Addressing the problem of preventable drug related morbidity (PDRM) in primary care is a challenge for health care systems internationally. The increasing implementation of clinical information systems in the UK and internationally provide new opportunities to systematically identify patients at risk of PDRM for targeted medication review. The objectives of this study were (1) to develop a set of explicit medication assessment criteria to identify patients with sub-optimally effective or high-risk medication use from electronic medical records and (2) to identify medication use topics that are perceived by UK primary care clinicians to be priorities for quality and safety improvement initiatives.For objective (1), a 2-round consensus process based on the RAND/UCLA Appropriateness Method (RAM) was conducted, in which candidate criteria were identified from the literature and scored by a panel of 10 experts for 'appropriateness' and 'necessity'. A set of final criteria was generated from candidates accepted at each level. For objective (2), thematically related final criteria were clustered into 'topics', from which a panel of 26 UK primary care clinicians identified priorities for quality improvement in a 2-round Delphi exercise.(1) The RAM process yielded a final set of 176 medication assessment criteria organised under the domains 'quality' and 'safety', each classified as targeting 'appropriate/necessary to do'  or 'inappropriate/necessary to avoid' (safety) medication use. Fifty-two final 'quality' assessment criteria target patients with unmet indications, sub-optimal selection or intensity of beneficial drug treatments. A total of 124 'safety' assessment criteria target patients with unmet needs for risk-mitigating agents, high-risk drug selection, excessive dose or duration, inconsistent monitoring or dosing instructions. (2) The UK Delphi panel identified 11 (23%) of 47 scored topics as 'high priority' for quality improvement initiatives in primary care.The developed criteria set complements existing medication assessment instruments in that it is not limited to the elderly, can be implemented in electronic data sets and focuses on drug groups and conditions implicated in common and/or severe PDRM in primary care. Identified priorities for quality and safety improvement can guide the selection of targets for initiatives to address the PDRM problem in primary care. Systematic reviews have demonstrated deficits in the quality and safety of medication use in primary care to an extent sufficient to constitute a public health threat. Three to four percent of all unplanned hospital admissions are due to preventable drug related morbidity (PDRM), with the majority attributed to high-risk prescribing and inconsistent monitoring -4. AntipThe 'Data-driven Quality Improvement in Primary care (DQIP)' research programme is designing and testing a complex intervention to improve the quality and safety of medication use in UK primary care. It is based on encouraging and facilitating primary care medical practices to systematically and continuously identify, correct or otherwise manage drug therapy risks that are potential pre-cursors to PDRM . The DQIA number of explicit medication assessment tools have been developed in recent years. The Beers criteria set  lists potopics, are perceived to be priorities for quality improvement by professionals working in UK primary care.The study had two aims. First, we aimed to develop and classify by clinical importance a set of up-to-date medication assessment criteria that can be implemented in routine primary care clinical datasets to identify instances of (a) sub-optimally effective medication use for conditions commonly encountered in primary care and (b) high-risk use of drugs that have been shown to either commonly cause harm and/or cause severe harm in primary care. Second, we aimed to elicit the extent to which thematically-related medication assessment criteria, subsequently referred to as The study was conducted in three stages. First, an extensive list of candidate medication assessment criteria was generated based on a structured literature review. Second, an expert panel participating in a modified RAND/UCLA (University College of Los Angeles) Appropriateness Method (RAM) study scored these items by clinical importance based on a summary of research evidence and their clinical judgement. Candidate criteria with high importance scores were translated into a final criteria set by removing redundancies (see below). Final criteria were characterised by the type of medication use targeted, informed by available taxonomies -15. ThirPrescribing is a ubiquitous feature of medical care which makes a systematic evaluation of the literature on prescribing quality or safety unfeasible in a single research project. We therefore focussed on medication use for conditions commonly encountered in primary care and drugs with clear evidence of significant benefit or harm. The literature review drew initially on UK national clinical guidelines, prescribing advice, and safety alerts, supplemented by European or other clinical guidelines and targeted primary literature review in selected areas as detailed below.Candidate medication assessment criteria either described potentially beneficial medication use  or the use of potentially harmful treatments ('safety'). Candidate 'quality' criteria targeted common conditions where there are compelling indications for drug therapy based on UK and European guidelines -25. CommIn order to identify candidate safety criteria, the drug groups reported to be most frequently implicated in PDRM hospital admissions were identified from systematic reviews and large scale studies -4,34. FoThe RAND/University of California Los Angeles (UCLA) appropriateness method is a rigorous way of combining research evidence with expert opinion , and hasimproper' not to be offered or avoided, whereas 'appropriate' is a more neutral balancing of net benefit or harm [The questionnaire aimed to classify candidate medication assessment criteria derived from the literature as either 'necessary' or 'appropriate' care table . 'Necess or harm -45. Foll or harm ,46.and three or more ratings of 1 to 3) on the appropriateness scale were rejected. Those items with median ratings of 7 to 9 were accepted as 'appropriate' and those with median ratings of 1 to 3 as 'inappropriate'.All candidate quality and safety assessment criteria were scored for 'appropriateness'. Candidate criteria with a median rating of 4 to 6 ('uncertain') or disagreement  were accepted. Candidate safety assessment criteria were additionally scored on a 'necessary to avoid' scale, where items with a median rating of 1 to 3 (= clearly necessary to avoid) were accepted. Candidate criteria with median ratings of < 7 on the 'necessary to do' and > 3 on the necessary to avoid scale and those showing disagreement (defined as above) were rejected. The concept of 'necessary to avoid' was an extension to the original RAM method to differentiate between prescribing that is 'generally not worthwhile' from 'improper' in safety terms (see box 2).The ten RAM panel members were emailed the first round questionnaire and a summary of the supporting evidence base. Panellists were asked to rate each item with reference to an 'average' patient consulting an 'average' primary care clinician in 2009 based on both the evidence summary and their clinical judgement . PanelliA random sample of general medical practitioners (GPs) and eligible pharmacists in Scotland and England was invited to participate by e-mail. In order to be eligible, pharmacists had to have experience of working in medicines governance, as a prescribing advisor or as a practice pharmacist. Twenty three (64%) GPs and 13 (36%) pharmacists agreed to participate.'The described topic is a priority for collaborative quality improvement in primary care'. The term 'collaborative' was used in order to emphasise that the intended purpose of this study was to identify priority topics for quality improvement rather than measures for judging practitioners or practices as part of performance management.The Delphi questionnaire listed the medication improvement topics to be scored together with a short summary of the scientific rationale for each topic. For each item, panellists were asked to state their level of agreement with the statement and 30% or more ratings of 7 to 9) were accepted as 'priority', with median ratings of 8 or 9 defined as 'high priority'. All findings reported in this paper refer to second round ratings.As in the RAM study, all ratings used an ordinal scale of 1 to 9 (1 = strongly disagree and 9 = strongly agree). Panellists were instructed to rate topics in relation to primary care in general, rather than their own practice. The first round ratings were summarised and returned to participants by email for a second round of scoring. Topics with second round median ratings of 7 to 9 without disagreement  candidate assessment criteria. Upon completion of the second rating round, 318 (82%) candidates  were accepted at the 'appropriate' and 275 (71%) items  at the 'necessary' level. A number of candidate criteria were duplicates, in the sense that they were designed to determine thresholds beyond which care was judged appropriate and necessary. For example, 18 candidate quality assessment criteria related to glycated haemoglobin (HbA1c) levels beyond which treatment intensification was appropriate or necessary. Removing redundant candidate criteria yielded 52 quality and 124 safety assessment criteria to be included in the final set. Forty (77%) final quality assessment criteria and 107 (86%) final safety assessment criteria were derived from candidates accepted at the 'necessary' level. The results of the RAM study are summarised in tables Table Similarly, table The majority of safety assessment criteria are drug-focussed (74%), either targeting drugs reported to be frequently implicated in PDRM hospital admissions (54% - section A) or others implicated in severe preventable harm (20% - section B). The remainder (26% - section C) target medication use in particularly vulnerable groups, namely the elderly 15%), patients with heart failure (8%) and children (4%). Over a third (36%) of final safety assessment criteria focus on potentially harmful use of NSAIDs, antiplatelets, anticoagulants and diuretics, the drug groups most frequently implicated in PDRM hospital admissions [5%, patieOver half (52%) of safety criteria target the selection of high-risk drugs (MS2 to 4), either for indications where safer  alternatives exist (MS2) or in patients particularly susceptible to adverse reactions because of age/co-morbidity (MS3) or co-prescription (MS4). A further 15 (12%) criteria target omissions of drugs indicated to mitigate the risk of adverse events from high-risk treatments (MS1), while twenty (16%) criteria target inconsistent laboratory monitoring (MS8). Two (2%) criteria focus on prescribing that may jeopardise patient compliance with methotrexate dosing schedules (MS7).The majority of quality (81%) and safety (71%) assessment criteria are not restricted to the elderly (patients aged 65 years or older).Grouping of thematically related assessment criteria that were derived from candidates accepted at the 'necessary' level yielded a total of 47  medication improvement topics to be rated by the Delphi panel. Thirty-six Delphi study participants completed a first round and 26 (73%) a second round questionnaire  and high-risk use, inconsistent monitoring or patient instructions for drugs implicated in preventable harm (medication use safety) in primary care. All items are classified by clinical importance (appropriateness and necessity) as the output of an extended RAM process. Key professionals in UK primary care identified eleven clusters of thematically related medication assessment criteria (topics) as 'high priority' for quality improvement initiatives. The three highest rated topics related to methotrexate dosing instructions, high-risk prescribing of NSAIDs and antiplatelets and underuse of corticosteroids in asthma.The RAM approach had advantages over the Delphi technique as an initial step in the criteria development process, because the face-to-face meeting ensured the necessary commitment of panellists to place ratings on an extensive and thematically broad list of candidate criteria that were grounded in the evidence base. The original RAM approach was extended in this study by introducing the concept of 'necessary to avoid', in order to distinguish between inappropriate ('not worthwhile') and 'improper' medication use in safety terms  results in the UK are often held in bespoke systems which hinder the implementation of meaningful measures for monitoring anticoagulant use -4. SimilAlthough the DQIP criteria set has been developed for application in UK primary care, the drug groups reported to be implicated in PDRM events in primary care are similar internationally -52, and The Delphi approach allowed stakeholders in primary care to prioritise the chosen medication use topics for improvement initiatives in UK primary care. The Delphi panel was deliberately chosen to include both day to day prescribers  and those involved in prescribing governance and improvement (predominately pharmacists but including GPs with a more strategic role). A limitation is that our focus on professionals involved in primary care prescribing meant that we did not seek to include either specialist or patient/public perspectives in the Delphi panel. Since there is evidence that practitioners' perceptions of a targeted behaviour as meaningful is a pre-requisite to changing behaviour  we aimedIt is important to note that even those topics that were not considered to be priorities  contain individual criteria that were agreed to be 'necessary' to do or avoid by the RAM panel. Examples are 'inadequate dose titration of ACEI, ARBs and beta blockers in chronic heart failure', and the 'using of warfarin without a compelling indication in atrial fibrillation with low risk of stroke'. These should therefore not be neglected. Lower priority ratings nevertheless indicate that changing and improving the corresponding medication use aspects may require targeted effort (or resources) in order to influence prescribing behaviour.NCT01425502).The DQIP medication assessment criteria set presented here has been developed using established consensus methods and complements existing medication assessment instruments by not being limited to the elderly and by targeting a wide spectrum of medication use practices implicated in common and/or severe PDRM events in primary care. As all previously published explicit medication assessment tools, the criteria set presented here does not, however, provide comprehensive coverage of all situations that put patients at risk of PDRM, reflecting the large scope and high complexity of medication use in primary care and the limitations of current UK clinical information systems. The best choice of criteria set will therefore depend on the main purpose to be addressed and will be guided by local priorities. Informed by the priority ratings of a panel of UK primary care professionals, we have selected a subset of the DQIP criteria to serve as outcome measures in a cluster randomised trial evaluating the effectiveness of a complex intervention to improve prescribing safety  and the extent to which any improvements in prescribing or monitoring translate into improved patient outcomes  therefore deserve further study.ACEI: Angiotensin converting enzyme inhibitor; ACOVE: Assessing care of vulnerable elders; ACS: Acute coronary syndrome; ADE: Adverse drug event; AF: Atrial fibrillation; ARB: Angiotensin receptor blocker; BB: Beta blocker; BP: Blood pressure; CCB: Calcium channel blocker; 2: CHADSScore for stroke risk assessment in atrial fibrillation based on the following risk factors: cardiac failure, hypertension, age > 75, diabetes and stroke; CHD: Coronary heart disease; CHD: Coronary Heart Disease; CHF: Chronic heart failure; CKD: Chronic kidney disease; CKD: Chronic kidney disease; COPD: Chronic obstructive pulmonary disease; COX: Cyclo-oxygenase; CVD: Cardiovascular disease; DM: Diabetes mellitus; DMARD: Disease modifying antirheumatic drug; DQIP: Data driven quality improvement in primary care; eGFR: Estimated glomerular filtration rate; FBC: Full blood count; GIP: Gastro-intestinal protective agents; GP: General practitioner; HbA1c: Glycated haemoglobin; HIV: Human immunodeficiency virus; HRT: Hormone replacement therapy; HTN: Hypertension; INR: International normalised ratio; IT: Information technology; MI: Myocardial infarction; MTX: Methotrexate; NSAID: Non-steroidal anti-inflammatory drug (includes non-selective and COXII selective agents unless stated otherwise); NS NSAID: Non-selective NSAID NYHA: New York Heart Association; PDRM: Preventable drug related morbidity; RAM: RAND appropriateness method; SABA: Short acting beta 2 receptor agonist; SD: Standard deviation; START: Screening tool to alert doctors to right treatment; STOPP: Screening tool of older person's prescriptions; TCA: Tricyclic antidepressant; TIA: Transient ischaemic attack; U&E: Urea and electrolytes; UCLA: University College of Los Angeles; UK: United Kingdom; Z-drug: zopiclone, zolpidem or zaleplone.The authors declare that they have no competing interests.The study is part of the Data-driven Quality Improvement in Primary Care (DQIP) research programme, which is led by BG. TD led the literature review, conduct of RAM and Delphi studies, data analysis and wrote the first draft of the manuscript. All co-authors contributed to subsequent drafts. All authors have read and approved the final manuscript.The pre-publication history for this paper can be accessed here:http://www.biomedcentral.com/1472-6904/12/5/prepub


The purpose of this study was to determine whether the duration of antiarrhythmic therapy after discharge from the hospital following abdominal surgery is related to the incidence of atrial fibrillation (AF) recurrence in elderly patients with the occurrence of peri-operative AF.The occurrence of peri-operative AF after abdominal surgery is a clinical condition burdened by several complications, especially in the elderly -3. When The recurrence of AF needs different kind of treatment. Medical therapy includes various antiarrhythmic drugs to control heart rate and restore sinus rhythm and anticoagulation to reduce the tromboembolic risk . OveractA pilot study was conducted in 19 elderly patients (age > 65 years) who underwent abdominal surgery  and with occurrence of peri-operative AF that successfully reverted to sinus rhythm. They were prospectively randomized at dismissal to receive antiarrhythmic therapy for 1 week (six patients in group A), 3 weeks (seven patients in group B), or 6 weeks (six patients in group C). Patients were followed up for an additional 4 weeks after discontinuation of antiarrhythmic therapy for detection of recurrence of AF.There was no significant difference in the recurrence of AF among groups .In elderly patients with peri-operative AF after abdominal surgery, converted to normal sinus rhythm before hospital discharge, have a benign course and the duration of antiarrhythmic therapy shorter than one week is appropriate.


To the Editor: The biologic role of amino acid variants at position 222 of the hemagglutinin (HA) gene of pandemic (H1N1) 2009 virus in severe infections has been extensively discussed. A recent series of studies , confirming the clustering of all the worldwide 222E variants in a well-defined subclade . The same analysis showed that D222G variants could be reconciled with 2 different phylogenetic patterns. The first less frequent pattern includes sequences appearing sparsely throughout the tree, confirming the mentioned hypothesis of sporadic mutation. In contrast, the second pattern (the majority) relates to small groups of sequences appearing in monophyletic microclusters. Among these microclusters, 2 are particularly interesting because they include only 222G sequences, isolated in different parts of the world  to 2,492 complete HA sequences from the Global Initiative on Sharing All Influenza Data database (expanded Figure online,


Early results of an international RCT comparing drainage, irrigation &fibrinolytic therapy (DRIFT)for prematurity-associated post-ventricular dilatation (PHVD), with standard treatment suggested no benefit in terms of avoidance of shunt dependency. There was also an increased rate of secondary bleeds in those having DRIFT. The longer term benefits or otherwise of DRIFT treatment were unknown.We randomly allocated 77 preterm infants with PHVD to either DRIFT or standard treatment . Severe disability was assessed at 2 years’ corrected age.Of 39 infants assigned to DRIFT, 21 (54%) died or were severely disabled versus 27 of 38 (71%) in the standard group . Among the survivors, 11 of 35 (31%) in the DRIFT group had severe cognitive disability versus 19 of 32 (59%) in the standard group . Median Mental Development Index was 68 with DRIFT and_50 with standard care.Despite inducing an increased rate of secondary intraventricular bleeding, DRIFT reduced severe cognitive disability in survivors and overall rates of death or severe disability. A modification of DRIFT involving simple ventricular lavage and fibrinolytic therapy (limited to unblocking clogged-up drainage catheters) will now be employed in clinical practice at our unit.


When patients with a rheumatic disorder reach adulthood, transfer to adult-focused care is advocated (1). A successful continuation of rheumatology follow-up beyond childhood depends on how clinicians think about transfer and transition (2).To explore the attitudes of rheumatology practitioners on transfer and transition from pediatric to adult health care of adolescents with a rheumatic disorder.A survey  among rheumatology practitioners attending the Pediatric Rheumatology European Society congress in 2010. Overall, 138 individuals participated, of which 59 were medical doctors (MDs) with paediatric and rheumatology background; 30 MDs with pediatric background; and 21 MDs with rheumatology background.Participants judged that, when patients with active rheumatic disorder reach adulthood, they should receive medical follow-up from an adult rheumatologist (87%). Only 19% thought that patients should stay under surveillance of a paediatric rheumatologist. Several initiating factors for transfer were marked as important: readiness of the patient according to the caregiver (62%), age (61%) and psychosocial maturity (49%). A transfer meeting with the patient (76%), a referral letter (73%), or a medical transfer file (64%) were most preferred transfer communication tools with adult healthcare. Joint outpatient clinic, phone calls, and transfer meetings without the patient were considered to be less useful. The pediatric (94%) or adult rheumatologist (83%), parents (81%) and the nurse specialist (74%) were stated as most important active participants in the transition process.This study emphasized the importance of transfer to specialized rheumatology care of adolescents with an active rheumatic disease and highlighted transfer initiators and transfer communication tools.


We analysed the responses using descriptive statistics; 660 individuals responded to the public and 303 individuals responded to the expert survey. We found evidence of public skepticism about the use of neuroimaging for applications such as lie detection or to determine consumer preferences and considerable disquiet about use by employers or government and about how their data would be stored and used. While also somewhat skeptical about new applications of neuroimaging, experts grossly underestimated how often neuroimaging had been used as evidence in court. Although both the public and the experts rated highly the importance of a better informed public in limiting the inappropriate uses to which neuroimaging might be put, opinions differed on the need for, and mechanism of, actual regulation. Neuroscientists recognized the risks of inaccurate reporting of neuroimaging capabilities in the media but showed little motivation to engage with the public. The present study also emphasizes the need for better frameworks for scientific engagement with media and public education.Emerging applications of neuroimaging outside medicine and science have received intense public exposure through the media. Media misrepresentations can create a gulf between public and scientific understanding of the capabilities of neuroimaging and raise false expectations. To determine the extent of this effect and determine public opinions on acceptable uses and the need for regulation, we designed an electronic survey to obtain anonymous opinions from as wide a range of members of the public and neuroimaging experts as possible. The surveys ran from 1 Am I politically right or left wing? Which of sex or money interests me most? Should I take an fMRI lie detector test? Can other people see my dreams? Can neuroimaging help me chose the right career? Can imaging identify future criminals amongst young children? The potential for new technologies to improve the health, living and economic prospects of society quite rightly attract public curiosity We are exposed, almost weekly, to reports of new, thrilling and increasingly fantastical applications of neuroimaging to unravel the complexities of our minds – Misinterpretations of neuroimaging research by the public may arise in several ways: the distortion of findings by scientists or the media to enflame interest, through commercial interests, or poor engagement of the public by researchers. Promotion of the level of public understanding of neuroscience concepts Motivated by the need for evidence-based solutions to these challenges, we surveyed the public for their thoughts on the current uses, communication of, and need for regulation of, neuroimaging research. In parallel, we also surveyed experts in neuroimaging to provide a benchmark of the scientifically accepted uses and identify where public and professional opinions might differ.We designed two short questionnaires, one for the public and one for neuroimaging experts, suitable for delivery online or on paper. The public and expert surveys were modified to reflect the respondents' likely knowledge of and degree of personal relevance in determining their opinion on each issue. There were 10 questions in the public survey and 21 questions in the expert survey , websites , tweets , and science blogs  as well as to attendees at meetings (e.g. Scottish Parliament Futures Forum) and societies for public engagement (Beltane http://www.publicengagement.ac.uk/about/beacons/edinburgh-beltane). The Expert Survey was distributed via university mailing lists , professional interest websites  and individual imaging research centre websites) to target individuals who routinely used neuroimaging methods . Ethics approval was not sought. A level one self-assessment indicated that there was no risk to the respondent or the researcher.The survey was posted online, using the Survey Monkey Pro web survey template, between 31p<0.05). All available data were included. Percentages are based on samples of completed response, as opposed to the entire potential sample. After main effects were examined, we examined for trends within demographic profiles. The public sample was divided into groups based on scientific interest. The expert sample was divided based on the capacity in which they used neuroimaging technology .We used descriptive statistics to characterize the composition of the sample, t-tests to examine differences between the samples and chi-squared tests to analyse main effects  and the majority described themselves as professionals (60%), students (16.5%), administrator (9.8%), not working (9.8%), skilled manual (2.7%) or manual workers (1.1%). The highest educational attainment was university undergraduate degree (30%), a Masters degree (21.3%), a PhD (15.9%), a professional qualification (15.5%), minimum school leaver's exam (8.9%) or final school leaver's exam (8.1%). Most respondents to the Expert Survey were neuroscientists (27%), followed by psychologists (17%), neuroradiologists (12.5%), psychiatrists (12.5%) or medical physicists (10.5%); had been using neuroimaging for 5–10 years (31%), <5 years (28%) or 10–20 years (26%); and were based in Scotland (34%), elsewhere in the United Kingdom (32%), Europe (16%) or the United States (15%).n = 39, 5%) who said that their professions involve neuroimaging and may therefore not be representative of the public profile was identified, but no differences were found between the groups (p>0.05).Almost half the sample reported themselves as at least ‘a little’ aware of neuroimaging uses (47%), followed by ‘quite aware’ (26%), ‘no awareness’ (17%) and ‘very aware’ (10%). A subgroup , but few had the same confidence in use of neuroimaging to diagnose mental illness , some thought that neuroimaging could detect lies  or consumer preferences , but had less confidence in detecting racial attitudes , political views  or reading minds , Most respondents reported that they would be comfortable having their brain scanned for medical purposes (96%), for scientific research (90%), but were less inclined as part of a criminal investigation (36%), for insurance purposes (9%), for marketing research (16%) or as part of a job interview (11%) Most respondents were ‘very’ or ‘quite’ concerned about the confidentiality and storage of scans (82%) and that ‘people would know what they were thinking’ (55%), being forced to have a scan (70%) or that there would be ‘something wrong with their brain’ (57%). Most were ‘not at all ‘or’ only a little ‘worried that having a scan would make them vulnerable to thought control (61%), Most respondents reported that newspapers (65%) and popular science publications (64%) were the most common sources for information about neuroimaging, followed by television documentary (62%), television or film fiction (59%) and online sources (46%). Of those who reported using online sources, twitter and blogs were the most popular. When asked how often they encountered information on brain imaging, responses were highly varied with about a third reporting a little , often  and very often .When asked about the regulation of brain imaging, most public respondents chose legislation (85%) and professional regulatory bodies (86%), followed by increased public awareness (84%). The least favoured response was the self-regulation of brain imaging research by the scientists themselves (53%), followed by the licensing of the scientists to conduct neuroimaging research (66%).Most expert survey respondents reported using neuroimaging for neuroscience research (66%), followed by clinical research (47%), medical diagnosis (35%), neuromarketing (4%), forensic purposes (3%) and security purposes (1%). The majority of respondents were aware of the use of neuroimaging outside traditional research (86%), particularly for neuromarketing (67%) and lie-detection (68%). Moderate awareness was reported for legal uses (48%), neuroaesthetics (56%), cognitive enhancement (55%) and mind reading (45%). Few were aware that neuroimaging was being used for security purposes (11%).Most respondents report specialist peer-reviewed literature as their main source of information on neuroimaging (84%), followed by general literature (65%), popular science media (50%), research conferences (54%), science blogs (25%), newspapers (25%) and popular science websites (23%).Most respondents thought that neuroimaging could improve the understanding of cognition , or improve treatment of psychiatric illness . However, most experts thought that the ability of neuroimaging to detect lies, read minds, understand consumer or criminal behaviour was largely a thing of the future or unlikely ever to occur : 29% of When asked how often neuroimaging evidence has been presented in U.S. courts in the last 5 years, most believed it was between 10 and 30 times (24%), followed closely by 6–10 times (22%) and between 1 and 5 times (23%). The lowest rates were reported for greater than 100 times (15%), between 30–60 times (8%) and between 60 and 100 times (8%).development of new applications, changing the legal system, changing views on responsibility for criminal behavior and enhancement of mental abilities, although changing the legal system and enhancing mental abilities were considered the least likely amongst these options (innovative applications (45%), cognitive enhancement (46%), change views of criminal responsibility (34%) and change the legal system (47%).Most respondents thought that wider uses of neuroimaging would, to some extent, lead to  options . These vIn terms of strategies for regulating use of neuroimaging, respondents ranked professional guidelines first (66% ranked first or second choice), followed by more funding for interdisciplinary projects integrating neuroscience and law (50%) and improved public awareness (51%), with legal regulation (16%) and self regulation (15%) being placed last.A substantial majority (87%) felt that neuroimaging was not accurately portrayed in the media. When asked why, most cited poor journalism (54%) as either a quite or very important factor, followed by exaggerated results by researchers (51%), a lack of effective scientific communication (38%) and poorly conducted research (44%). Most respondents believe that increased exposure to neuroimaging research would raise its public profile as a beneficial technology (96%), whereas 80% foresaw a public backlash on the widespread use of the technology. 92% predicted an increase in funding opportunities, but 44% anticipated the opposite trend. 69% feared the over regulation of neuroimaging in research.Having said that, although most respondents thought it was important to communicate with the public (69%), most do not regularly engage with the media (52%). Of those that do communicate their results to the public, most used media and peer-reviewed publication to do this (78%), followed by public seminars (49%), the internet (39%) and television (28%). Among those that did not engage with the public, the majority gave little opportunity (58%), little incentive (38%) and distrust of media (27%) as their main reasons, followed by a lack of public speaking training (15%) and a fear of criticism (5%). When asked how this situation might be improved, most rated as ‘quite’ or ‘very effective’: professional credit for public engagement (75%), making public communication a funding requirement (72%), increased public exposure of research (76%), and outlets for a career in the media (25%).This survey of members of the public and neuroimaging experts revealed a range of similarities and differences of opinions. About a third of the public saw articles on neuroimaging in the media once or twice a month indicating, in line with previous research Although the public and experts were generally in agreement about some points, e.g. both had little faith in uses of neuroimaging in non-medical applications and both agreed on some methods to use for regulation, the results also suggest that extensive media coverage has moderated viewpoints and awareness differentially among the public and experts. The public were concerned about data protection issues. The experts had little awareness of how often neuroimaging had been used in court in the USA  and one in three experts reporting no familiarity with neuromarketing or commercial lie-detection, although the public were aware of these uses. Although the experts thought that neuroimaging results were not well communicated in the media, and recognised the dangers of public loss of trust, few experts seemed motivated to improve the situation.Who can say which, if any, of the current legal, commercial or governmental uses of neuroimaging may become in future years routine, reliable tools? Neuroimaging is a rapidly evolving discipline; insights gained from well-conducted neuropsychological imaging research are in turn influencing knowledge of human behavioural traits on which many of society's attitudes are based. Consequently, it is entirely possible that society will need to adjust its views on behaviour, culpability, consciousness, etc. in future. However, a major focus of the present study was to determine how the public perceived the current capability of neuroimaging. Limited confidence in diagnosing psychiatric illness, lie-detection and neuromarketing was found, and to a far lesser degree in revealing racial and political attitudes and inner thoughts. Interestingly, the evidence base that complex social behaviours, such as racial attitudes and basic forms of mental representation, can be inferred using neuroimaging  especially with regards privacy and human rights (e.g. “Anything that is voluntary is acceptable. Mandatory brain scans are not…”). The public distrust of other technologies used to extract sensitive information for non-health related uses has been echoed in a public survey on the acceptability of genetic testing in insurance and employment settings One of the clearest trends identified in the survey is the public disapproval of the use of neuroimaging in non-medical or scientific settings, such as in marketing research, and in employment screening. Indeed, respondents who made specific comments cited moral and ethical grounds as explanations for this standpoint Click here for additional data file.


The recommendations are presented with supporting level of evidence.In this report, guidelines for the evaluation, medical and surgical management of transitional cell carcinoma of testicular germ cell tumors is presented. It is categorized according to the stage of the disease using the tumor node metastasis staging system, 7 A total of 38 cases have been diagnosed in 2006, with an age standardized rate (ASR) of 0.5 cases per 100,000 representing 1% of all diagnosed cancer in Saudi Arabia  High level: well-conducted phase III randomized trials or meta-analysis.(EL2) intermediate level: good phase II trials or phase III with limitations.(EL3) low level: Observational/retrospective studies or expert opinion.thedition 2010) was used.The American Joint Committee on Cancer (AJCC) TNM staging for testis cancer (7 The international Germ Cell Cancer Collaborative Group Risk Classification should bWill depend on the histological subtype as follows:


Most patients undergoing cardiac surgeries are exposed to red blood cell (RBC) transfusions, in the operating room or in the postoperative period. One of the main beliefs of this therapy is the ability of the RBCs to improve tissue perfusion through oxygen supply. However, recently, this concept is being questioned by some evidence as RBC storage lesion and adverse outcomes in transfused patients. The aim of this study was to determine if RBC transfusion after cardiac surgery results in improvement of tissue perfusion.2) were collected immediately at the beginning and end of the procedure, immediately postoperative (POI), after 24 hours (1PO), 48 hours (2PO), 72 hours (3PO) and at ICU discharge. Mean values of these above-mentioned parameters were compared in patients exposed to RBC transfusions and patients not exposed through repeated-measures variance analysis.From February 2009 to February 2010, a total of 502 patients underwent cardiac surgery with cardiopulmonary bypass at InCor - University of São Paulo. Arterial lactate, standard base deficit (SBD), arterial bicarbonate and oxygen central venous saturation (ScVOHemoglobin values were different between groups since before surgery until just before ICU discharge and in all periods, the group not exposed to RBC transfusions presented higher values compared with the exposed group (see Figure In this prospective study, red blood transfusion did not result in improvement of tissue perfusion parameters. This finding brings to discussion the real role of blood transfusion in cardiac patients.


We sought to determine if a common polymorphism can influence vulnerability to LDL cholesterol, and thereby influence the clinical benefit derived from therapies that reduce LDL cholesterol.We conducted a meta-analysis of the association between a common Trp719Arg polymorphism in the kinesin-like protein 6 (KIF6) gene and the risk of cardiovascular disease (CVD), and a meta-regression analysis to measure the effect modification of this polymorphism on the association between LDL cholesterol and the risk of CVD. We used this measure of genetic effect modification to predict the expected difference in clinical benefit among KIF6 719Arg allele carriers and non-carriers in response to therapies that reduce LDL cholesterol. We then conducted a meta-analysis of statin trials to compare the expected difference in clinical benefit with the observed difference during treatment with a statin.In a meta-analysis involving 144,931 participants, the KIF6 719Arg allele was not associated with the relative risk (RR) of CVD . Meta-regression analysis involving 88,535 participants, however, showed that the 719Arg allele appears to influence the effect of LDL cholesterol on the risk of CVD. KIF6 carriers experienced a 13% greater reduction in the risk of CVD per mmol/L decrease in LDL cholesterol than non-carriers. We interpreted this difference as the expected difference in clinical benefit among KIF6 carriers and non-carriers in response to therapies that lower LDL cholesterol. The difference in clinical benefit predicted by the increased vulnerability to LDL cholesterol among KIF6 carriers  agreed very closely with the observed difference among 50,060 KIF6 carriers and non-carriers enrolled in 8 randomized trials of statin therapy .The KIF6 719Arg allele increases vulnerability to LDL cholesterol and thereby influences the expected clinical benefit of therapies that reduce LDL cholesterol. A common single nucleotide polymorphism (rs20455) results in an argine to tryptophan substitution at position 719 (Trp719Arg) in the gene encoding kinesin-like protein 6 (KIF6). Some One possible explanation is that the KIF6 719Arg allele may increase the risk of cardiovascular disease (CVD), and that treatment with a statin may ameliorate this increased risk through a pleiotropic effect A biologically plausible hypothesis that can potentially reconcile these apparently conflicting findings is that the KIF6 719Arg allele may increase vulnerability to LDL cholesterol. If carriers of the 719Arg allele are more vulnerable to the deleterious effects of LDL cholesterol, then they should experience a greater increase in the risk of CVD per unit increase in LDL, and a correspondingly greater reduction in the risk of CVD per unit decrease in LDL, as compared to non-carriers. Therefore, if KIF6 carriers are more biologically vulnerable to LDL cholesterol, they should derive a greater clinical benefit per unit reduction in LDL cholesterol than non-carriers in response to therapies that lower serum LDL cholesterol levels.The primary aim of our study was to test the hypothesis that a common polymorphism can influence biological vulnerability to LDL cholesterol, and thereby influence the expected clinical benefit of therapies that reduce serum LDL cholesterol levels. To test this hypothesis, we first conducted a systematic review and meta-analysis of the association between the KIF6 719Arg allele and the risk of CVD. We then conducted a meta-regression analysis to measure the effect of the KIF6 719Arg allele on the association between LDL cholesterol on the risk of CVD. We used this measure of effect modification to estimate the expected difference in clinical benefit among KIF6 carriers and non-carriers in response to therapies that lower serum LDL cholesterol level. We then conducted a systematic review and meta-analysis of the effect of the KIF6 719Arg allele on the clinical efficacy of statin therapy. Finally, we compared the expected difference in clinical benefit predicted by the meta-regression analysis with the observed difference in clinical benefit among KIF6 carriers and non-carriers in the meta-analysis of statin trials.We searched Pubmed, Embase, Index of Science, HUGEnet, and Google Scholar, using the search terms: KIF6, rs20455, 719Arg, and Trp719Arg without language restriction; we also checked the abstracts and presentations from major cardiovascular medicine meetings held from 2004 to present First, we conducted an updated systematic review and meta-analysis of the association between KIF6 719Arg carrier status and the risk of CVD. For this analysis, we included case-control studies, epidemiologic cohort studies, and the treatment allocation arms of randomized trials of statin therapy that have reported data separately among KIF6 719Arg allele carriers and non-carriers, but excluded family-based studies. We included both the placebo allocation and statin allocation arms of the placebo controlled trials, as well as both the less intensive and more intensive treatment allocation arms of the trials that compared different treatment intensities of statin therapy. Each treatment allocation arm was considered as a separate prospective cohort study for the purposes of this analysis. For study samples that were included in more than one report, we used data from only the most recent report. In each study, Hardy-Weinberg equilibrium was assessed, as appropriate, using a chi-square test. For each prospective cohort study , we extracted the total number of KIF6 719Arg allele carriers and non-carriers, as well as the number of KIF6 carriers and non-carriers who experienced an incident CVD event during study follow-up. From each case-control study, we extracted the total number of prevalent cases and controls for both KIF6 719Arg allele carriers and non-carriers. We used these data to create 2×2 tables to calculate the unadjusted relative risk (RR), and 95% confidence interval (CI), for each prospective cohort study, and the unadjusted odds ratio (OR), and 95% CI, for each case-control study (as an approximation of the RR) 2) by the effect modification was calculated as the difference in tau2 between the regression model with and without a regression term, expressed as a percentage.Next, we performed a random effects meta-regression analysis to measure the effect modification of study baseline LDL cholesterol level on the association between KIF6 carrier status (and separately KIF6 genotype) and the risk of CVD Finally, we conducted an updated systematic review and meta-analysis of statin trials that have reported results stratified by KIF6 carrier status (or KIF6 genotype). From the report of each randomized trial, we extracted data on the number of persons assigned to either treatment allocation arm and the number of incident CVD events that occurred during study follow-up in each arm, stratified by KIF6 carrier status. We used these data to create 2×2 tables to calculate an unadjusted RR (and 95% CI) for the effect of statin therapy as compared to placebo (or more intensive statin therapy as compared to less intensive statin therapy) on the risk of incident CVD events, separately among KIF6 carriers and non-carriers in each study. All analyses were by the intent-to-treat principle. We then standardized the estimate of the effect of statin therapy per mmol/L reduction in LDL cholesterol across trials by multiplying the natural logarithm of the RR for each trial (and its standard error) by 1÷ Y, where Y is difference in average LDL cholesterol during follow-up between the two treatment allocation arms . We then combined the adjusted RR for each trial using an inverse variance weighted random effects model We standardized the effect of statin therapy per mmol/L reduction in LDL cholesterol in order to compare the observed difference in clinical benefit in the meta-analysis with the expected difference in clinical benefit estimated from the meta-regression analysis using the same scale. Standardizing the effect of statin therapy also allowed us to include both the trials that compared statin with placebo and the trials that compared more intensive and less intensive statin therapy. In the meta-analysis, therefore, the primary measure of effect is not the clinical benefit of statin therapy per se, but rather the clinical benefit per mmol/L reduction in LDL cholesterol during treatment with a statin.2 metric Heterogeneity was measured using Cochran's Q, and the IA total of 37 case-control studies, prospective cohort studies, or randomized trial treatment allocation arms (each considered as a separate cohort), including 144,931 participants and 27,465 CVD events, were included in our meta-analysis of the association between the KIF6 719Arg allele and the risk of CVD  [1]–[6],2 = 58.7%).Among the 37 study samples included in our meta-analysis, 19 prospective cohort studies  involving a total of 88,535 participants and 9,692 incident CVD events, provided information on the average baseline LDL cholesterol level of the population under study and therefore were included in our meta-regression analysis Relying on the symmetrical nature of the effect modification between any two epidemiologic exposures A total of 8 randomized trials, involving 50,060 participants and 7,307 incident cardiovascular events, were included in our meta-analysis of the effect of statin therapy, stratified by KIF6 carrier status  [1]–[6],The magnitude of the difference in clinical benefit per mmol/L reduction in LDL cholesterol observed among KIF6 719Arg carriers and non-carriers in response to treatment with a statin  was nearly identical with the expected difference in clinical benefit per mmol/L reduction in LDL cholesterol predicted by the increased vulnerability to LDL cholesterol among KIF6 719Arg carriers from the meta-regression analysis .Our study demonstrates that the common KIF6 719Arg allele appears to increase vulnerability to LDL cholesterol, and thereby influences the expected clinical benefit from therapies that lower serum LDL cholesterol levels. Persons who inherit one or more copies of the 719Arg allele appear to be more biologically vulnerable to the deleterious effects of LDL cholesterol than non-carriers. As a result, KIF6 719Arg carriers appear to derive a greater reduction in the risk of CVD per mmol/L reduction in LDL cholesterol than non-carriers. The results of our study resolve several apparently conflicting lines of evidence to provide a biologically plausible explanation for how KIF6 carriers can derive a greater clinical benefit from treatment with a statin than non-carriers, even though this polymorphism does not have any impact on the LDL cholesterol lowering effect of statins, and is not independently associated with the risk of CVD.More generally, our study demonstrates that a common polymorphism can influence vulnerability to LDL cholesterol, and thereby influence the expected clinical benefit from therapies designed to lower serum levels of LDL cholesterol. The concept of genetically-mediated differential vulnerability to LDL cholesterol is illustrated in Our study shows that, on average, KIF6 719Arg allele carrier status is not associated with the risk of CVD. Our study also shows, however, that this association varies significantly according to the average baseline LDL cholesterol level of the population under study . FurtherOur findings do not challenge the results of a large-scale meta-analysis of statin randomized trials that found that the clinical benefit of statin therapy is determined largely by the magnitude of LDL cholesterol reduction achieved during treatment Importantly, our study shows that the KIF6 719Arg allele increases vulnerability to LDL cholesterol even though it is not, on average, associated with an increased risk of CVD. This means that all 2.5 million directly genotyped and imputed polymorphism measured in previously conducted genome-wide association studies of CVD, very few of which have been shown to be reliably associated with the risk of CVD Our study has several limitations. Most importantly, we did not have any data on the association between LDL cholesterol and the risk of CVD, stratified by KIF6 carrier status. Therefore, we could not estimate the effect modification of KIF6 on the association between LDL cholesterol and the risk of CVD directly. Instead, we estimated this effect from the meta-regression estimate of the effect modification of LDL cholesterol on the association between the KIF6 719Arg allele and the risk of CVD. We believe this approach is valid because these two estimates of effect modification are equivalent as shown in In conclusion, we found that the common KIF6 719Arg allele appears to increase vulnerability to LDL cholesterol, and thereby influences the expected clinical benefit from therapies that lower serum LDL cholesterol levels. More generally, our study demonstrates that a common polymorphism can influence vulnerability to a common modifiable risk factor for CVD even if it is not independently associated with the risk of CVD. Our study provides a roadmap for further research that could potentially lead to the discovery of polymorphisms that cause large differences in vulnerability to common modifiable risk factors for CVD and thus lead to correspondingly large differences in the clinical benefit derived from therapies used to treat these risk factors. Incorporating such polymorphism into the care of individual patients may help to guide the selection of optimal medical therapies for each individual person based on their genetic background and therefore introduce a practical clinical strategy to personalize the prevention of CVD.Table S1A list of studies included in the meta-analysis of the KIF6 719Arg allele and the risk of cardiovascular disease. The included studies are Atherosclerotic Disease, Vascular Function, and Genetic Epidemiology study (ADVANCE); Acute Myocardial Infarction Gene Study, Dortmund Health Study (AMI Gene Study); CATHGENE Research Project (CATHGENE); deCODE CAD Study (deCODE); National FINRISK studies (FINRISK); German Myocardial Infarction Family Study I (GerMIFS I); German Myocardial Infarction Family Study II (GerMIFS II); Heart Attack in Puget Sound study (HARPS); International Heart Study (INTERHEART); Malmo Diet and Cancer Study (MDS); Washington Hospital Center catheterization study (MEDSTAR); Massachusetts General Hospital study of premature CAD (MGH PCAD); Mid-America Heart Institute (MAHI); Penn-CATH study; Registre Gironi de Cor (REGIGOR); Verona Heart Study (VHS); Wellcome Trust Case Control Consortium CAD Study (WTCCC); Ottawa Heart Study; Cholesterol and Recurrent Events (CARE); West of Scotland Coronary Prevention Study (WOSCOPS); Pravastatin or Atorvastatin Evaluation and Infection Therapy: Thrombolysis in Myocardial Infarction 22 (PROVE-IT TIMI22); Prospective Study of Pravastatin in the Elderly at Risk (PROSPER); Heart Protection Study (HPS); Justification for the Use of Statins in Primary Prevention trial: An Intervention Trial Evaluating Rosuvastatin (JUPITER); Treat to New Targets (TNT); Incremental Decrease in Events through Aggressive Lipid Lowering (IDEAL); Atherosclerosis Risk in Communities study (ARIC); Cardiovascular Health Study (CHS); Women's Health Study (WHS).(PDF)Click here for additional data file.Table S2A description of the baseline characteristics of the randomized trials included in the meta-analysis of the clinical benefit of statin therapy stratified by KIF6 carrier status. The Difference in average LDL (mmol/L) is the difference between either treatment allocation arm. Study acronyms are defined in (PDF)Click here for additional data file.Text S1Equivalent measures of effect modification.(PDF)Click here for additional data file.


Despite the use of hepatitis B virus (HBV) vaccines and preventive mDespite the use of hepatitis B virus (HBV) vaccines and preventive measures, infection with HBV remains a major global health problem. Patients with chronic kidney disease (CKD) are at an increased risk of acquiring HBV infections from shared dialysis equipment, increased exposure to blood products, and immunodeficiency associated with CKD 2]. In a. In a2].


Differentiation of acute heart failure from infection in patients with respiratory symptoms and a history of congestive heart failure (CHF) is challenging due to overlap of clinical symptoms and X-ray findings. The BACH study found higher mortality rates if patients presenting with dyspnea were treated with antibiotics and their procalcitonin (PCT) levels were low indicating absence of bacterial infection. Yet the BACH study was observational and causal inference cannot be drawn. Herein, we analyzed the effects of PCT-guided antibiotic stewardship in CHF patients from a previous trial (ProHOSP).This is a secondary analysis of a previous randomized trial of adult ED patients with respiratory symptoms and a history of CHF. Patients were randomized to administration of antibiotics based on a PCT algorithm (PCT group) or standard guidelines without knowledge of PCT levels (control group). The primary endpoint of this analysis is the risk of adverse outcome defined as death or ICU admission within 30 days after ED admission.n = 110), PCT-guided patients had a significant reduction in antibiotic exposure , P <0.001). Furthermore, PCT-guided patients had a significant lower risk for death and ICU admission , P = 0.02). See Figure A total of 233 patients met the inclusion criteria, with 116 in the PCT-guided group and 117 in the control group. In the subgroup of patients with low initial PCT levels <0.25 ng/l (In CHF patients with suspicion of respiratory infection, use of a PCT protocol resulted in a significant decrease of antibiotic exposure and significantly improved outcomes in patients with low PCT levels indicating absence of bacterial infection. Whether inadequate antibiotic therapy in these CHF patients requiring diuretic treatment explains this difference in clinical outcomes needs verification.


Anser albifrons) in South Korea. Phylogenetic analyses of the complete genome sequence showed that polymerase acidic (PA) and neuraminidase (NA) genes belonged to the Eurasian lineage AIV, but polymerase basic 2 (PB2), PB1, hemagglutinin (HA), nucleoprotein (NP), matrix (M), and nonstructural (NS) genes belonged to the North-American lineage AIV. These data are beneficial for understanding the ecology and epidemiology of AIVs.In 2007, we isolated a natural recombinant H9N2 avian influenza virus (AIV) from the fecal droppings of a white-fronted goose ( Influenzavirus A genus of the Orthomyxoviridae family (Avian influenza virus (AIV) belongs to the e family . The AIVe family .Anser albifrons) in South Korea in 2007. Species identification of the fecal droppings was done by DNA barcoding using the cytochrome oxidase I gene, as described previously  was isolated from the fecal droppings of a white-fronted goose  virus were derived from North American lineages but that PA and NA genes were derived from Eurasian lineages. It seems that continuous reshuffling of AIV genes between North American and Eurasian lineages has been occurring in wild bird populations, producing the reassortant North American-Eurasian lineage AIV.In the present study, our results provide crucial information on the epidemiology of AIV in wild birds by increasing understanding of the role of migratory birds in exchanging AIV genes between the Eurasian and North American continents. Enhanced surveillance of the wild bird population is required to improve our understanding of the natural history of AIV in wild birds.KC693639 to KC693646.The genome sequences of A/white-fronted goose/Korea/20-36/2007 (H9N2) have been deposited in GenBank under accession no.


Geologic process, including tectonics and global climate change, profoundly impact the evolution of life because they have the propensity to facilitate episodes of biogeographic differentiation and influence patterns of speciation. We investigate causal links between a dramatic faunal turnover and two dominant geologic processes operating within Laurentia during the Late Ordovician: the Taconian Orogeny and GICE related global cooling. We utilize a novel approach for elucidating the relationship between biotic and geologic changes using a time-stratigraphic, species-level evolutionary framework for articulated brachiopods from North America. Phylogenetic biogeographic analyses indicate a fundamental shift in speciation mode—from a vicariance to dispersal dominated macroevolutionary regime—across the boundary between the Sandbian to Katian Stages. This boundary also corresponds to the onset of renewed intensification of tectonic activity and mountain building, the development of an upwelling zone that introduced cool, nutrient-rich waters into the epieric seas of eastern Laurentia, and the GICE isotopic excursion. The synchronicity of these dramatic geologic, oceanographic, and macroevolutionary changes supports the influence of geologic events on biological evolution. Together, the renewed tectonic activity and oceanographic changes facilitated fundamental changes in habitat structure in eastern North America that reduced opportunities for isolation and vicariance. They also facilitated regional biotic dispersal of taxa that led to the subsequent establishment of extrabasinal (=invasive) species and may have led to a suppression of speciation within Laurentian faunas. Phylogenetic biogeographic analysis further indicates that the Richmondian Invasion was a multidirectional regional invasion event that involved taxa immigrating into the Cincinnati region from basins located near the continental margins and within the continental interior. Late Ordovician (~450 Ma) shallow marine deposits of Laurentia record a series of dramatic faunal and biogeographic shifts including a rapid change from predominantly warm to cool water adapted taxa during the middle Mohawkian (late Sandbian) and a major regional biotic immigration event into the Cincinnati Basin during the early Richmondian (Katian) ,2. TheseDuring the Late Ordovician, the Taconian orogeny developed along the eastern margin of Laurentia as a result of diachronous collisions with an island arc and/or microplates –5 , carbonate deposits of eastern Laurentia reflect a heterozoan assemblage typified by cool water and/or non-photosynthetic organisms, whereas tropical-style carbonates became restricted to the continental interior and marginal basins ,7. This  sequenceAlso during the M5 sequence, faunas of the Appalachian Basin transitioned from predominantly warm to cool water adapted. The biotic overturn was accomplished primarily through immigration of extra-basinal taxa into a region following regional generic extinction . For exaThe Richmondian Stage   was employed as described in Wiley & Lieberman . SpeciesBecause different geologic processes operated before and after the M4/M5 sequence boundary, LBPA was employed in a time-stratigraphic framework to more accurately discern patterns of biogeographic differentiation. Stratigraphic dimension was incorporated based on equal phylogenetic split using first and last appearances of species at the level of depositional sequence. Cladogenetic events were separated into Time Slice 1 (T1), approximately corresponding to the Blountian tectophase (W-b through M4 sequences), and Time Slice 2 (T2), approximately corresponding to the Taconic tectophase (M5 though C6 sequences) . AdditioGlyptorthis indicates that the genus originated in the Southern Midcontinent region and subsequently underwent a major dispersal event into the Appalachian and Central Basins  with frequent episodes of speciation via dispersal into neighboring tectonic basins during T2 , whereas the other tree is consistent with a more recent exchange of taxa between Anticosti Island and the Midcontinent region (supported by biogeographic affinities of Psubquadratus. ). These data indicate that both dispersal pathways were operational during the Richmondian Invasion.The diametrically opposed patterns of speciation and biogeographic differentiation between T1 and T2 reflect different biogeographic controls operating before and after the M4/M5 sequence boundary. This discrepancy is clearly illustrated by the divergent topology of vicariance trees between times slices and the dissimilarity of the geodispersal and vicariance trees within each time slice , 3. The The level of congruence between vicariance and geodispersal trees is indicative of the style of geologic processes impacting macroevolutionary patterns . CongrueDuring T1, the Blountian tectophase introduced siliciclastics into eastern Laurentia yet did not inhibit the production of tropical carbonates ,6. The hDispersal became the dominant mode of speciation during the Taconic tectophase of the Taconian Orogeny T2). The combination of flexural downwarping of the craton and GICE related oceanographic changes facilitated the upwelling of cool, nutrient rich water into the shallow epieric seas of eastern Laurentia ,5,9. Thi. The comThe shift in speciation regime across the M4/M5 boundary is significant because high rates of vicariance are typically associated with high rates of speciation, whereas high rates of dispersal are associated with reduced speciation . The negThe main pulse of the Richmondian Invasion into the Cincinnati Basin occurred during the C5 sequence . The retPhylogenetic biogeographic analysis of species-level patterns in Middle to Late Ordovician brachiopods indicates a shift in biogeographic patterns and macroevolutionary dynamics between the Blountian and Taconic tectophases of the Taconian orogeny . These patterns were driven largely by geologic processes related to the Taconian Orogeny and global cooling at the boundary between the Sandbian and Katian stage boundary (M4/M5 sequence boundary). Prior to the M5 sequence, tectonic activity associated with the Blountian tectophase produced increased habitat fragmentation in tropical carbonate environments, thereby facilitating high levels of vicariant speciation. In contrast, the post-M5 sequence interval corresponds to a shift in paleoceanographic conditions related to the combined effect of structural deformation from the Taconic tectophase and GICE related global cooling, whereby cool, nutrient rich water from the Sebree Trough was introduced into shallow intracratonic seas. Dispersal became the dominant form of speciation and the increased number of invasive species may have led to increased faunal homogenization between basins. High rates of dispersal in the T2 interval and a return to tropical conditions within the Cincinnati Basin facilitated the Richmondian Invasion into the Cincinnati Basin. Species-level phylogenetic biogeographic patterns suggest both intracratonic seas and marginal basins supplied invasive species into the Cincinnati Basin.Figure S1The division between TS1 and TS2 is indicated by the horizontal line.(TIF)Click here for additional data file.Table S1Numbered columns refer to nodes and tips on the stratocladograms . Coding as in Wiley & Lieberman [31].(PDF)Click here for additional data file.


Dear Editor,We read with great interest the report of Alcelik et al.This 64-year old woman with chronic kidney disease due to nephrosclerosis, commencing on hemodialysis before 3 years, desired to candidate for kidney transplantation. Her medical history limited to hypertension of 10 years duration and end stage renal disease with well controlled secondary hyperparathyroidism [intact parathyroid hormone (iPTH): 17O pg/ml], but increased calcium phosphorus product (about 62 over the last 6 months) due to non compliance with absorption of phosphorus binders. No signs of osteoporosis have been identified on Dexa scan. Pretransplantation evaluation of the hands (plain radiograph) identified subperiostal bone resorption at the inner part of the terminal thumb phalanges symmetrically at both hands, with preservation of peripheral cortex . MoreovePublished data point out that the earliest subperiostal bone resorption due to secondary hyperparathyroidism is demonstrated at the phalangeal tufts and the radial aspects of the middle phalanges of the second and the third hand fingers. Whereas previously, radiologic findings were more intense, improvements in therapeutic management of secondary hyperparathyroidism in hemodialysis result in significant decrease of radiologic evidence.


The current methods utilized to track stem cells by cardiac MR are affected by important limitations, and new solutions are needed.In vivo cine and in vivo and ex vivo multiecho T2 weighted images were obtained by 9.4T cardiac MR.We tested human ferritin heavy chain (hFTH) tagged with both myc and green fluorescence protein (GFP) as a reporter gene for in vivo tracking of stem cells by cardiac MR. Rat mesenchymal stem cells (rMSCs) were transduced with lentiviurs to overexpress hFTH. Myocardia infarction was induced by cryoinjury in rats, and the animals were immediately subjected to intramyocardial injection of 100 µl of 106 rMSCs (experiment group) or buffer solution (control group) in the border zone. ex vivo images was significantly decreased in the infarct area compared to remote normal myocardium in the experiment group, but not in the control group. GFP and myc immune-staining confirmed the presence of differentiated rMSCs around infarct area in the experiment group.Four-week follow-up cine MR showed that marked left ventricular remodeling developed in the control group. T2 relaxation time of in vivo and hFTH can be used as a MR reporter gene to track dividing and differentiating stem cells in the beating heart while simultaneously monitoring cardiac morpho-functional changes.This study was supported by a grant of the Korean Health Technology R&D Project, Ministry of Health & Welfare, Republic of Korea (No.A100131) and partly by the National Research Foundation of Korea (NRF) grant funded by the Korean government (MEST) .


Chronic progressive external ophthalmoplegia (CPEO) is a classical mitochondrial ocular disorder characterised by bilateral progressive ptosis and ophthalmoplegia. These ocular features can develop either in isolation or in association with other prominent neurological deficits (CPEO+). Molecularly, CPEO can be classified into two distinct genetic subgroups depending on whether patients harbour single, large-scale mitochondrial DNA (mtDNA) deletions or multiple mtDNA deletions secondary to a nuclear mutation disrupting mtDNA replication or repair. The aim of this magnetic resonance imaging (MRI) study was to investigate whether the ophthalmoplegia in CPEO is primarily myopathic in origin or whether there is evidence of contributory supranuclear pathway dysfunction.POLG, PEO1, OPA1, and RRM2B. All subjects underwent a standardised brain and orbital MRI protocol, together with proton magnetic resonance spectroscopy in two voxels located within the parietal white matter and the brainstem.Ten age-matched normal controls and twenty patients with CPEO were recruited nine patients with single, large-scale mtDNA deletions and eleven patients with multiple mtDNA deletions secondary to mutations in There was evidence of significant extraocular muscle atrophy in patients with single or multiple mtDNA deletions compared with controls. There was no significant difference in metabolite concentrations between the patient and control groups in both the parietal white matter and brainstem voxels. Volumetric brain measurements revealed marked cortical and cerebellar atrophy among patients with CPEO+ phenotypes.The results of this study support a primary myopathic aetiology for the progressive limitation of eye movements that develops in CPEO. Chronic progressive external ophthalmoplegia (CPEO) is a slowly progressive extraocular muscle disorder characterised by bilateral, usually symmetrical, limitation of eye movements and ptosis The aim of this magnetic resonance imaging (MRI) study was to investigate whether the limitation of eye movements in CPEO is myopathic in origin or whether there is evidence of contributory brainstem dysfunction with magnetic resonance spectroscopy (MRS). Brain volumetric measurements were also performed to assess the extent of central nervous system involvement and whether this correlated with the development of extraocular neurological features in patients with CPEO+ phenotypes.The study cohort included: (i) nine patients with single, large-scale mtDNA deletions  = 3.0 years), (ii) eleven patients with multiple mtDNA deletions , and (iii) ten age-matched normal controls  Table 1.POLG mutations (n = 4) PEO1 mutations (n = 3) OPA1 mutations (n = 1) RRM2B mutations (n = 1) c oxidase-deficient fibres in addition to prominent ragged red fibres.All patients were assessed by an experienced multi-disciplinary team of neurologists and ophthalmologists to define the clinical phenotype and disease severity Table 1.Text S1), with a high degree of intra- and inter-observer reliability (Table S1).MRI and MRS data were acquired on a 3-Tesla Philips Achieva clinical MR system using an 8-channel head coil. Proton MRS analysis was performed by an experienced research physicist (FES) to derive brain metabolite concentrations in the parietal white matter and brainstem regions: (i) choline; (ii) creatine; (iii) total glutamate and glutamine (Glx); (iv) myo-inositol; and (v) N-acetyl-aspartate (NAA). Extraocular and brain compartment volumes were measured using standardised semi-automated segmentation protocols . Group comparisons were made using the unpaired t-test. Intra-observer and inter-observer variability for the measurement of extraocular muscle and brain volumes was assessed with Pearson correlation coefficient (r). and  and There was a significant reduction in extraocular muscle volumes in the CPEO group compared with normal controls for all four recti muscles Figure 1There was no significant difference in metabolite concentrations between the patient and control groups in both the parietal white matter and brainstem voxels Table 4.Carlow and colleagues observed a significant reduction in extraocular muscle volumes for the inferior rectus, medial rectus and lateral rectus Ortube and colleagues measured extraocular muscle volumes in five patients with a clinical diagnosis of CPEO Ortube and colleagues, including the relatively small number of patients that were recruited and the lack of a confirmatory molecular diagnosis. Furthermore, only a relatively small portion of the extraocular muscle belly was sampled, which could have underestimated the degree of atrophy in the recti muscles.This study has shown clear evidence of significant extraocular muscle atrophy involving all four recti muscles in CPEO. This striking observation was noted for both patients with single, large-scale mtDNA deletions and for those harbouring multiple mtDNA deletions secondary to an underlying nuclear genetic defect. In a previous report of nine patients with CPEO, Our study is the first to demonstrate objectively that the extent of extraocular muscle atrophy in CPEO is highly symmetrical between the two eyes of the same patient, reflecting the ophthalmoplegia pattern observed clinically A variable decrease in N-acetyl-aspartate (NAA) has previously been reported in the brain of patients with the Kearns-Sayre syndrome – a particularly severe clinical phenotype characterised by the development of CPEO and pigmentary retinopathy before the age of twenty years, often in association with progressive cardiac conduction block Patients with CPEO+ features had significantly reduced total grey matter and cerebellar volumes compared with controls, in keeping with the higher burden of neurological disease in this specific patient population. The prominent degree of atrophy seen in the cerebellum further highlights the particular vulnerability of this specific brain region to the deleterious consequences of mtDNA defects This comprehensive magnetic resonance study of patients with molecularly confirmed CPEO has revealed a number of important findings that are directly relevant to our understanding of the underlying pathophysiology in this mitochondrial ocular disorder. In addition to marked generalised extraocular muscle atrophy, there was clear evidence of significant CNS involvement in this disorder, which becomes clinically manifest in a subgroup of patients.Figure S1Voxel placements and proton magnetic resonance spectra.(PDF)Click here for additional data file.Figure S2Boundary delineation of extraocular muscle cross-sections.(PDF)Click here for additional data file.Figure S3Measurement protocol for brainstem and cerebellar volumes.(PDF)Click here for additional data file.Table S1Validation of extraocular muscle and brain volume measurements.(DOC)Click here for additional data file.Text S1Supplementary methods.(DOC)Click here for additional data file.


Malaria prevalence has recently declined markedly in many parts of Tanzania and other sub-Saharan African countries due to scaling-up of control interventions including more efficient treatment regimens (e.g. artemisinin-based combination therapy) and insecticide-treated bed nets. Although continued molecular surveillance of malaria parasites is important to early identify emerging anti-malarial drug resistance, it is becoming increasingly difficult to obtain parasite samples from ongoing studies, such as routine drug efficacy trials. To explore other sources of parasite DNA, this study was conducted to examine if sufficient DNA could be successfully extracted from malaria rapid diagnostic tests (RDTs), used and collected as part of routine case management services in health facilities, and thus forming the basis for molecular analyses, surveillance and quality control (QC) testing of RDTs.Plasmodium species detection. Additionally, 165 archived RDTs obtained from ongoing malaria studies were analysed to determine the amplification success and test applicability of RDT for QC testing.One hyper-parasitaemic blood sample  was serially diluted in triplicates with whole blood and blotted on RDTs. DNA was extracted from the RDT dilution series, either immediately or after storage for one month at room temperature. The extracted DNA was amplified using a nested PCR method for DNA was successfully extracted and amplified from the three sets of RDT dilution series and the minimum detection limit of PCR was <1 asexual parasite/μl. DNA was also successfully amplified from (1) 70/71 (98.6%) archived positive RDTs (RDTs and microscopy positive) (2) 52/63 (82.5%) false negative RDTs (negative by RDTs but positive by microscopy) and (3) 4/24 (16.7%) false positive RDTs (positive by RDTs but negative by microscopy). Finally, 7(100%) negative RDTs (negative by RDTs and microscopy) were also negative by PCR.This study showed that DNA extracted from archived RDTs can be successfully amplified by PCR and used for detection of malaria parasites. Since Tanzania is planning to introduce RDTs in all health facilities , availability of archived RDTs will provide an alternative source of DNA for genetic studies such as continued surveillance of parasite resistance to anti-malarial drugs. The DNA obtained from RDTs can also be used for QC testing by detecting malaria parasites using PCR in places without facilities for microscopy. Malaria has for a long time remained a major cause of morbidity and mortality in most endemic countries particularly in sub-Saharan Africa. However, recent reports have indicated that malaria prevalence has declined drastically in some endemic countries -4. The dFollowing changes of malaria treatment guidelines in Tanzania in 2006, by introducing an ACT containing artemether and lumefantrine (ALu) ,8. HowevPlasmodium falciparum specific histidine rich protein 2 (PfHRP-2), Plasmodium lactate dehydrogenase (pLDH) or aldolase  districts of Tanga region, Tanzania.This study was conducted at Amani Biomedical Research Laboratory (AMBRELA) of the National Institute for Medical Research (NIMR) at Tanga Centre in Tanga region, northeastern Tanzania. Archived RDTs were obtained from ongoing studies in Muheza  and Korogwe . Other 94 RDT samples (on ParaHIT®f), included 24 RDT false positive (positive by RDT but negative by microscopy), 63 false negatives (negative by RDTs but positive by microscopy) and seven negative samples (negative by both RDTs and microcopy) obtained from a cross-sectional survey conducted in May 2010 in four villages in Muheza district where high level of false positive and negative RDT results were observed in previous surveys . All 165 RDT samples were stored at room temperature in the field or laboratory for a duration of 6 - 95 days (Table The archived RDT samples included 71 RDTs (positive for both RDT and microcopy) collected at health facilities in Muheza and Korogwe districts and stored without any preservatives at room temperature (temperature ranging from 25 - 34°C). Out of these, 26 RDTs were obtained from patients enrolled in InterACT project in Muheza  were ParaHIT®f (Table ®f), which failed to amplify by PCR, had a low parasitaemia detected by microscopy . Fifty-two (82.5%) of the 63 false negative RDTs  were positive by PCR. Eleven (17.4%) of the 63 false negative RDT samples which failed to amplify by PCR had low parasite density detected by microscopy .Table ®f Table . One posPfdhfr gene as a marker of pyrimethamine resistance using DNA extracted from RDTs was also successful and the results were similar to those observed when DNA was obtained from other sources such as cultured parasites or filter papers.Detection of microscopically negative samples was equally successful whereby, 20 out of 24 (83.3%) RDT false positive samples (which were positive by RDTs but negative by microscopy) were confirmed to be negative by PCR. Fourteen out of the 20 RDT false positive samples with negative PCR results were obtained from individuals who had taken anti-malarials (within seven days) before the survey. All seven negative RDTs (negative by both RDTs and microscopy) were also negative by PCR Table . Detectiet al, personal communication) and most likely, the decline is apparent in other parts of Tanzania as well . However, because of this current positive trend, recruitment of malaria positive patients for various studies, such as malaria drug efficacy trials has proved to be difficult. Since the region has been known as a hotspot for the emergence of anti-malarial drug resistance in Eastern Africa [Previously, malaria has consecutively been shown to be the main cause of morbidity and mortality in Tanga region, as for the rest of Tanzania -31. Howen Africa -36, it iIn this context, the study presented here has shown that archived RDTs used for malaria diagnosis in routine practice can be used as an alternative source of DNA for detection of malaria parasites and other molecular analyses. The RDTs can also be used as source of DNA as part of QC testing of RDTs instead of microscopic examination of Giemsa-stained blood smears, by comparing RTD results produced by health workers with those obtained in the laboratory based on detection of malaria parasite by PCR. Thus, after deployment of RDTs in the field, archived RDTs can also be used for QC of RDTs in addition to using Giemsa-stained blood smears, particularly in health facilities without capacity for performing malaria diagnosis by microscopy or lacking the skills and experience to prepare good blood smears as recently shown in Eastern Tanzania by McMorrow and colleagues ,25.Storage of RDTs (that were prepared in the laboratory) for one month either with or without silica gel as preservatives did not affect the quality of extracted DNA as determined by the detection limit of PCR in the diluted samples and band intensity of amplified products in comparison to known standard DNA samples which were used as control. Similarly, field samples stored at room temperature without preservatives for over 3 months were equally amplified. However, the impact of long term storage of RDTs beyond this duration on the quantity and quality of DNA is not clearly known, particularly if they are stored at room temperature without preservatives and under high ambient temperature, and humidity (as in most parts of Tanzania) which support growth of moulds that can degrade the parasite DNA. The period of three months or more shown in this and a previous study  should b®f and Paracheck Pf®) examined provided DNA of sufficient quality with similar amplification success although the quantity of DNA was not assessed. This is in concordance to a previous study which showed that the type/brand of RDTs did not affect the quality of extracted DNA [Both types of PfHRP-2 based RDTs (ParaHITcted DNA . Furthercted DNA . Among tcted DNA .This study showed that DNA can be extracted from routinely used and archived RDTs, and successfully amplified by PCR; thus providing an alternative source of DNA for detection of malaria parasites and possibly other molecular analyses such as surveillance of parasite resistance to anti-malarial drugs and monitoring of insidious parasite reservoirs in different interventions, such as malaria elimination programmes. Furthermore, the RDTs can be used for QC testing of RDTs deployed in the field for malaria diagnosis based on comparing the results obtained by PCR genotyping of DNA extracted from archived RDTs with the test results produced by health workers, particularly from remotely located health facilities without the capacity of microscopic examination of blood smears. Thus, availability of archived RDTs after the tests are deployed in all health facilities in Tanzania as planned by the Ministry of Health and Social Welfare will provide a good source of parasite DNA for laboratory studies.Plasmodium falciparum; PfHRP-2: Plasmodium falciparum histidine rich protein 2; RDTs: Rapid diagnostic tests; WBCs: white blood cells; WHO: World Health Organisation; μl: microlitre.ACT: artemisinin combination therapy; DNA: deoxyribonucleic acid; pLDH: Plasmodium lactate dehydrogenase; NIMR: National Institute for Medical Research; NMCP: National Malaria Control Programme; PCR: polymerase chain reaction; Pf: The authors declare that they have no competing interests.DSI, MA conceived of the study, designed the experiments, supervised the laboratory analyses and wrote the manuscript. NN, OP and LV participated in study design and supplied archived RDTs while SL and RAM conducted the laboratory analyses. MML and ICB participated in study design and supervised the study. All authors read and approved the manuscript.


In contrast to the RNA viruses, the genome of large DNA viruses such as herpesviruses have been considered to be relatively stable. Intra-specific recombination has been proposed as an important, but underestimated, driving force in herpesvirus evolution. Recently, two distinct field strains of infectious laryngotracheitis virus (ILTV) have been shown to have arisen from independent recombination events between different commercial ILTV vaccines. In this study we sequenced the genomes of additional ILTV strains and also utilized other recently updated complete genome sequences of ILTV to confirm the existence of a number of ILTV recombinants in nature. Multiple recombination events were detected in the unique long and repeat regions of the genome, but not in the unique short region. Most recombinants contained a pair of crossover points between two distinct lineages of ILTV, corresponding to the European origin and the Australian origin vaccine strains of ILTV. These results suggest that there are two distinct genotypic lineages of ILTV and that these commonly recombine in the field. Herpesviridae have genomes ranging in size from 124 to 241 kbp Alpha-, Beta- and Gammaherpesvirinae. Classically, sequences obtained from a single gene or multiple concatenated genes have been used for phylogenetic analyses of herpesviruses. More recently developments in high-throughput sequencing techniques have resulted in the availability of complete genome sequences for comparative complete genomic analyses of herpesviruses Herpesviruses are highly host-specific double stranded DNA viruses that have been isolated from most animal species, including mammals, birds, reptiles and fish. The Alphaherpesvirinae (genus Iltovirus) and causes acute upper respiratory tract disease in chickens Iltovirus genus (including psittacid herpesvirus 1 (PsHV1) and ILTV) separated from the mammalian alphaherpesviruses around 200 million years (Mys) ago, in agreement with the theory that herpesviruses have co-evolved with their hosts AlphaherpesvirinaeInfectious laryngotracheitis virus  belongs to the subfamily In Australia three commercial live ILTV vaccines  are registered for use. The SA2 vaccine strain  is an attenuated ILTV field strain isolated from South Australia in 1950 Australian ILTV field strains and vaccine strains have been classified into nine genotypes (classes 1 to 9) using polymerase chain reaction and restriction fragment length polymorphism (PCR-RFLP) analysis th century) In a previous report we analysed and compared the complete genome sequences of two Australian origin ILTV vaccines and a European origin ILTV vaccine strain. The genome sequences of the two Australian origin vaccines differed by only 23 nucleotide substitutions, even though one of the strains was derived from the other after more than twenty-five serial passages in cell culture and embryonated eggs. Although similar to each other, these two Australian vaccine strains showed considerable divergence (0.8% nucleotide difference) from the genomic sequence of the European origin ILTV vaccine strain. This is higher than the 0.1–0.5% nucleotide difference reported between the most distantly related strains of varicella zoster viruses (VZV), which is a human alphaherpesvirus with the same genomic sequence arrangement as ILTV The aim of this study was to better understand the molecular epidemiology of ILTV by analysing a broader range of ILTV sequence data, including currently available complete or partial genomic sequences of ILTV. The complete genome sequences of two Australian ILTV field strains (V1-99 and CSW-1) were determined using high-throughput sequencing and included in these analyses. Additionally, we aimed to identify the origins of the parent strain of the Australian ILTV vaccines by searching for similar genotypes among publically available nucleotide sequences from ILTV strains isolated in other countries.de novo and mapped to the complete genome sequence of the Serva vaccine strain. Sequence gaps were filled and ambiguous sequences clarified using Sanger sequencing. The size of the ILTV genomes ranges from 150,335 to 153,952 bp . The size of the strain V1-99 genome was 153,631 bp, similar to that of the Serva strain . The V1-99 and Serva strains shared 99.6% nucleotide sequence identity. The CSW-1 strain genome was somewhat smaller  due to three large deletions within the unique long region  and both the internal and terminal repeat regions (463 bp) (Sequencing data from the V1-99 and CSW-1 strains of ILTV obtained by high-throughput sequencing were assembled (463 bp) . Excludi(463 bp) .In order to further understand the relationship between ILTV vaccine strains and Australian field strains of ILTV, complete genome sequence alignments were performed and phylogenetic trees were inferred using the six complete genomic sequences of ILTV currently available. This analysis included a European origin vaccine (Serva), an Australian origin vaccine (SA2), two Australian field strains (V1-99 and CSW-1) and two strains from the USA (63140 and USDAref). The sequences were examined in order to identify relevant genes that could be used to differentiate between all six strains .All predicted ORFs were extracted from the complete genome alignments and separate phylogenetic trees were inferred from the nucleotide sequences of each ORF. Similarly, alignments of amino acid sequences for all the predicted ORFs were performed separately and phylogenetic trees were inferred. The UL21, 32, 34 and 43 genes of all six ILTV strains were found to share 100% nucleotide and amino acid sequence identity. The UL3, 4, 13, 14, 18, 24, 31, 33, 40 and 49 genes of the six strains had differing nucleotide sequences, but were predicted to have 100% amino acid sequence identity. The ICP4, UL27, UL36, US5 and US8 genes showed the most variability within the six strains of ILTV and the six strains could be distinguished using the nucleotide and/or amino acid sequences of these genes . The UL2All available nucleotide sequences for the ICP4 and UL27 genes of ILTV from the NCBI database were aligned and phylogenetic analyses were performed . The EurTo confirm the close relationship between the Australian origin vaccine strain and strains isolated from backyard flocks in the USA, concatenated nucleotide and amino acid sequences were generated using all available sequences for each of these strains. This included the complete sequences of the UL27, UL32 and UL10 genes from the unique long region, the UL47 and US4 genes from the unique short region, and the complete sequence of the ICP4 gene from the repeat region. Surprisingly, one of the strains isolated from backyard flocks in the USA (25/H/88/BCK) shared 100% amino acid sequence identity  with the concatenated sequence of the Australian origin vaccine. Another strain isolated from backyard flocks in the USA (12/D/02/BCK) differed in only two amino acid residues from the Australian origin vaccine strain . These rA possible mosaic pattern of relationship between the genome sequences of the Australian field strains of ILTV was detected in the phylogenetic analyses of the UL27 and ICP4 genes . In addiTo confirm the mosaic patterns of these relationships and detect the suspected recombination events responsible, we performed additional phylogenetic analyses on the nucleotide sequences of the UL47 and US4 genes . As expeIn order to define recombination crossover points, Bootscan analyses were performed using complete genome alignments of five ILTV strains . Bootscan analyses of the 1874C5 and CSW-1 strains revealed pairs of crossover points in the repeat region, while the 81658 strain had multiple tightly grouped pairs of crossover points in the unique long region . Most ofA history of recombination was not detected in the sequences located in the unique short region of the genome of the eight strains of ILTV for which complete genomic sequence was available, so the sequences of this region were used to investigate the evolutionary history of ILTV. In the resulting trees, all Australian strains of ILTV clustered together and separately from European and USA strains of ILTV . The timin vivo. In this study, we sought to perform broader phylogenetic analyses of ILTV in order to understand the evolution and molecular epidemiology of the virus. We found that the parent strains of Australian origin vaccine and Australian field strains may have been imported into Australia from the USA. Moreover, with the exception of field isolates derived from bird-to-bird passage of commercial vaccines, most field strains of ILTV isolated in Australia and in other geographic areas of the world show evidence of historical recombination events between two distinct viral lineages, represented by the Serva and SA2 strains.Genome wide comparative sequence analyses of closely related strains of ILTV, or of strains from specific geographical regions, have been reported previously The Australian origin SA2 vaccine was produced using an Australian field isolate that induced comparatively smaller pocks on the chorioallantoic membrane of embryonated hen eggs In order to search for an SA2-like strain of ILTV in other geographical areas, we compared the phylogenetic tree inferred from alignments of complete genome sequences with those inferred from alignments for all individual predicted ORFs. The ICP4, UL27, UL36, US5 and US8 genes were found to be sufficiently divergent to allow differentiation between distinct strains of ILTV . ComparaThe European origin Serva vaccine strain was recently registered in Australia and rapidly became widely used by the Australian poultry industry within a relatively short timeframe The Australian field strains V1-99 and CSW-1 and the USA field strain 874C5 shared a distinct sequence segment in the unique long region. This suggests that the Australian field strains and the 1874C5 strain may have shared the same (or very similar) parent strain(s) before recombination, or that these strains may have obtained this distinctive sequence segment from some other strain in independent recombination events.Evidence of historical intra-specific recombination in alphaherpesviruses has been described previously With the exception of the USDAref and 81658 ILTV strains, which each contained three recombination crossover points, all other ILTV recombinants contained a single pair of recombination crossover points between the Serva-like and the SA2-like parental strains, as detected by bootscan analyses. Recombination networks further supported these findings, confirming that recombination events occurred in the unique long and the repeat regions, but not in the unique short region  and 7. TIn conclusion, the origins of Australian strains of ILTV have been determined using complete genomic sequence analyses of multiple strains of ILTV and wider phylogenetic analyses. Moreover, evidence for significant recombination events in most field strains of ILTV was identified by recombination network and bootscan analyses. We found that the Serva-like and SA2-like lineages of ILTV have recombined frequently over the history of ILTV evolution. This, taken together with data from our previous studies Pock purified V1-99 (six passages from isolation) and a laboratory stock of CSW-1 ILTV (approximately twenty passages from isolation) de novo assembled contigs and to produce a consensus sequence using the Serva ILTV genomic sequence (Genbank accession HQ_630064) as reference. Any sequence gaps or ambiguous regions of sequence were confirmed by Sanger sequencing using BDT version 3.1 (Applied Biosystems). The complete genome sequences of the V1-99 and CSW-1 strains of ILTV have been deposited in the NCBI GenBank database under the accession numbers JX646898 and JX646899.Total viral genomic DNA preparation, high-throughput sequencing and genome assembly were performed as previously described Complete genome sequences, nucleotide sequences and amino acid sequences used in this study are summarized in To detect evidence of historical recombination events between ILTV strains, recombination networks were generated using SplitsTree 4 To estimate the time points of divergence between ILTV strains, the Minimum Evolution Table S1ILTV genome and gene sequences used in this study.(DOC)Click here for additional data file.