Standards for clinical trials for treating TB.

BACKGROUND: The value, speed of completion and robustness of the evidence generated by TB treatment trials could be improved by implementing standards for best practice.METHODS: A global panel of experts participated in a Delphi process, using a 7-point Likert scale to score and revise draft standards until consensus was reached.RESULTS: Eleven standards were defined: Standard 1, high quality data on TB regimens are essential to inform clinical and programmatic management; Standard 2, the research questions addressed by TB trials should be relevant to affected communities, who should be included in all trial stages; Standard 3, trials should make every effort to be as inclusive as possible; Standard 4, the most efficient trial designs should be considered to improve the evidence base as quickly and cost effectively as possible, without compromising quality; Standard 5, trial governance should be in line with accepted good clinical practice; Standard 6, trials should investigate and report strategies that promote optimal engagement in care; Standard 7, where possible, TB trials should include pharmacokinetic and pharmacodynamic components; Standard 8, outcomes should include frequency of disease recurrence and post-treatment sequelae; Standard 9, TB trials should aim to harmonise key outcomes and data structures across studies; Standard 10, TB trials should include biobanking; Standard 11, treatment trials should invest in capacity strengthening of local trial and TB programme staff.CONCLUSION: These standards should improve the efficiency and effectiveness of evidence generation, as well as the translation of research into policy and practice.

Premature ovarian insufficiency: the need for evidence on the effectiveness of hormonal therapy.

Premature ovarian insufficiency (POI) - the loss of ovarian function before the age of 40 years, a decade before natural menopause - is a life-changing diagnosis for women. POI causes significant short-term and long-term morbidity related to estrogen deficiency. The condition is managed by providing exogenous estrogen replacement, usually as the oral contraceptive pill or hormone therapy. These preparations have different estrogen formulations and may have differing benefits and risks. At present, there are no robust data to inform clinical recommendations and women's decision-making about treatment that they may be taking for many years. The POISE study (Premature Ovarian Insufficiency Study of Effectiveness of hormonal therapy) has been designed to determine whether hormone therapy is superior to combined oral contraceptives on important clinical outcomes and patient-reported symptoms, based on the hypothesis that hormone therapy provides more physiological continuous hormone supplementation with natural estrogens. The study is an open and pragmatic, parallel, randomized controlled trial. The primary outcome is absolute bone mineral density assessed by dual-energy X-ray absorptiometry of the lumbar spine after 2 years of treatment. The study will also investigate cardiovascular markers, symptom relief and acceptability of treatment, and will continue to collect long-term data on fractures and cardiovascular events. Results will inform future guidance on management of POI.

Review: Capripoxvirus Diseases: Current Status and Opportunities for Control.

Lumpy skin disease, sheeppox and goatpox are high-impact diseases of domestic ruminants with a devastating effect on cattle, sheep and goat farming industries in endemic regions. In this article, we review the current geographical distribution, economic impact of an outbreak, epidemiology, transmission and immunity of capripoxvirus. The special focus of the article is to scrutinize the use of currently available vaccines to investigate the resource needs and challenges that will have to be overcome to improve disease control and eradication, and progress on the development of safer and more effective vaccines. In addition, field evaluation of the efficacy of the vaccines and the genomic database available for poxviruses are discussed.

Genomic cartography of varicella-zoster virus: a complete genome-based analysis of strain variability with implications for attenuation and phenotypic differences.

In order to gain a better perspective on the true variability of varicella-zoster virus (VZV) and to catalogue the location and number of differences, 11 new complete genome sequences were compared with those previously in the public domain (18 complete genomes in total). Three of the newly sequenced genomes were derived from a single strain in order to assess variations that can occur during serial passage in cell culture. The analysis revealed that while VZV is relatively stable genetically it does posses a certain degree of variability. The reiteration regions, origins of replication and intergenic homopolymer regions were all found to be variable between strains as well as within a given strain. In addition, the terminal viral sequences were found to vary within and between strains specifically at the 3' end of the genome. Analysis of single nucleotide polymorphisms (SNPs) identified a total of 557 variable sites, 451 of which were found in coding regions and resulted in 187 different in amino acid substitutions. A comparison of the SNPs present in the two gE mutant strains, VZV-MSP and VZV-BC, suggested that the missense mutation in gE was primarily responsible for the accelerated cell spread phenotype. Some of the variations noted with high passage in cell culture are consistent with variations seen in the IE62 gene of the vaccine strains (S628G, R958G and I1260V) that may help in pinpointing variations essential for attenuation. Although VZV has been considered to be one of the most genetically stable human herpesviruses, this initial assessment of genomic VZV cartography provides insight into ORFs with previously unreported variations.

Use of modified U1 snRNAs to inhibit HIV-1 replication.

Control of RNA processing plays a central role in regulating the replication of HIV-1, in particular the 3' polyadenylation of viral RNA. Based on the demonstration that polyadenylation of mRNAs can be disrupted by the targeted binding of modified U1 snRNA, we examined whether binding of U1 snRNAs to conserved 10 nt regions within the terminal exon of HIV-1 was able to inhibit viral structural protein expression. In this report, we demonstrate that U1 snRNAs complementary to 5 of the 15 regions targeted result in significant suppression of HIV-1 protein expression and viral replication coincident with loss of viral RNA. Suppression of viral gene expression is dependent upon appropriate assembly of a U1 snRNP particle as mutations of U1 snRNA that affect binding of U1 70K or Sm proteins significantly reduced efficacy. However, constructs lacking U1A binding sites retained significant anti-viral activity. This finding suggests a role for these mutants in situations where the wild-type constructs cause toxic effects. The conserved nature of the sequences targeted and the high efficacy of the constructs suggests that this strategy has significant potential as an HIV therapeutic.

Poxviruses: past, present and future.

The analysis of poxvirus genomes is complex, in part, because of their size (130-360 kb) and the fact that gene content is variable; a common set of 49 genes has been found in all sequenced poxviruses and an additional 41 genes are also present in all sequenced orthopoxviruses. As a group, poxviruses have a very broad range of eukaryotic hosts (including mammals, birds, reptiles and insects) and many poxvirus genes are associated with blocking host anti-viral responses. One consequence of this is that many poxvirus genes are not essential for growth in tissue culture and that extensive passaging in vitro results in the accumulation of mutations, including deletions that result in loss of gene function. Here, we review various comparative analyses of the poxviruses including gene prediction, gene conservation and function, genome organization, and poxvirus taxonomy and evolution.

Complete coding sequences of the rabbitpox virus genome.

Rabbitpox virus (RPXV) is highly virulent for rabbits and it has long been suspected to be a close relative of vaccinia virus. To explore these questions, the complete coding region of the rabbitpox virus genome was sequenced to permit comparison with sequenced strains of vaccinia virus and other orthopoxviruses. The genome of RPXV strain Utrecht (RPXV-UTR) is 197 731 nucleotides long, excluding the terminal hairpin structures at each end of the genome. The RPXV-UTR genome has 66.5 % A + T content, 184 putative functional genes and 12 fragmented ORF regions that are intact in other orthopoxviruses. The sequence of the RPXV-UTR genome reveals that two RPXV-UTR genes have orthologues in variola virus (VARV; the causative agent of smallpox), but not in vaccinia virus (VACV) strains. These genes are a zinc RING finger protein gene (RPXV-UTR-008) and an ankyrin repeat family protein gene (RPXV-UTR-180). A third gene, encoding a chemokine-binding protein (RPXV-UTR-001/184), is complete in VARV but functional only in some VACV strains. Examination of the evolutionary relationship between RPXV and other orthopoxviruses was carried out using the central 143 kb DNA sequence conserved among all completely sequenced orthopoxviruses and also the protein sequences of 49 gene products present in all completely sequenced chordopoxviruses. The results of these analyses both confirm that RPXV-UTR is most closely related to VACV and suggest that RPXV has not evolved directly from any of the sequenced VACV strains, since RPXV contains a 719 bp region not previously identified in any VACV.

A systematic review to examine the impact of psycho-educational interventions on health outcomes and costs in adults and children with difficult asthma.

OBJECTIVES: Prior research has highlighted the importance of psychosocial factors in 'difficult' asthma. This study aimed to review the content, effectiveness and cost-effectiveness of psycho-educational interventions designed to address these factors in patients with severe and difficult asthma. DATA SOURCES: Thirty-two electronic databases and other sources were searched for studies of educational, self-management, psychosocial and multifaceted interventions. REVIEW METHODS: Abstracts were screened in duplicate, against prior definitions, to identify eligible interventions targeted to patients with forms of or risk factors for difficult asthma. Studies were classified by patient group (child, adult) and graded along two dimensions related to study design and relevance in terms of the degree to which they were judged to have targeted difficult asthma. Detailed data were extracted from studies meeting a minimum design and relevance threshold. Characteristics of studies were tabulated and results qualitatively synthesised. Where sufficiently similar studies reported adequate data about comparable outcomes, quantitative syntheses of results were undertaken using a random effects approach to calculate pooled relative risks (RR) or standardised mean differences (SMD), with 95% confidence intervals (CI). RESULTS: Searches identified over 23,000 citations. After initial screening and removal of duplicates, 4240 possibly relevant abstracts were assessed. Papers associated with 188 studies were initially obtained and classified. Fifty-seven studies including control groups and those that were judged to have at least 'possible' targeting of difficult asthma (35 in children, 21 in adults, 1 in both) were selected for in-depth review. The delivery, setting, timing and content of interventions varied considerably even within broad types. Reporting of interventions and methodological quality was often poor, but studies demonstrated some success in targeting and following up at-risk patients. Studies reporting data suitable for calculation of summary statistics were of higher quality than those that did not. There was evidence from these that, compared to usual or non-psycho-educational care, psycho-educational interventions reduced admissions when data from the latest follow-ups reported were pooled across nine studies in children (RR = 0.64, CI = 0.46-0.89) and six studies with possible targeting of difficult asthma in adults (RR = 0.57, CI = 0.34-0.93). In children, the greatest and only significant effects were confined to individual studies with limited targeting of difficult asthma and no long-term follow-up. Limited data in adults also suggested effects may not extend to those most at risk. There was no evidence of pooled effects of psycho-educational interventions on emergency attendances from eight studies in children (RR = 0.97, CI = 0.78-1.21) and four in adults (RR = 1.03, CI = 0.82-1.29). There were overall significant reductions in symptoms, similar in different sub-groups of difficult asthma, across four paediatric studies that could be combined (SMD = -0.45, CI = -0.68 to -0.22), but mixed results across individual adult studies. A few individual studies in children showed mainly positive effects on measures of self-care behaviour, but with respect to all other outcomes in adults and children, studies showed mixed results or suggested limited effectiveness of psycho-educational interventions. No studies of psychosocial interventions were included in any quantitative syntheses and it was not possible to draw clear conclusions regarding the relative effectiveness of educational, self-management and multifaceted programmes. Data on costs were very limited. Of the two well-designed economic evaluations identified, both of multifaceted interventions, one in children suggested an additional cost of achieving health gain in terms of symptom-free days. Provisional data from the other study suggested that in adults the significantly increased costs of providing an intervention were not offset by any short-term savings in use of healthcare resources or associated with improvements in health outcomes. CONCLUSIONS: There was some evidence of overall positive effects of psycho-educational interventions on hospital admissions in adults and children, and on symptoms in children, but limited evidence of effects on other outcomes. The majority of research and greatest effects, especially in adults, were confined to patients with severe disease but who lacked other characteristics indicative of difficult asthma or likely to put them at risk. A lack of good-quality research limited conclusions about cost-effectiveness. Although psycho-educational interventions may be of some benefit to patients with severe disease, there is currently a lack of evidence to warrant significant changes in clinical practice with regard to the care of patients with more difficult asthma. Further research is needed to: (1) standardise reporting of complex interventions; (2) extend and update this review; (3) improve identification of patients at risk from their asthma; (4) develop and test appropriate outcome measures for this group; and (5) design and evaluate, via the conduct of high-quality pragmatic RCTs, more powerful psycho-educational interventions that are conceptualised in terms of the ways in which psychosocial factors and asthma interact.

Genome segment RNA-1 of a flat apple isolate of Cherry rasp leaf virus: nucleotide sequence analysis and RT-PCR detection.

The sequence of the RNA-1 of a flat apple isolate of Cherry rasp leaf virus (CRLV-FA) was determined using overlapping cDNA fragments. CRLV-FA RNA-1 consists of 6992 nucleotides (nt), excluding a 3' poly (A) tail. A single open reading frame (ORF) consisting of 6705 nt was identified. This ORF encodes a putative polyprotein consisting of 2235 amino acid (aa) residues, approximately 249.6 kDa. When compared to CRLV-pot (potato isolate) RNA-1 ORF, 2 deletions of 5 aa and 10 aa (total 15 aa) were observed at the variable N-terminus of the protease cofactor of CRLV-FA. Non-coding regions were identified at the 5'-(142 nt) and 3'-end (145 nt). CRLV-FA and CRLV-pot are isolates of the same virus with identity levels for the RNA-1 associated nt and deduced aa of 94% and 95%, respectively. RT-PCR targeting CRLV-FA RNA-1 appear to be of similar sensitivity and just as reliable as RT-PCR targeting RNA-2.

Emergency asthma inhalers in school.

Despite there being a lack of direct evidence of the effectiveness of providing emergency inhalers to schools, the balance of evidence at present suggests the benefits outweigh any possible harm. However, unless UK prescribing law or its interpretation is changed, this will remain an action which opens teachers, nurses, and doctors to possible legal and professional sanctions, and may nullify their institutional or professional indemnity. As a consequence, provision will remain patchy and research into the value of emergency inhalers will be inhibited. A position statement from one or more responsible organisations such as the Royal College of Paediatrics and Child Health, the British Thoracic Society, or the British Paediatric Respiratory Society could persuade a reassessment from the Medicines Control Agency. This is also an issue which could be addressed in the forthcoming National Service Framework for children.

Nucleotide sequence analysis of RNA-2 of a flat apple isolate of Cherry rasp leaf virus with regions showing greater identity to animal picornaviruses than to related plant viruses.

RNA-2 of a flat apple isolate of Cherry rasp leaf virus (CRLV-FA) appears to consist of 3274 nucleotides, excluding a 3' poly (A) tail. The data supports re-classification of CRLV in a new genus in the family Comoviridae. A single open reading frame (ORF) encoding a putative 108 kDa polyprotein was identified. Potential protease cleavage sites were identified which would result in the production of a putative movement protein (41 kDa), and 3 capsid protein subunits (24, 20, and 22 kDa, respectively). A 5'-UTR and 3'-UTR were identified, 248 nt and 146 nt long, respectively. The genome organisation of CRLV-FA RNA-2 is similar to that of Apple latent spherical virus (ALSV) RNA-2, a new member of the family Comoviridae. The Vp25 amino acid sequences were unique to CRLV-FA and ALSV (54% identity), with no relationship identified to any other virus. CRLV-FA Vp20 and Vp24 amino acid sequences were closely related to ALSV (59 and 65%, respectively) but the only other relationships identified were with a range of animal ssRNA positive-strand viruses.

Lack of detection of retroviral particles (HIAP-1) in the H9 T cell line co-cultured with thyrocytes of Graves' disease.

Evidence for a possible aetiopathogenetic role of endogenous and/or exogenous retroviruses (RVs) in organ- and non-organ-specific autoimmune diseases is circumstantial in both humans and animal models. Intracisternal A type particles, antigenically related to HIV, have been reported in H9 cells co-cultured with homogenates of salivary glands obtained from patients with Sjögren syndrome and with synovial fluid of patients with rheumatoid arthritis. In order to identify a possible transfer of a putative 'infective RV agent' involved in the pathogenesis of human thyroid autoimmune disease, the H9 T cell line was co-cultured not only with thyroid homogenates, but also with viable thyrocytes, both prepared from glands of patients with Graves' disease. At the end of a prolonged co-culture period (24 weeks), no RV particles could be detected by electron microscopy in the H9 cells co-cultured with both thyroid preparations. These data seem to exclude the involvement of HIAP-1 in the aetiopathogenesis of human autoimmune thyroid disease.

Avoiding drug errors.

Although no statistics are available, medication errors occur relatively frequently and should be a target for Clinical Governance activities. Organisations need to create a culture where risk management regarding medication is incorporated.

Viral genome organizer: a system for analyzing complete viral genomes.

The viral genome organizer (VGO) is designed to simplify the characterization and annotation of complete viral genomes (particularly those of large poxviruses) and to help researchers discover new genes and detect gene fragmentation. VGO is based on Genotator [Harris, N.L., 1997. Genome Res. 7, 754-762], an annotation workbench designed for the analysis of eukaryotic genomic sequences. VGO automates a number of database search routines (FASTA, BLASTP, PSI-BLAST and TBLASTN), processes the results through a multiple-alignment viewer (MView; [Brown, N.P., Leroy, C., Sander, C. , 1998. Bioinformatics 14, 380-381]) and serves to manage the hundreds of DNA, protein and database search results files that must be organized when dealing with large complete poxviral genomes. It also directs the generation a self-dotplot of the genome by Dotter [Sonnhammer, E.L.L., Durbin, R., 1995. A dot-matrix program with dynamic threshold control suited for genomic DNA and protein sequence analysis. Gene 167: GC1-10. http://www.sanger.ac. uk/Software/Dotter/] to uncover repeated genes and sequences and provides Internet links to programs for generation of restriction maps and analysis of potential PCR primers. The user-friendly graphical interface displays DNA and protein sequences, links to search results, ORFs, stop-start codons, restriction sites and flags of database searches. Currently, VGO and associated programs run in an X-windows environment on commonly available UNIX machines.

Nucleotide sequence and genome organisation of cherry mottle leaf virus and its relationship to members of the Trichovirus genus.

The nucleotide sequence of cherry mottle leaf virus (CMLV) was determined and compared to sequences of a number of plant viruses including the type member of the Trichovirus genus (apple chlorotic leafspot virus, ACLSV), and members of the Vitivirus genus including grapevine virus B, (GVB). The CMLV genome was determined to consist of 8003 nt excluding the poly(A) tail at the 3' end of the genome. The overall A + U content of CMLV genomic RNA was 59.1%, which is similar to ACLSV, but significantly different from GVB. Four putative open reading frames were identified (ORFs 1, 2, 3, and 4) encoding proteins of M(r) 215.8 kDa, 47 kDa, 21.6 kDa, and 15.3 kDa, respectively. This differs from ACLSV which has 3 ORFS, and GVB which has 5 ORFs. Protein database searches showed no matches of CMLV ORF4 with ACLSV sequences, but found similarities between ORF4 of CMLV and ORF5 of GVB, suggesting that this may be a nucleic acid-binding protein. CMLV and ACLSV formed a common virus clade in phylogenetic analysis of the coat protein amino acid sequence and except for CMLV's ORF4, these viruses show high levels of similarity throughout the genome. CMLV appears to be a member of the Trichovirus genus.

Conformationally-restricted ligands for the histamine H1 receptor.

Potent H1-antagonistic activity in a series of novel indeno[2,1-c]pyridines and their 4-arylpiperidinol precursors is reported; one compound shows an in vitro activity four times that of the standard mepyramine that it was screened against. Their failure to translate this protection to in vivo tests is discussed.

Screening predicted coding regions in poxvirus genomes.

The amino acid composition and pI values were calculated for the predicted proteins of a series of complete poxvirus genomes. Many of the vaccinia virus (strain Copenhagen) minor ORFs, thought not to be functional genes, were found to have significantly more or less of several amino acids than a set of the largest 150 vaccinia virus proteins. Very high isoelectric point (pI) values were also correlated with a group of the minor ORFs. Analysis of molluscum contagiosum virus ORFs by amino acid composition and pI identified a number of ORFs previously denoted as doubtful and highlighted several others that could be similarly classified. The use of amino acid composition and pI appears to be a generally applicable tool to aid identification of viral ORFs that are unlikely to be functional genes.

Viral Genome DataBase: storing and analyzing genes and proteins from complete viral genomes.

SUMMARY: The Viral Genome DataBase (VGDB) contains detailed information of the genes and predicted protein sequences from 15 completely sequenced genomes of large (&100 kb) viruses (2847 genes). The data that is stored includes DNA sequence, protein sequence, GenBank and user-entered notes, molecular weight (MW), isoelectric point (pI), amino acid content, A + T%, nucleotide frequency, dinucleotide frequency and codon use. The VGDB is a mySQL database with a user-friendly JAVA GUI. Results of queries can be easily sorted by any of the individual parameters. AVAILABILITY: The software and additional figures and information are available at http://athena.bioc.uvic.ca/genomes/index.html .

Analysis of host response modifier ORFs of ectromelia virus, the causative agent of mousepox.

From the right-hand end of the ectromelia virus (strain Moscow) genome, 32318 bps have been sequenced, and characterized to include a total of 18 open reading frames (ORFs) and six regions which apparently no longer code for functional proteins. At least six of the ORFs appear to be involved in blocking the inflammatory/immune host response to infection, and therefore probably contribute significantly to the virulence of this virus in its natural host, the mouse. One of these genes encoded an isolog of the poxvirus chemokine binding protein, and was shown to be the most abundant protein secreted from ectromelia virus infected cells. Two regions were found to have significant similarity to poxvirus genes encoding tumor necrosis factor (TNF) binding proteins. Both are distinct from cytokine response modifier (crm)B and crmC but only one is predicted to encode a functional TNF binding protein. A novel similarity between the C-terminal domain of poxvirus TNF binding proteins and several other poxvirus proteins is also presented. The results are discussed in the context of ectromelia virus pathogenesis of mice.