The methodological quality of nurse-sensitive indicators in Dutch hospitals: A descriptive exploratory research study.

OBJECTIVE: Nurse-sensitive indicators (NSIs) are increasingly being developed and used to establish quality of nursing care in Western countries. The objective was to gain insights into the methodological quality of mandatory NSIs in Dutch hospitals, including indicators for pain, wound care, malnutrition and delirium. DESIGN: A descriptive exploratory design was used, starting with desk research into publicly available documents and reports describing the development of the NSIs included in this study. We used the validated Appraisal of Indicators through Research and Evaluation (AIRE) instrument to evaluate the methodological quality. RESULTS: Although the purpose and relevance of each individual NSI have been described, no detailed information about the criteria for selecting these topics is available. It is not clear which specific stakeholders participated and how their input was used. We found no information about the process of collecting and compiling scientific evidence. It is unclear whether and to what extent the usability of NSIs has been tested. CONCLUSION: The methodological quality of NSIs used in Dutch hospitals is less than optimal in various ways and it is therefore questionable if the indicators are accurate enough to identify changes or improve nursing practice. Our study also provides an example of how the methodological quality of NSIs can be assessed systematically, which is relevant considering the increasing use of NSIs in various countries.

Mapping the Dutch SNOMED CT subset to Omaha System, NANDA International and International Classification of Functioning, Disability and Health.

BACKGROUND: Nurses register data in electronic health records, which can use various terminology and coding systems. The net result is that information cannot be exchanged and reused properly, for example when a patient is transferred from one care setting to another. A nursing subset of patient problems was therefore developed in the Netherlands, based on comparable and exchangeable terms that are used throughout the healthcare sector and elsewhere (semantic interoperability). The purpose of the current research is to develop a mapping between the subset of patient problems and three classifications in order to improve the exchangeability of data. Those classifications are the Omaha System, NANDA International, and ICF (the International Classification of Functioning, Disability and Health). METHOD: Descriptive research using a unidirectional mapping strategy. RESULTS: Some 30%-39% of the 119 SNOMED CT patient problems can be mapped one-to-one from the subset onto each separate classification. Between 6% and 8% have been mapped partially to a related term. This is considered to be a one-to-one mapping, although the meanings do not correspond fully. Additionally, 23%-51% of the patient problems could be mapped n-to-one, i.e. more specifically than the classification. Some loss of information will always occur in such exchanges. Between 1% and 4% of the patient problems from the subset are defined less specifically than the problems within the individual classifications. Finally, it turns out that 9%-32% of the terms from the subset of patient problems could not be mapped onto a classification, either because they did not occur in the classification or because they could not be mapped at a higher level. CONCLUSION: To promote the exchange of data, the subset of patient problems has been mapped onto three classifications. Loss of information occurs in most cases when the patient problems are transformed from the subset into a classification. This arises because the classifications are different in structure and in the degree of detail. Structural cooperation between suppliers, healthcare organisations and the experts involved is required in order to determine how the mapping should be used within the electronic health records, and whether it is usable in day-to-day practice.

The development of a nursing subset of patient problems to support interoperability.

BACKGROUND: Since the emergence of electronic health records, nursing information is increasingly being recorded and stored digitally. Several studies have shown that a wide range of nursing information is not interoperable and cannot be re-used in different health contexts. Difficulties arise when nurses share information with others involved in the delivery of nursing care. The aim of this study is to develop a nursing subset of patient problems that are prevalent in nursing practice, based on the SNOMED CT terminology to assist in the exchange and comparability of nursing information. METHODS: Explorative qualitative focus groups were used to collect data. Mixed focus groups were defined. Additionally, a nursing researcher and a nursing expert with knowledge of terminologies and a terminologist participated in each focus group. The participants, who work in a range of practical contexts, discussed and reviewed patient problems from various perspectives. RESULTS: Sixty-seven participants divided over seven focus groups selected and defined 119 patient problems. Each patient problem could be documented and coded with a current status or an at-risk status. Sixty-six percent of the patient problems included are covered by the definitions established by the International Classification of Nursing Practice, the reference terminology for nursing practice. For the remainder, definitions from either an official national guideline or a classification were used. Each of the 119 patient problems has a unique SNOMED CT identifier. CONCLUSIONS: To support the interoperability of nursing information, a national nursing subset of patient problems based on a terminology (SNOMED CT) has been developed. Using unambiguously defined patient problems is beneficial for clinical nursing practice, because nurses can then compare and exchange information from different settings. A key strength of this study is that nurses were extensively involved in the development process. Further research is required to link or associate nursing patient problems to concepts from a nursing classification with the same meaning.

A single amino acid substitution in the group 1 Trypanosoma brucei gambiense haptoglobin-hemoglobin receptor abolishes TLF-1 binding.

Critical to human innate immunity against African trypanosomes is a minor subclass of human high-density lipoproteins, termed Trypanosome Lytic Factor-1 (TLF-1). This primate-specific molecule binds to a haptoglobin-hemoglobin receptor (HpHbR) on the surface of susceptible trypanosomes, initiating a lytic pathway. Group 1 Trypanosoma brucei gambiense causes human African Trypanosomiasis (HAT), escaping TLF-1 killing due to reduced uptake. Previously, we found that group 1 T. b. gambiense HpHbR (TbgHpHbR) mRNA levels were greatly reduced and the gene contained substitutions within the open reading frame. Here we show that a single, highly conserved amino acid in the TbgHpHbR ablates high affinity TLF-1 binding and subsequent endocytosis, thus evading TLF-1 killing. In addition, we show that over-expression of TbgHpHbR failed to rescue TLF-1 susceptibility. These findings suggest that the single substitution present in the TbgHpHbR directly contributes to the reduced uptake and resistance to TLF-1 seen in these important human pathogens.

Tandemly repeated DNA is a target for the partial replacement of thymine by beta-D-glucosyl-hydroxymethyluracil in Trypanosoma brucei.

In the DNA of African trypanosomes a small fraction of thymine is replaced by the modified base beta-D-glucosyl-hydroxymethyluracil (J). The function of this large base is unknown. The presence of J in the silent variant surface glycoprotein gene expression sites and the lack of J in the transcribed expression site indicates that DNA modification might play a role in control of gene repression. However, the abundance of J in the long telomeric repeat tracts and in subtelomeric arrays of simple repeats suggests that J may also have specific functions in repetitive DNA. We have now analyzed chromosome-internal repetitive sequences in the genome of Trypanosoma brucei and found J in the minichromosomal 177-bp repeats, in the long arrays of 5S RNA gene repeats, and in the spliced-leader RNA gene repeats. No J was found in the rDNA locus or in dispersed repetitive transposon-like elements. Remarkably, the rDNA of T. brucei is not organized in long arrays of tandem repeats, as in many other eukaryotes. T. brucei contains only approximately 15-20 rDNA repeat units that are divided over six to seven chromosomes. Our results show that J is present in many tandemly repeated sequences, either at a telomere or chromosome internal. The presence of J might help to stabilize the long arrays of repeats in the genome.

Base J originally found in kinetoplastida is also a minor constituent of nuclear DNA of Euglena gracilis.

We have analyzed DNA of EUGLENA: gracilis for the presence of the unusual minor base beta-D-glucosyl-hydroxymethyluracil or J, thus far only found in kinetoplastid flagellates and in DIPLONEMA: Using antibodies specific for J and post-labeling of DNA digests followed by two-dimensional thin-layer chromatography of labeled nucleotides, we show that approximately 0.2 mole percent of EUGLENA: DNA consists of J, an amount similar to that found in DNA of Trypanosoma brucei. By staining permeabilized EUGLENA: cells with anti-J antibodies, we show that J is rather uniformly distributed in the EUGLENA: nucleus, and does not co-localize to a substantial extent with (GGGTTA)(n) repeats, the putative telomeric repeats of EUGLENA: Hence, most of J in EUGLENA: appears to be non-telomeric. Our results add to the existing evidence for a close phylogenetic relation between kinetoplastids and euglenids.

The modified base J is the target for a novel DNA-binding protein in kinetoplastid protozoans.

DNA from Kinetoplastida contains the unusual modified base beta-D-glucosyl(hydroxymethyl)uracil, called J. Base J is found predominantly in repetitive DNA and correlates with epigenetic silencing of telomeric variant surface glycoprotein genes in Trypanosoma brucei. We have now identified a protein in nuclear extracts of bloodstream stage T.brucei that binds specifically to J-containing duplex DNA. J-specific DNA binding was also observed with extracts from the kinetoplastids Crithidia fasciculata and Leishmania tarentolae. We purified the 90 kDa C.fasciculata J-binding protein 50 000-fold and cloned the corresponding gene from C.fasciculata, T.brucei and L.tarentolae. Recombinant proteins expressed in Escherichia coli demonstrated J-specific DNA binding. The J-binding proteins show 43-63% identity and are unlike any known protein. The discovery of a J-binding protein suggests that J, like methylated cytosine in higher eukaryotes, functions via a protein intermediate.

Biosynthesis and function of the modified DNA base beta-D-glucosyl-hydroxymethyluracil in Trypanosoma brucei.

beta-D-Glucosyl-hydroxymethyluracil, also called J, is a modified DNA base conserved among kinetoplastid flagellates. In Trypanosoma brucei, the majority of J is present in repetitive DNA but the partial replacement of thymine by J also correlates with transcriptional repression of the variant surface glycoprotein (VSG) genes in the telomeric VSG gene expression sites. To gain a better understanding of the function of J, we studied its biosynthesis in T. brucei and found that it is made in two steps. In the first step, thymine in DNA is converted into hydroxymethyluracil by an enzyme that recognizes specific DNA sequences and/or structures. In the second step, hydroxymethyluracil is glucosylated by an enzyme that shows no obvious sequence specificity. We identified analogs of thymidine that affect the J content of the T. brucei genome upon incorporation into DNA. These analogs were used to study the function of J in the control of VSG gene expression sites. We found that incorporation of bromodeoxyuridine resulted in a 12-fold decrease in J content and caused a partial derepression of silent VSG gene expression site promoters, suggesting that J might strengthen transcriptional repression. Incorporation of hydroxymethyldeoxyuridine, resulting in a 15-fold increase in the J content, caused a reduction in the occurrence of chromosome breakage events sometimes associated with transcriptional switching between VSG gene expression sites in vitro. We speculate that these effects are mediated by the packaging of J-containing DNA into a condensed chromatin structure.

beta-D-glucosyl-hydroxymethyluracil is a conserved DNA modification in kinetoplastid protozoans and is abundant in their telomeres.

The unusual DNA base beta-D-glucosyl-hydroxymethyluracil, called "J, " replaces approximately 0.5-1% of Thy in DNA of African trypanosomes but has not been found in other organisms thus far. In Trypanosoma brucei, J is located predominantly in repetitive DNA, and its presence correlates with the silencing of telomeric genes. Using antibodies specific for J, we have developed sensitive assays to screen for J in a range of organisms and have found that J is not limited to trypanosomes that undergo antigenic variation but is conserved among Kinetoplastida. In all kinetoplastids tested, including the human pathogens Leishmania donovani and Trypanosoma cruzi, J was found to be abundantly present in the (GGGTTA)n telomere repeats. Outside Kinetoplastida, J was found only in Diplonema, a small phagotrophic marine flagellate, in which we also identified 5-MeCyt. Fractionation of Diplonema DNA showed that the two modifications are present in a common genome compartment, which suggests that they may have a similar function. Dinoflagellates appear to contain small amounts of modified bases that may be analogs of J. The evolutionary conservation of J in kinetoplastid protozoans suggests that it has a general function, repression of transcription or recombination, or a combination of both. T. brucei may have recruited J for the control of genes involved in antigenic variation.

The role of transferrin-receptor variation in the host range of Trypanosoma brucei.

Trypanosoma brucei is a unicellular parasite transmitted between African mammals by tsetse flies. T. brucei multiplies freely in the bloodstream of many different mammals, and survives by antigenic variation of the main component of its surface coat, variant surface glycoprotein (VSG). Trypanosomes take up transferrin through a heterodimeric transferrin receptor, the genes for which are expressed in telomeric expression sites along with the VSG gene. There are up to 20 of these expression sites per trypanosome nucleus, but usually only one is active at a time. Different expression sites encode transferrin receptors that are similar but not identical. Here we show that these small differences between transferrin receptors can have profound effects on the binding affinity for transferrins from different mammals, and on the ability of trypanosomes to grow in the sera of these mammals. Our results suggest that the ability to switch between different transferrin-receptor genes allows T. brucei to cope with the large sequence diversity in the transferrins of its hosts.

Localization of the modified base J in telomeric VSG gene expression sites of Trypanosoma brucei.

African trypanosomes such as Trypanosoma brucei undergo antigenic variation in the bloodstream of their mammalian hosts by regularly changing the variant surface glycoprotein (VSG) gene expressed. The transcribed VSG gene is invariably located in a telomeric expression site. There are multiple expression sites and one way to change the VSG gene expressed is by activating a new site and inactivating the previously active one. The mechanisms that control expression site switching are unknown, but have been suggested to involve epigenetic regulation. We have found previously that VSG genes in silent (but not active) expression sites contain modified restriction endonuclease cleavage sites, and we have presented circumstantial evidence indicating that this is attributable to the presence of a novel modified base beta-D-glucosyl-hydroxymethyluracil, or J. To directly test this, we have generated antisera that specifically recognize J-containing DNA and have used these to determine the precise location of this modified thymine in the telomeric VSG expression sites. By anti J-DNA immunoprecipitations, we found that J is present in telomeric VSG genes in silenced expression sites and not in actively transcribed telomeric VSG genes. J was absent from inactive chromosome-internal VSG genes. DNA modification was also found at the boundaries of expression sites. In the long 50-bp repeat arrays upstream of the promoter and in the telomeric repeat arrays downstream of the VSG gene, J was found both in silent and active expression sites. This suggests that silencing results in a gradient of modification spreading from repetitive DNA flanks into the neighboring expression site sequences. In this paper, we discuss the possible role of J in silencing of expression sites.

A ribosomal DNA promoter replacing the promoter of a telomeric VSG gene expression site can be efficiently switched on and off in T. brucei.

Trypanosoma brucei survives in the mammalian blood-stream by regularly changing its variant surface glycoprotein (VSG) coat. The active VSG gene is located in a telomeric expression site, and coat switching occurs either by replacing the transcribed VSG gene or by changing the expression site that is active. To determine whether VSG expression site control requires promoter-specific sequences, we replaced the active VSG expression site promoter in bloodstream-form T. brucei with a ribosomal DNA (rDNA) promoter. These transformants were fully infective in laboratory animals, and the rDNA promoter, which is normally constitutively active, was efficiently inactivated and reactivated in the context of the VSG gene expression site. As there is no sequence similarity between the VSG expression site promoter and the rDNA promoter, VSG expression site control does not involve sequences specific to the VSG expression site promoter. We conclude that an epigenetic mechanism, such as telomeric silencing, is involved in VSG expression site control in bloodstream-form T. brucei.

Genomic organization of an invariant surface glycoprotein gene family of Trypanosoma brucei.

The genomic organization of a gene family for the invariant surface glycoprotein, ISG75 (invariant surface glycoprotein with a molecular mass of 75 kDa), from Trypanosoma brucei is described. In T. brucei strain 427 ISG75 genes are present in tandem arrays at two loci, A and B, containing 5 and 2 copies, respectively. At the 3'-end of locus A, a single gene was identified that encodes a structural isoform of ISG75. This isoform contains a unique amino-terminal domain, whereas the rest of the protein is nearly identical to the polypeptides encoded by the other genes. This isoform is transcribed into a stable mRNA, but the expression of the derived polypeptide was below the detection limit. The ISG75 gene clusters are present on chromosomal bands 9' and 10, supporting the hypothesis of Gottesdiener et al. [25] that these bands contain allelic chromosomes. The total number of ISG75 genes is strain dependent, but at least one copy of the unique isoform is present in every variant tested.

VSG gene expression site control in insect form Trypanosoma brucei.

When the African trypanosome Trypanosoma brucei is taken up from mammals by a tse-tse fly, it replaces its variant surface glycoprotein (VSG) coat by a procyclin coat. Transcription of VSG genes stops in the fly, but transcription of sequences derived from the promoter area of the VSG expression site(s) remains high. Whether this is due to continuing high activity of one promoter or to low activity of many promoters was unclear. We have used the small differences between the sequences of different expression sites to show that multiple expression site promoters are active in insect form trypanosomes. This is confirmed by the low expression of single copy marker genes introduced into the transcribed area. However, if the expression site promoter is removed from the genomic location of the expression site and inserted in the non-transcribed spacer of the ribosomal DNA (rDNA), it is derepressed. Derepression of transcription can also be accomplished by replacing the promoter of an expression site by an rDNA promoter. We conclude that the down-regulation of VSG gene expression site promoters in insect form trypanosomes is affected by both the DNA sequence of the promoter and the genomic context in which it resides.

Reconstitution of a surface transferrin binding complex in insect form Trypanosoma brucei.

In the bloodstream of the mammalian host, Trypanosoma brucei takes up host transferrin by means of a high-affinity uptake system, presumably a transferrin receptor. Transferrin-binding activity is seen in the flagellar pocket and is absent in insect form trypanosomes. By transfection we have reconstituted a transferrin-binding complex in insect form trypanosomes. Formation of this complex requires the products of two genes that are part of a variant surface glycoprotein expression site, expression site-associated gene (ESAG) 6 (encoding a protein with GPI-anchor) and ESAG 7 (encoding a protein without any obvious membrane attachment). This complex can be precipitated by transferrin-Sepharose and by an antibody directed only against the ESAG 6 protein. Transfection of ESAG 6 or 7 alone did not result in transferrin binding. In the transfected trypanosomes, the products of ESAG 6 alone and the combination of ESAG 6 and 7 did not exclusively localize to the flagellar pocket, but were present all over the surface of the trypanosome. The reconstituted transferrin-binding complex also did not result in the uptake of transferrin. Additional proteins present in bloodstream trypanosomes, but not in sufficient amounts in insect form trypanosomes, may therefore be required for the correct routing of the transferrin-binding complex to the flagellar pocket, and for its rapid internalization after ligand binding.

Stable transformation of Trypanosoma brucei.

We have further analyzed parameters affecting stable transformation of Trypanosoma brucei. Linear DNA was much more efficient than circular DNA and in the vast majority of transformants analyzed the plasmid DNA had inserted into the chromosomes by homologous recombination. The presence of non-homologous (vector) DNA at one or both ends of linear constructs inhibited transformation efficiency. Less than 1 kb of homologous flanking sequence was sufficient for efficient targeting of a marker gene into the tubulin gene array. When transformants with a single neomycin phosphotransferase (neo(r)) gene replacing a beta-tubulin gene were selected for higher levels of G418 resistance, the neo(r) gene was amplified and spread through the tubulin gene cluster. The additional neo(r) gene copies were adjacent in the tubulin gene array and were added to the array rather than replacing beta-tubulin genes. These results are compatible with asymmetric post-replication recombination (unequal sister chromatid exchange) as the mechanism for neo(r) gene amplification. Starting with a circular construct containing the neo(r) gene between tubulin intergenic regions, we obtained a single transformant that maintained the neo(r) genes as an extrachromosomal plasmid. We show this plasmid to consist of a circular pentamer of the input construct. All other attempts to derive a shuttle vector that replicates extrachromosomally in T. brucei were unsuccessful. Our experiments extend previous observations suggesting that T. brucei has a strong preference for chromosomal insertion of exogenous DNA by homologous recombination.

Insertion of the promoter for a variant surface glycoprotein gene expression site in an RNA polymerase II transcription unit of procyclic Trypanosoma brucei.

The variant-specific surface glycoprotein (VSG) genes of Trypanosoma brucei are invariably expressed near the ends of chromosomes (telomeres). We have targeted a VSG gene expression site (ES) promoter driving a selectable marker gene (neomycin phosphotransferase) into a chromosome-internal transcription unit, the tubulin gene array of procyclic trypanosomes. To avoid read through transcription of the marker gene from the tubulin promoter, we targeted the ES promoter in inverse orientation relative to tubulin gene transcription. The only correctly targeted transformant obtained contained the marker gene close to the border of the tubulin gene array, and expression of this gene was relatively low. Possible reasons for the low targeting efficiency and expression level are discussed.

A ribosomal RNA gene promoter at the telomere of a mini-chromosome in Trypanosoma brucei.

The parasitic protozoan Trypanosoma brucei has some hundred mini-chromosomes of 50-150 kb, which mainly consist of telomeric repeats, sub-telomeric repeats and internal 177-bp repeats. Their primary function seems to be to expand the repertoire of non-transcribed sub-telomeric variant surface glycoprotein (VSG) genes. Here we report that two of the smaller mini-chromosomes (55 and 60 kb) contain sequences homologous to the ribosomal RNA gene promoter region. We have targeted by homologous recombination the neomycin phosphotransferase (neo(r)) gene behind the promoter on the 55 kb chromosome and show that this promoter mediates the efficient synthesis of properly trans-spliced and polyadenylated neo mRNA. The resulting high resistance to G418 (a neo analogue) is stable in the absence of drug showing that mitotic segregation of this mini-chromosome is precise. Downstream of the transcription start the wild-type version of the ribosomal promoter is flanked by telomeric repeats. The absence of the sub-telomeric repeats found in other T.brucei chromosome ends suggests that the rDNA-telomeric junction has been formed by de novo addition of telomeric repeats to a broken chromosome end (healing). Our results provide a plausible explanation for the alpha-amanitin-resistant transcription of telomeric repeats in T.brucei reported by Rudenko and Van der Ploeg and they show that trypanosomes can efficiently use RNA polymerase I for the expression of sub-telomeric genes, supporting the notion that the alpha-amanitin-resistant transcription of sub-telomeric VSG genes may also be catalyzed by this enzyme.

Alpha-amanitin-resistant transcription units in trypanosomes: a comparison of promoter sequences for a VSG gene expression site and for the ribosomal RNA genes.

Transcription of the predominant surface antigen genes in Trypanosoma brucei is unusual in its resistance to the RNA polymerase inhibitor alpha-amanitin, a property typical for rDNA transcription in eukaryotes. Transcription of most other protein-coding genes in trypanosomes is sensitive to alpha-amanitin. To investigate whether RNA polymerase I, the polymerase that transcribes rRNA genes, can give rise to functional mRNAs in trypanosomes, we have fused the putative promoter of the T.brucei rRNA genes to the chloramphenicol acetyl transferase (CAT) gene and determined CAT activity after transient expression of chimeric constructs in procyclic trypanosomes. We show here that the rRNA promoter yields the same high CAT activity as the promoters for the two predominant surface antigen genes of trypanosomes, the Variant-specific Surface Glycoprotein (VSG) gene of bloodstream trypanosomes and the procyclin gene of insect-form trypanosomes, both of which are also transcribed by an alpha-amanitin-insensitive RNA polymerase. RNA polymerase I of trypanosomes seems therefore able to synthesize pre-mRNAs that are effectively processed into translatable mRNAs. Dissection of the promoter segments showed the minimal elements for a VSG gene expression site promoter to be confined to a segment of -60 to +77 bp, overlapping the most 5' putative transcription start sites as determined in vivo by RNase protection experiments. For the ribosomal promoter region a segment of -258 to +200 bp relative to the putative transcription start site was sufficient for maximal CAT activity. There is a precise requirement for specific nucleotides at the rRNA transcription start site. We detect no homology between the sequences required for promoter function of the three alpha-amanitin-resistant transcription units, rRNA, VSG and procyclin (parp) genes. This suggests that the sequence-specific recognition of these promoters either occurs by common factors detecting sequence homologies that escape us, or by separate factors that bind to different DNA sequences but interact with a common alpha-amanitin-resistant RNA polymerase.