell-mediated transfer of HACs enabled the expression of those fluorescent proteins in recipient cells. We next demonstrated that GLVs could be introduced into a HAC one-by-one via reciprocal usage of recombinase/integrase. Lastly, we introduced a fourth GLV into a HAC after simultaneous integration of three GLVs by FLP-mediated DNA recombination. The SIM system expands the applicability of HAC vectors and is useful for various biomedical studies, including cell reprogramming.     

Central line bundle implementation in US intensive care units and impact on bloodstream infections

Background

Central line-associated bloodstream infections (CLABSI) represent a serious patient safety issue. To prevent these infections, bundled interventions are increasingly recommended. We examine the extent of adoption of Central Line (CL) Bundle elements throughout US intensive care units (ICU) and determine their effectiveness in preventing CLABSIs.

Methodology/Principal Findings

In this cross-sectional study, National Healthcare Safety Network (NHSN) hospitals provided the following: ICU-specific NHSN-reported rates of CLABSI/1,000 central line days; policies and compliance rates regarding bundle components; and other setting characteristics. In 250 hospitals the mean CLABSI rate was 2.1 per 1000 central line days and 49% reported having a written CL Bundle policy. However, of those that monitored compliance, only 38% reported very high compliance with the CL Bundle. Only when an ICU had a policy, monitored compliance, and had ≥95% compliance did CLABSI rates decrease. Complying with any one of three CL Bundle elements resulted in decreased CLABSI rates (β = -1.029, p = 0.015). If an ICU without good bundle compliance achieved high compliance with any one bundle element, we estimated that its CLABSI rate would decrease by 38%.

Conclusions/Significance

In NHSN hospitals across the US, the CL Bundle is associated with lower infection rates only when compliance is high. Hospitals must target improving bundle implementation and compliance as opposed to simply instituting policies.     

Dechlorination of individual congeners in aroclor 1248 as enhanced by chlorobenzoates, chlorophenols, and chlorobenzenes

Previous investigations showed that three classes of haloaromatic compounds (HACs; chlorobenzoates, chlorophenols, and chlorobenzenes) enhanced the reductive dechlorination of Aroclor 1248, judging from the overall extent of reduction in Cl atoms on the biphenyl. In the present study, we further investigated the kind of polychlorinated biphenyl (PCB) congeners involved in the enhanced dechlorination by four isomers belonging to each class (2,3-, 2,5-, 2,3,5-, and 2,4,6-chlorobenzoates; 2,3-, 3,4-, 2,5-, and 2,3,6-chlorophenols; and 1,2-, 1,2,3-, 1,2,4-, and penta-chlorobenzenes). Although the PCB congeners involved in the enhanced dechlorination varied with the HACs, the enhancement primarily involved paradechlorination of the same congeners (2,3,4'-, 2,3,4,2'- plus 2,3,6,4'-, 2,5,3',4'- plus 2,4,5,2',6'-, and 2,3,6,2',4'- chlorobiphenyls), regardless of the HACs. These congeners are known to have low threshold concentrations for dechlorination. To a lesser extent, the enhancement also involved meta dechlorination of certain congeners with high threshold concentrations. There was no or less accumulation of 2,4,4'- and 2,5,4'-chlorobiphenyls as final products under HAC amendment. Although the dechlorination products varied, the accumulation of orthosubstituted congeners, 2-, 2,2'-, and 2,6-chlorobiphenyls, was significantly higher with the HACs, indicating a more complete dechlorination of the highly chlorinated congeners. Therefore, the present results suggest that the enhanced dechlorination under HAC enrichment is carried out through multiple pathways, some of which may be universal, regardless of the kind of HACs, whereas others may be HAC-specific.     

Advances in human artificial chromosome technology

Human artificial chromosome (HAC) technology has developed rapidly over the past four years. Recent reports show that HACs are useful gene transfer vectors in expression studies and important tools for determining human chromosome function. HACs have been used to complement gene deficiencies in human cultured cells by transfer of large genomic loci also containing the regulatory elements for appropriate expression. And, they now offer the possibility to express large human transgenes in animals, especially in mouse models of human genetic diseases.     

Immunophenotypic analysis of human articular chondrocytes: changes in surface markers associated with cell expansion in monolayer culture

Cartilage tissue engineering relies on in vitro expansion of primary chondrocytes. Monolayer is the chosen culture model for chondrocyte expansion because in this system the proliferative capacity of chondrocytes is substantially higher compared to non-adherent systems. However, human articular chondrocytes (HACs) cultured as monolayers undergo changes in phenotype and gene expression known as "dedifferentiation." To gain a better understanding of the cellular mechanisms involved in the dedifferentiation process, our research focused on the characterization of the surface molecule phenotype of HACs in monolayer culture. Adult HACs were isolated by enzymatic digestion of cartilage samples obtained post-mortem. HACs cultured in monolayer for different time periods were analyzed by flow cytometry for the expression of cell surface markers with a panel of 52 antibodies. Our results show that HACs express surface molecules belonging to different categories: integrins and other adhesion molecules (CD49a, CD49b, CD49c, CD49e, CD49f, CD51/61, CD54, CD106, CD166, CD58, CD44), tetraspanins (CD9, CD63, CD81, CD82, CD151), receptors (CD105, CD119, CD130, CD140a, CD221, CD95, CD120a, CD71, CD14), ectoenzymes (CD10, CD26), and other surface molecules (CD90, CD99). Moreover, differential expression of certain markers in monolayer culture was identified. Up-regulation of markers on HACs regarded as distinctive for mesenchymal stem cells (CD10, CD90, CD105, CD166) during monolayer culture suggested that dedifferentiation leads to reversion to a primitive phenotype. This study contributes to the definition of HAC phenotype, and provides new potential markers to characterize chondrocyte differentiation stage in the context of tissue engineering applications.     

Human Artificial Chromosome with a Conditional Centromere for Gene Delivery and Gene Expression

Human artificial chromosomes (HACs), which carry a fully functional centromere and are maintained as a single-copy episome, are not associated with random mutagenesis and offer greater control over expression of ectopic genes on the HAC. Recently, we generated a HAC with a conditional centromere, which includes the tetracycline operator (tet-O) sequence embedded in the alphoid DNA array. This conditional centromere can be inactivated, loss of the alphoid^tet-O (tet-O HAC) by expression of tet-repressor fusion proteins. In this report, we describe adaptation of the tet-O HAC vector for gene delivery and gene expression in human cells. A loxP cassette was inserted into the tet-O HAC by homologous recombination in chicken DT40 cells following a microcell-mediated chromosome transfer (MMCT). The tet-O HAC with the loxP cassette was then transferred into Chinese hamster ovary cells, and EGFP transgene was efficiently and accurately incorporated into the tet-O HAC vector. The EGFP transgene was stably expressed in human cells after transfer via MMCT. Because the transgenes inserted on the tet-O HAC can be eliminated from cells by HAC loss due to centromere inactivation, this HAC vector system provides important novel features and has potential applications for gene expression studies and gene therapy.     

Efficient assembly of <Emphasis Type="Italic">de novo</Emphasis>human artificial chromosomes from large genomic loci

Background  

Human Artificial Chromosomes (HACs) are potentially useful vectors for gene transfer studies and for functional annotation of the genome because of their suitability for cloning, manipulating and transferring large segments of the genome. However, development of HACs for the transfer of large genomic loci into mammalian cells has been limited by difficulties in manipulating high-molecular weight DNA, as well as by the low overall frequencies of de novo HAC formation. Indeed, to date, only a small number of large (>100 kb) genomic loci have been reported to be successfully packaged into de novo HACs.     

Functional Complementation of a Genetic Deficiency with Human Artificial Chromosomes

We have shown functional complementation of a genetic deficiency in human cultured cells, using artificial chromosomes derived from cloned human genomic fragments. A 404-kb human-artificial-chromosome (HAC) vector, consisting of 220 kb of alphoid DNA from the centromere of chromosome 17, human telomeres, and the hypoxanthine guanine phosphoribosyltransferase (HPRT) genomic locus, was transferred to HPRT-deficient HT1080 fibrosarcoma cells. We generated several cell lines with low-copy-number, megabase-sized HACs containing a functional centromere and one or possibly several copies of the HPRT1 gene complementing the metabolic deficiency. The HACs consisted of alternating alphoid and nonalphoid DNA segments derived only from the input DNA (within the sensitivity limits of FISH detection), and the largest continuous alphoid segment was 158-250 kb. The study of both the structure and mitotic stability of these HACs offers insights into the mechanisms of centromere formation in synthetic chromosomes and will further the development of this human-gene-transfer technology.     

Heat-regulated production and secretion of insulin from a human artificial chromosome vector

Human artificial chromosomes (HACs) behave as independent minichromosomes and are potentially useful as a way to achieve safe, long-term expression of a transgene. In this study, we sought to elucidate the potential of HAC vectors carrying the human proinsulin transgene for gene therapy of insulin-dependent diabetes mellitus (IDDM) using non-beta-cells as a host for the vector. To facilitate the production of mature insulin in non-beta-cells and to safely regulate the level of transgene expression, we introduced furin-cleavable sites into the proinsulin coding region and utilized the heat shock protein 70 (Hsp70) promoter. We used Cre-loxP-mediated recombination to introduce the gene cassettes onto 21DeltapqHAC, a HAC vector whose structure is completely defined, present in human fibrosarcoma HT1080 cells. We observed long-term expression and stable retention of the transgene without aberrant translocation of the HAC constructs. As expected, the Hsp70 promoter allowed us to regulate gene expression with temperature, and the production and secretion of intermediates of mature insulin were made possible by the furin-cleavable sites we had introduced into proinsulin. This study can be an initial step on the application of HAC vectors on the gene delivery to non-beta-cells, which might provide a direction for future treatment for diabetes.     

Assessing the Necessity of the Standardized Infection Ratio for Reporting Central Line-Associated Bloodstream Infections

This brief article presents results that support the contention that risk adjustment via the standardized infection ratio (SIR) for the reporting of central line-associated bloodstream infections (CLABSIs) may be no more predictive than standard rate adjustments utilizing CLABSIs per central line days (i.e., CLABSI rates). Recent data posted on the U.S. Department of Health and Human Services’ Hospital Compare website showed that nearly 70% of 1721 reporting hospitals with at least 1000 central line days had five or fewer infections during 2011. These hospitals had 39.3% of the total central line days and a significantly lower SIR than poorer performing hospitals with six or more CLABSIs (p<0.0001). In addition, 19 hospitals are presented which had central line days between 9000 to over 22,000 that also had zero to three CLABSIs. Some of these hospitals were university referral centers and inner city facilities. There was great variation of CLABSI cases among US hospitals. Evidence is mounting that all hospitals should be able to achieve a near zero incidence of CLABSIs and that these infections may in fact be near ‘never events’, which begs whether risk adjustment with the SIR is needed and whether it adds more information than does rate adjustment using CLABSI rates.     

Global indicators of biological invasion: species numbers, biodiversity impact and policy responses

Aim Invasive alien species (IAS) pose a significant threat to biodiversity. The Convention on Biological Diversity’s 2010 Biodiversity Target, and the associated indicator for IAS, has stimulated globally coordinated efforts to quantify patterns in the extent of biological invasion, its impact on biodiversity and policy responses. Here, we report on the outcome of indicators of alien invasion at a global scale. Location Global. Methods We developed four indicators in a pressure‐state‐response framework, i.e. number of documented IAS (pressure), trends in the impact of IAS on biodiversity (state) and trends in international agreements and national policy adoption relevant to reducing IAS threats to biodiversity (response). These measures were considered best suited to providing globally representative, standardized and sustainable indicators by 2010. Results We show that the number of documented IAS is a significant underestimate, because its value is negatively affected by country development status and positively by research effort and information availability. The Red List Index demonstrates that IAS pressure is driving declines in species diversity, with the overall impact apparently increasing. The policy response trend has nonetheless been positive for the last several decades, although only half of countries that are signatory to the Convention on Biological Diversity (CBD) have IAS‐relevant national legislation. Although IAS pressure has apparently driven the policy response, this has clearly not been sufficient and/or adequately implemented to reduce biodiversity impact. Main conclusions For this indicator of threat to biodiversity, the 2010 Biodiversity Target has thus not been achieved. The results nonetheless provide clear direction for bridging the current divide between information available on IAS and that needed for policy and management for the prevention and control of IAS. It further highlights the need for measures to ensure that policy is effectively implemented, such that it translates into reduced IAS pressure and impact on biodiversity beyond 2010.