This Personal Account describes the author's involvement in the field of microwave‐assisted organic synthesis (MAOS) from the late 1990’s starting out with kitchen microwave ovens right through to the development of a reactor in 2016 that – although not using microwave technology – in many ways mimics the performance of a modern laboratory microwave. The reader is taken along a journey that has spanned two decades of intense research on various aspects of microwave chemistry, and, at the same time, was intimately linked to key innovations regarding equipment design and development. A “behind the scenes” approach is taken in this article to share – from a very personal point of view – how specific projects and research ideas were conceived and developed in my research group, and how in general the field of microwave chemistry has progressed in the last two decades. Since my research activities have shifted away from microwave chemistry some time ago, my first instinct was to decline. On second thoughts I found the opportunity to reflect on “my twenty years” in the field of microwave chemistry quite appealing and I ultimately accepted the invitation. This was perhaps motivated, at least in part, by the fact that one of my postdoc advisors, Albert Padwa, had written an inspirational Perspectives Article for The Journal of Organic Chemistry in 2009, where he most elegantly reflected on how and why his own research interests had progressed and changed quite significantly during the span of close to 50 years of research in organic synthesis.When I received the invitation from the Editors of why things happened the way they did, focusing more on a political “behind the scenes” perspective, and hopefully will be of interest and an entertaining read for the many “microwave chemists” that were active during that time period. As with most Personal Accounts of this nature, the contributions of other scientists in the field will only be mentioned where this is needed to understand the context of our own work. In order not to disrupt the flow of the manuscript too much many of my personal comments have been placed in the reference section and technical jargon is kept to a minimum so the manuscript can be easily followed by the non‐expert reader.The present article is therefore not a standard scientific review but rather a very Personal Account that describes how my research group got involved in specific research topics within the general area of microwave chemistry over a time period of roughly two decades. This somewhat nostalgic journey puts a strong emphasis on Upon reflecting on the many different turns our research in microwave chemistry has taken over the years I thought it would be most appropriate to divide this article into different sections based on the type of reactor technology that was used at the time. This also allows a more or less chronological and logical discussion of our research.22.1My involvement in the field of microwave chemistry started rather abruptly in the summer of 1998 and it was not a planned affair at all. At that time I had just completed the requirements for obtaining my “Habilitation”As my research involved heterocycles I decided to attend and to present a lecture on my results at a regional conference on heterocyclic chemistry in nearby Hungary.The lecture by Raj Varma in Hungary was an eye opener for me and I was intrigued by what appeared to be the quite significant potential of this new and enabling technology. I was keen to enter this field as soon as possible and to investigate, as a start, the Biginelli reaction under microwave conditions. I was therefore very pleased that Raj Varma agreed to collaborate with me, so soon after I came back from Hungary I enthusiastically started the first experiments at the University of Graz.Synthesis in October of 1999.After considering our options, we decided that the most straightforward approach would be to adapt a protocol for the Biginelli reaction using polyphosphate ester (PPE) as reaction mediator and dehydrating agent that we had published earlier that year using THF as solvent under reflux conditions.2.2cf. Figure In mid‐1999, I obtained a grant from an Austrian funding institution to purchase a dedicated microwave reactor with proper temperature and microwave power control, also featuring magnetic stirring.While I perhaps had expected a somewhat more “exciting” outcome from our first study using a dedicated microwave reactor, the MLS multimode instrument has subsequently been utilized in our group for many years, mainly in those instances where other microwave instruments that we acquired (see below) could not be used, i. e., for open vessel processing (involving solid‐phase organic synthesis in single and multiple vessels),The main limitation of the MLS ETHOS system was, however, that it was not designed for small scale (<5 mL) organic synthesis, but had been adapted from a microwave digestion unit. In microwave digestion the reaction mixture, typically a Brønsted acid, is almost always strongly microwave absorbing.33.1Early in the year 2000, I heard rumors that a company from Sweden would launch a new revolutionary microwave synthesizer. I knew that a group at Uppsala University  had published a series of articles since 1996 on high‐speed microwave chemistry involving transition‐metal catalyzed transformations.The mysterious new system, initially called “Smith Synthesizer” Figure a, was ulThe vision of Personal Chemistry however did go far beyond microwave chemistry itself. The philosophy under which the system was marketed was called Coherent Synthesis and incorporated reaction planning systems, automation, distributed system access, searchable databases and reaction optimization kits.Clearly, the microwave system and overall concept was designed having the pharmaceutical industry and drug discovery in mind. Nevertheless, the Smith Synthesizer appeared extremely attractive to me as it would allow us to run synthetic transformations on small scale (up to 5 mL reaction volume) under controlled temperature conditions and using some degree of automation. The hefty price tag made it obvious that no academic group could ever afford such an instrument.nd International Conference on Microwave Chemistry, in Antibes/Juan‐les‐Pins on the French Riviera and we both presented the results from our recently published work.In September of 2000, along with my PhD student Alexander Stadler, I attended the 2cf. Figure Journal of Combinatorial Chemistry.In order to evaluate the automation and robotic features of the Smith Synthesizer to their full potential we first set out to synthesize a library of dihydropyrimidines using – again – the venerable Biginelli reaction as model transformation utilizing ethanol as a solvent .The success of these early trials ultimately led to the launch of a multimode reactor specifically designed for larger scale organic synthesis named Synthos 3000 by Anton Paar in October of 2004 Figure .42 At th4In my opinion, the “single‐mode revolution” of 2000 has introduced what I call the “golden decade” of microwave‐assisted organic synthesis (2001–2010). The benefits of controlled microwave heating quickly became evident not only to synthetic organic chemists but in particular also to the medicinal chemistry/drug discovery community as outlined in a 2001 review article by Mats Larhed and Anders Hallberg.In March of 2001, US‐based CEM Corporation introduced a new type of single‐mode microwave reactor named Discover. Similar to MLS/Milestone and Anton Paar, CEM had been active for many years in microwave digestion and related fields mainly using multimode instruments.With the entry of CEM in 2001 and Anton Paar in 2004, there were now four microwave equipment vendors on the market that served the organic synthesis/medicinal chemistry communities, with applications subsequently extending into solid‐phase peptide synthesis, nanoparticle generation, polymer chemistry and many more related scientific disciplines. A market survey released in 2005 predicted a 15–20 % growth rate of the instrument market for synthesis and also forecasted that within 5–6 years the chemical synthesis segment would overtake the analytical segment.With more and more academic groups getting involved in microwave chemistry, the number of publications started to skyrocket. Starting in 2005, we introduced a monthly lunchtime journal club where each member of the group – being responsible for one or more publishing houses – presented selected examples of MAOS from the recently published literature. Notably, only those publications where controlled microwave chemistry experiments involving dedicated equipment with online temperature monitoring had been reported were included.Nature was publishing a news feature on microwave chemistry in 2003, Chemical & Engineering News and Chemistry World followed with articles in 2004 and in subsequent years many similar news stories touting the benefits of MAOS appeared in print and online.On the Gartner hype curve, I believe that the “peak of inflated expectations” in microwave chemistry was reached somewhere between 2003 and 2005. Parallel to what happened on the academic front, pharma massively invested in the technology as well following to some extent the Coherent Synthesis philosophy. In order to speed up their drug discovery operations facilities containing multiple microwave reactors were installed and coupled with suitable robotics, automation, and, in particular, work‐up and purification strategies on the back‐end Figure . Given tShort Course on Microwave‐assisted Organic Synthesis (MAOS) series beginning in September of 2003. These two day events proved to be very popular over the years as in addition to formal talks we included lab sessions where the participants  could see and evaluate microwave reactors from different instrument manufacturers  in different parts of the world to attend a meeting on microwave chemistry.55.1In 2005, in the midst of the golden decade on MAOS, things were running rather smoothly in my laboratory. We were able to develop a number of new interesting synthetic applications and the scientific output and international recognition of our research group in Graz was growing.Christian Doppler Laboratory for Microwave Chemistry (CDLMC) initially were Anton Paar and piCHEM, a local company dedicated to peptide synthesis. In subsequent years, ThalesNano (2007), Lonza (2009) and Microinnova (2010) joined the consortium, all interested in the scale‐up aspects of this technology. The substantial amount of funds made available through this grant meant a significant step forward for the group.In order to address these challenges I submitted a grant proposal to the Christian Doppler Research Association (CDG) in November of 2005, an Austrian funding agency which promotes the cooperation between universities and industry by supporting application‐oriented basic research in research units termed Christian Doppler Laboratories for up to seven years. Our industrial partners in this grant, officially termed the 5.2One of the first issues that we needed to address was how to properly measure reaction temperature in a microwave‐heated experiment. We were quite pleased with the accuracy of our early investigations back in 2001 using the internal temperature sensor of the MLS multimode system Figure a, that wIn the true spirit of the Christian Doppler grant on application‐oriented basic research these findings were eventually reflected in the design and development of the Monowave 300 instrument, a single‐mode microwave reactor launched by Anton Paar in June of 2009 Figure .775.3not connected to a macroscopic bulk temperature effect .at the same measured bulk reaction temperature.Having sorted out how to accurately determine reaction temperature in microwave‐assisted transformations we then moved on to investigate the existence of so‐called specific or non‐thermal microwave effects. This topic has always been controversial. Since the first published reports on the use of microwave irradiation to “accelerate” organic chemical transformations dating back to 1986,Angewandte Chemie published a short highlight article by Nikolai Kuhnert entitled “Microwave‐assisted reactions in organic synthesis‐are there any nonthermal microwave effects?”.Angewandte Chemie, as the conclusion presented in this article sharply contradicted what had been presented in a review on microwave effects published the same year.applying the exact same adequately measured reaction temperature profiles we have never found any effect that ultimately could not be rationalized by a purely bulk temperature phenomenon.In 2001, Angewandte Chemie on microwave effects strongly indicated that erroneous temperature measurement was involved in the experiments carried out by the Dudley group . This was in part a consequence of the availability of single‐mode microwave technology that allowed the safe processing of small reaction volumes under sealed vessel conditions by microwave irradiation. It appeared to me that for microwave‐assisted synthesis to be become a fully accepted technology in the future there was a need to develop larger scale techniques, that could ultimately routinely provide products on a multi kg (or even higher) scale. At that time, two different philosophies for microwave synthesis on larger scale (>100 mL processing volume) had emerged. While some groups have employed larger batch‐type multimode reactors , others have used continuous flow techniques using single‐ and multimode instruments to overcome the inherent problems associated with microwave chemistry scale‐up.In 2007, ThalesNano joined the CDLMC, followed by Lonza in 2009 and Microinnova in 2010. All three companies had a strong interest in continuous flow chemistry, ThalesNano from the standpoint of developing reactors for labscale use, Lonza and Microinnova in the context of large‐scale manufacturing. The fast reactions possible under sealed vessel microwave batch conditions appeared extremely attractive from the manufacturing perspective, leading to potentially very high throughputs in properly designed high‐temperature flow reactors. The question that needed to be addressed first was: is there any benefit from using microwave heating for flow chemistry applications? We had experimented with our two CEM flow microwave reactors for some time,cf. Figure cf. Figure At the same time, as we went up in scale we also thought about miniaturization and parallelization, again a leftover of my longstanding interest in combinatorial chemistry. As outlined above using single‐mode processing aided by robotics proved extremely valuable for synthetic chemists both in academia and industry  ceramic material. Similar to the use of this material in a single‐mode microwave device,r Figure .98 Notabr Figure b allowed5.5Account of Chemical Research article, I further posed the question:After having performed a shear endless number of comparison experiments between microwave and conventionally heated reactions over the years,In 2009, we had introduced a 10 mL reaction vessel made out of silicon carbide (SiC) for use in single‐mode microwave reactors. disappeared from the market. Browsing through the relevant book chapters and reviews on microwave equipment,Nature in 2003 I had stated: “In 10–15 years, we will see a microwave reactor in every academic and industrial laboratory.”So the question I ask myself is: has MAOS failed? In the interview with cf. Figure st century.”Whatever the reasons, while I would not say that microwave chemistry has failed, the acceptance and penetration of this technology throughout the scientific community has been lower than expected. Although for discovery‐type operations in the pharmaceutical industry microwave chemistry has been fully embraced , I was forced to acquaint myself with scientific areas that were very new to me. Starting this journey in 1998, I could have never predicted that years later I would publish scientific articles on synthesizing inorganic nanocrystals, polymer synthesis, proteolytic digests, and on the extraction of caffeine from Nespresso capsules (in the context of forensic investigations we have even made heroin in a microwave).cf. Section 2.1.)? Would I have still started a career in microwave chemistry? Or would I have turned to something different? Obviously, I will never know the answer to this question but I do know that I have very much enjoyed “my twenty years in microwave chemistry”.The question that comes to my mind in looking back at the very beginnings of my involvement in microwave chemistry is: what would have happened if I had not attended the 1998 conference in Eger,C. Oliver Kappe is Professor of Chemistry at the University of Graz (Austria) and Scientific Director of the Center for Continuous Flow Synthesis and Processing (CC FLOW) at the Research Center Pharmaceutical Engineering GmbH (RCPE). He received his doctoral degree in organic chemistry from the University of Graz.. After periods of postdoctoral research work on reactive intermediates and matrix isolation spectroscopy with Curt Wentrup at the University of Queensland in Brisbane, Australia (1993–1994) and on synthetic methodology/alkaloid synthesis with Albert Padwa at Emory University in Atlanta, USA (1994–1996), he moved back to the University of Graz in 1996 to start his independent academic career. After a 20 year career in microwave chemistry his current research interests involve continuous‐flow chemistry, API manufacturing, and process intensification technologies. Since 2011 he has been Editor‐in‐Chief of the Journal of Flow Chemistry.As a service to our authors and readers, this journal provides supporting information supplied by the authors. Such materials are peer reviewed and may be re‐organized for online delivery, but are not copy‐edited or typeset. Technical support issues arising from supporting information (other than missing files) should be addressed to the authors.SupplementaryClick here for additional data file.


The presence of extraintestinal manifestations may be associated with the development of pouchitis in patients with ulcerative colitis after ileal pouch–anal anastomosis. The aim of this study was to assess this correlation.A systematic literature search was performed using MEDLINE and the Cochrane Library. Studies published in English up to 22 May 2017 investigating the association between extraintestinal manifestations and development of pouchitis in adults with ulcerative colitis were included. Case reports were excluded. The association of extraintestinal manifestations with the development of overall and chronic pouchitis was investigated using a random‐effects model.P = 0·001) and overall pouchitis .Of 1010 citations identified, 22 observational studies comprising 5128 patients were selected for analysis. The presence of extraintestinal manifestations was significantly associated with both chronic pouchitis (odds ratio 2·28, 95 per cent c.i. 1·57 to 3·32; The presence of extraintestinal manifestations is associated with development of pouchitis after ileal pouch–anal anastomosis. Several treatment options are currently available for patients with ulcerative colitisPouchitis is an idiopathic inflammation of the ileal pouch. First‐line treatment for pouchitis is antibiotics such as metronidazole and ciprofloxacinSeveral risk factors for pouchitis have been reported in small retrospective cohorts; they include the presence of extraintestinal manifestations (EIMs)This systematic review and meta‐analysis was conducted according to PRISMA guidelinesOvid MEDLINE and Cochrane Library databases were searched systematically to identify relevant articles published to May 2017. Studies investigating preoperative risk factors for the development of pouchitis were searched for using the search terms ‘ulcerative colitis’ and ‘pouchitis’ or ‘pouch surgery’ or ‘ileal pouch anal anastomosis’ or ‘IPAA’ and ‘complication’.The search was limited to RCTs and observational studies written in English. Studies with information on the presence of EIMs in relation to the development of pouchitis were included. Studies on familial adenomatous polyposis were excluded. The diagnostic criteria of pouchitis are essential, and it is recommendedPSC is one of the major EIMs in Western countries, and its association with pouchitis is the most frequently investigated among all EIMs. Thus, an additional attempt was made to elucidate the association of PSC and pouchitis.Two authors independently checked the titles and abstracts to exclude irrelevant studies and retrieve relevant articles. In the case of multiple publications from one institution, only studies for which the study periods did not overlap with one another were included, to avoid the duplication of cases. The reference lists of included articles were screened for additional studies.Data extraction was performed independently by two authors, and a third author revised and finalized any disagreements. The final data were presented in Excel®  format for statistical analysis. The corresponding author of one articlehttps://www.R‐project.org/) was used for statistical analysisP < 0·050 was considered statistically significant for the pooled OR. Heterogeneity was calculated using I2 values, and evaluated according to the method of Higgins et al.P < 0·100 was considered statistically significant for heterogeneity. To assess publication bias, funnel plots were created when ten or more studies were included, according to the recommendations of Sterne and colleaguesEZR (Easy R)® for the R free software environment for statistical computing and graphics Table S2, supporting information)Of 1010 citations identified, 22 articles were included .The definition of chronic pouchitis was diverse, with possible heterogeneity. EIMs included in each study varied from study to study. Most studies, however, included immune‐related EIMs such as joint disease , skin diseases (pyoderma gangrenosum and erythema nodosum), liver disease (PSC) and eye diseases (uveitis and episcleritis). In addition, some included thrombotic disorders and aphthous stomatitis. Regarding the presence of EIMs in relation to the timing of IPAA, 11 studies included exclusively preoperative EIMs, four included both preoperative and postoperative EIMs, and seven did not provide this information. The risk of bias of individual studies was assessed according to the Risk of Bias Assessment Tool for Nonrandomized Studies (RoBANS)Fig. The effect of EIMs on overall pouchitis was analysed using 11 papers comprising 2194 patients who received IPAA. Funnel and forest plots for overall pouchitis are shown in P < 0·001) with low heterogeneity .An integrated analysis showed that the presence of EIMs was significantly associated with overall pouchitis , with low heterogeneity .The association of EIM with chronic pouchitis was analysed using nine papers comprising 2222 patients. The forest plot for the effect of EIMs on chronic pouchitis is shown in Fig. P < 0·001), but with statistically significant heterogeneity .Two papers provided detailed and sufficient data on PSC of the 13 articles in the analysis of the effect of EIMs on the development of overall pouchitis. Eight other articles investigating the association of PSC with pouchitis were included. These ten publications comprised 2877 patients with ulcerative colitis who underwent IPAA. Funnel and forest plots for the effect of PSC on overall pouchitis are shown in Fig. P = 0·017), with considerable heterogeneity .Six papers comprising 1179 patients were included to assess the association between PSC and chronic pouchitis. The forest plot for the effect of PSC on chronic pouchitis is shown in Both overall and chronic pouchitis occurred significantly more often when EIMs were present. Patients with EIMs may benefit from preoperative and postoperative counselling on the need for long‐term follow‐up. Some RCTsThe authors have reported the association of EIMs and development of both overall and chronic pouchitis previouslyPSC was also associated with the development of pouchitis. Regarding other EIMs, such as arthritis and pyoderma gangrenosum, only a few publications have investigated the association with pouchitis, so their data could not be integrated. Although PSC is one of the most frequently occurring EIMs in Western countries, the incidence of PSC is reported to be lower in JapanIt should be stressed that diagnostic criteria for pouchitis are essential for the precise elucidation of the risk of pouchitis, because symptoms did not necessarily correlate with endoscopic and histological findings in the pouchThere were several limitations of this study. The design of all included studies was observational. Selection bias and heterogeneity is therefore inherently present. In particular, heterogeneity was statistically significant in the analysis of PSC. Several RCTs investigated the preventive effectiveness of medications such as probiotics, but data on the presence of EIMs were not detailed. The authors could not access Embase for this meta‐analysis, which might have captured some relevant publications. Most included papers used logistic rather than Cox regression in multivariable analysis. As the event of pouchitis occurred over time, it would have been better if time had been taken into account and expressed as a hazard ratio. The observation period in most studies, however, was long enough with an average of 5–10 years. The diagnosis of chronic pouchitis was not uniform among the studies. Although most papers adopted long‐term antibiotic use (more than 4 weeks) or unresponsiveness to antibiotic treatment as the definition of chronic pouchitis, the definition differed slightly from study to study.Table S1 Characteristics of included studies for extraintestinal manifestations and pouchitisTable S2 Characteristics of included studies for primary sclerosing cholangitis and pouchitisTable S3 Risk of bias of individual studies according to the RoBANSClick here for additional data file.


Systematic reviews with economic components are important decision tools for stakeholders seeking to evaluate technologies, such as breast cancer screening (BCS) programs. This overview of systematic reviews explores the determinants of the cost‐effectiveness of BCS and assesses the quality of secondary evidence. The search identified 30 systematic reviews that reported on the determinants of the cost‐effectiveness of BCS, including the costs of breast cancer and BCS. While the quality of the reviews varied widely, only four out of 30 papers were considered to be of a high quality. We did not identify publication bias in the original evidence on the cost‐effectiveness of mammography screening; however, we highlight a need for improved clarity in both reporting and data verification. The reviews consisted mainly of studies from high‐income countries. Breast cancer costs varied widely among the studies. Factors leading to higher costs included: time (diagnosis and last months before death), later stage or metastases, recurrence of the disease, age below 64 years and type of follow‐up . Overall, screening with mammography was considered cost‐effective in the age range 50‐69 years in Western European and Northern American countries but not for older or younger women. Its cost‐effectiveness was questionable for low‐income settings and Asia. Mammography screening was more cost‐effective with biennial screening compared to annual screening and single reading using computer‐aided detection vs double reading. No information on the cost‐effectiveness of ultrasonography was found, and there is much uncertainty on the cost‐effectiveness of CBE because of methodological limitations. Multiple reviews report on the determinants of the cost‐effectiveness of breast cancer screening. However, only a few of these are of high quality. These reviews suggest that while the cost‐effectiveness of screening mammography is favorable in general, it is impacted by population , screening , and setting  characteristics. The cost‐effectiveness of ultrasonography and clinical breast examination is questionable. No risk of publication bias in the original evidence was identified, though high variability and uncertainty in the original and secondary evidence may limit the value of these reviews. Cost‐effectiveness analysis is a comparative method, which combines relative costs and outcomes of different interventions into a single metric—the cost‐effectiveness ratio (CER) or incremental cost‐effectiveness ratio (ICER). The most frequently used outcome measures in cost‐effectiveness assessment of chronic illnesses, such as breast cancer, are quality‐adjusted life years  or disability‐adjusted life years . Depending on the viewpoint of the stakeholders, cost‐effectiveness analysis can consider a variety of costs, such as direct costs  and indirect costs (productivity loss for patient and caregiver). While multiple approaches to interpreting cost‐effectiveness exist,The cost‐effectiveness of BCS programs is dependent on economic and healthcare system settings as well as the methodological approaches toward evaluation. The factors that would affect the cost‐effectiveness of BCS in populations would include patient characteristics and epidemiological factors, screening accuracy, coverage and screening uptake, access to diagnosis and treatment, and costs of both breast cancer and implementation of screening programs. While multiple reviews on economic evaluations around BCS have been published over the last decades, no research to our knowledge has summarized these reviews’ findings on the cost‐effectiveness of the screening programs. In the current overview, we aim to explore determinants of the cost‐effectiveness of BCS according to existing systematic reviews.2The design of this study was reported in the published protocol, available open‐access online,The quality of the included reviews was assessed by using the Assessing the Methodological Quality of Systematic Reviews (AMSTAR) checklistTo analyze publication bias in the original evidence, we assessed the distribution of ICER per outcomes expressed in life years gained, QALYs, or DALYs in the reviews reporting on the cost‐effectiveness of screening mammography comparing to no screening. Similar to Bell et al 2006),06,20 we 3We identified 7768 abstracts through our database search, with 451 more gray literature sources reviewed  identified more than a 50% increase in cost of invitation for those women requiring a follow‐up reminder to come for screening.While mammography was a target intervention in most of the reviews, three publications reported BCS costs for the other screening approaches Table . Zelle e3.3The quality of the included reviews ranged from 1 to 7 (from a maximum possible of 9 in AMSTAR), of which 25 reviews had a quality score of three or above and were included in the data synthesis (Table P < .05), year of the search (P < .1), and reporting cost‐effectiveness parameters vs only cost parameters (P = .174). The model predicted 47% of variance in the AMSTAR score with residual standard error of 1.133 on 23 degrees of freedom.From the relevant AMSTAR criteria, most systematic reviews had a comprehensive literature search (81%) and reported study characteristics (84%). Only three reviews (10%) reported the conflict of interest for included original research, and only five (17%) reported having a protocol Figure . A stepwFour systematic reviews on the cost‐effectiveness of BCS in comparison to no screening reported the outcomes from 67 studies in total , patients’ characteristics , health provider characteristics , and health system characteristics .The cost and cost‐effectiveness outcomes related to screening were influenced by population characteristics , screening organization , and disease characteristics .The results of this review have a dual‐fold impact. Firstly, policy makers should take the factors listed above seriously, especially when new screening programs are designed. These factors are prerequisites for an optimal implementation of a cost‐effective screening program, even though the BCS of 50‐ to 69 year‐old‐women is considered to be, in general, a cost‐effective intervention. A comprehensive cost‐effectiveness evaluation of BCS with long‐term forecasted outcomes supported by the evidence from local pilots would support efficient program design and functioning.Secondly, the differences in healthcare systems, health providers, and populations as well as breast cancer costs and their determinants should be considered in estimations of the transferability of findings on the cost‐effectiveness of BCS. The transferability approaches suggest that, at a minimum, practice patterns and unit costs from the jurisdiction of interest should be considered.4.2Besides mammography, limited (for BSE and CBE) to no (for ultrasonography) information was reported on other screening modalities. Even though CBE and BSE are generally perceived as low‐investment approaches, they also require launch and maintenance costs related to the education and enrollment of women. Costs required for BSE education were reported in one review,4.3While the quality of the reviews varied widely, only four of them were considered to be of a high quality (scored 6 or above on AMSTAR). Meanwhile, there was no clear relation between the quality of the included systematic reviews and their conclusions. Reviews having a wide geographic focus (world rather than targeting a certain country or region) and more recent search tend to have higher AMSTAR scores. Our more in‐depth analysis of reporting from four systematic reviews has shown potential risk of search, selection, extraction, and reporting mistakes in the reviews rather than biases in these studies. Similarly, we did not identify publication biases in the original evidence. We consider that cost‐effectiveness analyses of public preventive programs, such as BCS, may be at less risk of publication bias than pharmaceutical treatments. To improve the quality, reliability, and applicability of systematic reviews, the reviewers should refer to the developed guidelines in their methods, provide more transparent reporting of programs and outcomes, and consider the transferability of their findings.4.4Our overview shows that more original trial‐based economic evaluations along with pilots of CBE and ultrasonography with evidence on clinical and economic benefits are required. In addition, more high‐quality field‐based studies and reviews on the cost‐effectiveness of mammography screening in low‐ and middle‐income countries as well as the cost‐effectiveness of mammography screening among older women are also required. Economic evaluations of BCS considering personal risk stratification would be an asset for health decision‐making. A better understanding of costs related to informal care and indirect screening program costs would help to decide how these finances should be considered in economic analyses of breast cancer. Standard and better structured collection and reporting of costs would improve comparability among the studies. The risk of bias tool designed for the reviews reporting cost outcomes would help to interpret the results of these studies and potentially simplify their use in healthcare decision making.4.5With the large scope of the searched literature, it is possible that we missed some of the important information, despite the comprehensive approach applied in this review. We also deviated from the protocol, including two reviews on which agreement between two raters was not reached. The AMSTAR tool was not fully applicable to assess the quality of the systematic reviews with cost and cost‐effectiveness outcomes. In addition, the applied transferability metric was not validated, and the use of a standard validated tool specific for systematic reviews of economic evaluations would be a preferable approach.5Screening mammography may be a potentially cost‐effective intervention, although how cost‐effective it is would depend on population characteristics (such as incidence and starting age for screening) and screening organization  as well as the direct and indirect costs of breast cancer and their determinants. No information on the determinants for the cost‐effectiveness of ultrasonography was retrieved, and the cost‐effectiveness of CBE is not certain because of methodological limitations. No risk of publication bias in the original evidence was identified, although high variability and uncertainty in both the original and secondary evidence may limit the value of these reviews. Click here for additional data file. Click here for additional data file. Click here for additional data file. Click here for additional data file. Click here for additional data file. Click here for additional data file.


HIV frequently relate to sexual activity, biting, or spitting. This includes instances in which no harm was intended, HIV transmission did not occur, and HIV transmission was extremely unlikely or not possible. This suggests prosecutions are not always guided by the best available scientific and medical evidence.Globally, prosecutions for non‐disclosure, exposure or transmission of HIV science by the criminal justice system. A detailed analysis of the best available scientific and medical research data on HIV transmission, treatment effectiveness and forensic phylogenetic evidence was performed and described so it may be better understood in criminal law contexts. Description of the possibility of HIV transmission was limited to acts most often at issue in criminal cases. The possibility of HIV transmission during a single, specific act was positioned along a continuum of risk, noting that the possibility of HIV transmission varies according to a range of intersecting factors including viral load, condom use, and other risk reduction practices. Current evidence suggests the possibility of HIV transmission during a single episode of sex, biting or spitting ranges from no possibility to low possibility. Further research considered the positive health impact of modern antiretroviral therapies that have improved the life expectancy of most people living with HIV to a point similar to their HIV‐negative counterparts, transforming HIV infection into a chronic, manageable health condition. Lastly, consideration of the use of scientific evidence in court found that phylogenetic analysis alone cannot prove beyond reasonable doubt that one person infected another although it can be used to exonerate a defendant.Twenty scientists from regions across the world developed this Expert Consensus Statement to address the use of HIV science that have occurred over the last three decades to ensure current scientific knowledge informs application of the law in cases related to HIV.The application of up‐to‐date scientific evidence in criminal cases has the potential to limit unjust prosecutions and convictions. The authors recommend that caution be exercised when considering prosecution, and encourage governments and those working in legal and judicial systems to pay close attention to the significant advances in  Thirty‐three countries are known to have applied other criminal law provisions in similar cases . Most prosecutions have related to perceived risk of HIV acquisition associated with sexual activity but prosecutions have also occurred for acts such as biting and spitting . These laws and prosecutions have not always been guided by the best available scientific and medical evidence In this context, 20 HIV scientists with expertise in scientific research, epidemiology, and patient care from regions across the world developed this Consensus Statement, prompted by concern that criminal law is sometimes applied in a manner inconsistent with contemporary medical and scientific evidence: including overstating both the risk of HIV transmission and also the potential for harm to a person's health and wellbeing. Such limited understanding of current HIV science reinforces stigma and may lead to miscarriages of justice. It may also undermine efforts to address the HIV epidemic This Consensus Statement aims to assist scientific experts considering individual criminal cases in which HIV non‐disclosure, (perceived or possible) exposure, or transmission has been alleged. It provides expert opinion regarding individual HIV transmission dynamics (i.e. the “possibility” of transmission), long‐term impact of chronic HIV infection (i.e. the “harm” of HIV), and the application of phylogenetic analysis as evidence. It describes the possibility of HIV transmission between individuals who have engaged in a specific act at a specific time under specific circumstance, as that is usually the focus of criminal cases, and aims to communicate current scientific evidence relating to HIV in a manner understandable to a non‐scientific audience. The Consensus Statement has been translated into French, Russian and Spanish  decided the contents and framing of this Consensus Statement. A detailed literature review was prepared based on a search for literature published in English using the PubMed online database up to April 2017. Specific search terms relating to the possibility of HIV transmission were used, including “HIV and viral load,” “HIV sexual transmission risk per act,” “oral sex HIV transmission,” “anal sex HIV transmission,” “vaginal sex HIV transmission condom per act,” “anal sex HIV transmission condom per act,” and “anal sex HIV transmission circumcision per act.” Key articles were used to search for related articles. Preference was given to meta‐analyses, reviews and important studies. Other sources were identified by the expert authors. Abstracts from scientific conferences were used as appropriate.The authors next engaged in multiple rounds of drafting and review, considering the best available scientific and medical research data according to the following hierarchy: systematic review of randomized clinical trials; randomized clinical trials; and comparative studies . Two teleconferences were held to discuss a preliminary draft, followed by three rounds of redrafting via electronic correspondence by all authors. National and international legal experts, including UNAIDS staff members, were consulted on the application of the criminal law in cases involving HIV. A second face‐to‐face meeting was convened in Paris (July 2017) to resolve outstanding data analysis issues. Further rounds of comment and redrafting were undertaken by the authors to ensure agreement that the Consensus Statement accurately relayed current scientific research related to HIV transmission, harms and the use of scientific evidence in court.et al. The authors considered numerical findings and statistical estimates from all studies cited herein, including data summaries from reports presented in systematic or table form  as a means to help people reduce the possibility of HIV transmission by comparing different acts.Although the simplicity of such public health terminology was originally intended to support effective, broad‐based public health education campaigns for HIV prevention, its generalized categories now pose real problems for those developing current HIV health promotion messaging based on up‐to‐date scientific evidence 2.1HIV is not easily transmitted from one person to another. It is a relatively fragile virus that is transmitted through specific well‐described routes. It is not passed on through airborne, droplet, fomite, contact or vector‐borne transmission routes and cannot penetrate intact human skin For HIV transmission to occur, certain basic conditions must exist:○There must be a sufficient amount of the virus in particular bodily fluids .○A sufficient quantity of at least one of those bodily fluids must come into direct contact with sites in the body of an HIV‐negative person where infection can be initiated. These are usually mucous membranes, damaged tissue or inflamed ulcers, but not intact skin.○The virus must overcome the person's innate immune defences so that infection can be established and propagated.Most everyday activities carry no risk of HIV transmission because these conditions are not met. Leaving aside parenteral or vertical transmission, intimate contact, such as sexual intercourse, is usually required for transmission. Even in those cases, the per‐act chance of transmission is zero to low (with estimates ranging from 0% to 1.4% per act) 2.2The possibility of HIV transmission associated with individual acts varies according to a range of intersecting factors. When multiple intersecting factors are present, their effect is minimized or amplified to various degrees Correct use of a condom prevents HIV transmissionCorrect use of a condom  prevents HIV transmission because the porosity of condoms is protective against even the smallest sexually transmissible pathogens, including HIV Population level studies have found that consistent condom use for anal or vaginal sex dramatically reduces the possibility of HIV transmission even when factoring in instances of incorrect use or breakage at least 80% lower, or 0.016%  Population‐level research is only relevant in cases where multiple sex acts have occurred and it is not known whether condoms were correctly used in each instance. The population level estimate of 80% condom effectiveness does not exist as a stand‐alone estimate of HIV transmission risk but must be applied against risk associated with different sex acts. For example, if the estimated risk of HIV transmission from an HIV‐positive man to a woman during a single episode of condomless vaginal sex is 0.08% To reiterate, HIV cannot be transmitted in individual cases where a condom has been used correctly (i.e. it was worn through the sex act in question and its integrity was not compromised). The population‐level estimates can only apply in situations where multiple instances of condom use have occurred, including occasional instances of incorrect use and breakage.Viral load that is low or “undetectable” significantly decreases or eliminates the possibility of HIV transmissionSoon after acquiring HIV, a person's viral load is very high but typically decreases over the first few weeks as their immune system responds. If a person does not commence treatment, their viral load remains fairly stable for some time, while the immune system is gradually depleted. In advanced HIV infection, viral load usually increases to higher levels again.Antiretroviral therapy prevents HIV from replicating, thereby significantly reducing the viral load in a person's bodily fluids. When effective antiretroviral therapy is commenced, viral load usually drops to levels that are undetectable by current standard laboratory blood tests within a few weeks or months. Testing availability and lower limits of detection vary in different parts of the world, with lower limits of detection ranging from around 20 viral copies/mL to 400 copies/mL. A small percentage of people living with HIV (often referred to as long‐term non‐progressors) have a low viral load without taking antiretroviral therapy because their immune systems are able to control HIV Reduced viral load improves immune function and dramatically decreases the long‐term likelihood of illness and death. It also greatly reduces the possibility of HIV transmission Recent analyses from key studies  involving both heterosexual and male couples of different HIV status have not identified any cases of sexual transmission from a person with an undetectable viral load In 2011, the HPTN052 trial , which investigated the impact of early treatment initiation, observed no HIV transmission from 1763 people on antiretroviral therapy who had a stable viral load below 400 copies/mL. Partners of HIV‐positive participants were followed for the equivalent of 8509 person‐years. The only transmission from people on treatment occurred either early in treatment  or when viral load was above 1000 copies/mL on two consecutive visits The PARTNER and Opposites Attract studies found no HIV transmission from people with a viral load below 200 copies/mL after more than 75,000 acts of condomless vaginal or anal sex A 2013 systematic review and meta‐analysis also found no transmission where viral load fell below a threshold of between 50 and 500 copies/mL (depending on the study) While short‐lived, small‐magnitude increases in viral load, known as “blips,” occur among many individuals adhering to their antiretroviral therapy Pre‐exposure Prophylaxis (PrEP) significantly decreases the possibility of HIV acquisitionPrEP describes the use of antiretroviral medication by HIV‐negative people prior to HIV exposure to prevent HIV acquisition Post‐exposure Prophylaxis (PEP) significantly decreases the possibility of HIV acquisitionPEP describes short‐term use of antiretroviral treatment by an HIV‐negative person after an exposure to HIV. If started within 72 hours of exposure and taken for 28 days with good adherence, PEP significantly reduces the likelihood of the person becoming HIV‐positive because it can stop HIV from establishing itself in a person's immune cells even after the virus has entered a person's body Medical Male Circumcision decreases the possibility of HIV transmission from women to menMedical male circumcision reduces the possibility of HIV transmission from HIV‐positive women to HIV‐negative men by approximately 50% Risk reduction practices such as withdrawal or strategic positioning decrease the possibility of HIV transmissionSome people living with HIV use risk reduction practices such as withdrawal prior to ejaculation or strategic positioning  when engaging in condomless sex with an HIV‐negative person or person of unknown serostatus Sexually Transmitted Infections (STIs) can increase the possibility of HIV transmission in some circumstancesor if the HIV‐negative person is taking PrEP The presence of some untreated STIs, particularly ulcerative STIs, in either partner has been associated with an increased likelihood of HIV transmission during sexual activity when the person living with HIV does not have a low viral load 2.3HIV transmission through sex usually occurs as a result of bodily fluids containing enough HIV coming into contact with mucous membranes located in: the foreskin or urethra of the penis; the cervix or vagina; the anus; or the rectum. HIV transmission is also possible through contact with oral mucous membranes but these are much less vulnerable to HIV transmission 2.3.1▫The possibility of HIV transmission from oral sex performed on an HIV‐positive person, including when the person does not have a low viral load and/or a condom is not used, varies from none to negligible depending on the context Oral sex is promoted as a safer sex option for partners of different HIV status wanting to engage in intimate sexual acts, with its practice reportedly very common.Oral sex is known to involve a much lower possibility of HIV transmission than vaginal or anal intercourse The few clinical studies investigating transmission through oral sex have failed to find any cases of HIV transmission ▫or a condom is properly used, or the HIV‐negative partner is taking PrEP There is no possibility of HIV transmission from oral sex performed on an HIV‐positive person when the HIV‐positive partner has a low viral load, While there are no studies investigating the impact of antiretroviral therapy or PrEP on the possibility of transmission during oral sex, it is our expert opinion that there is no possibility of HIV transmission associated with oral sex performed on an HIV‐positive individual on antiretroviral therapy, or performed by a person taking PrEP. Similarly, correct condom use reduces the likelihood of HIV transmission to zero.2.3.2▫and a condom is not used is low The possibility of HIV transmission from vaginal‐penile intercourse when the HIV‐positive partner does not have a low viral load Two meta‐analyses of heterosexual couples ▫or uses a condom or the HIV‐negative partner is taking PrEP varies from none to negligible depending on the context The possibility of HIV transmission from vaginal‐penile intercourse when the HIV‐positive partner has a low viral load Numerous studies, as discussed above, have shown that the possibility of HIV transmission from an HIV‐positive partner who has a low viral load during vaginal‐penile intercourse is none to negligible HIV cannot be transmitted when a condom is used correctly because HIV cannot pass through intact latex or polyurethane. Similarly, there is no possibility of HIV transmission when a person has an undetectable viral load.2.3.3▫and the HIV‐positive partner does not have a low viral load is low, whether the receptive partner is male or female The possibility of HIV transmission when a condom is not used Studies show that receptive condomless anal intercourse by heterosexual or same‐sex couples is associated with a higher likelihood of HIV transmission than receptive condomless vaginal intercourse Two systematic reviews (2010 and 2014) report a per‐act estimate of approximately 1.4%  for receptive anal sex (i.e. when the HIV‐positive person is the insertive partner) ▫or uses a condom, or the HIV‐negative partner is taking PrEP varies from none to negligible depending on the context The likelihood is similar whether the receptive partner is male or female The possibility of HIV transmission through anal‐penile intercourse when the HIV‐positive partner has a low viral load, There is negligible possibility of HIV transmission from an HIV‐positive partner who has a low viral load during anal‐penile intercourse. As discussed above, both the PARTNER study and the Opposites Attract study observed no transmission after approximately 39,000 acts of condomless anal sex when viral load was below 200 copies/mL HIV cannot be transmitted when a condom is used correctly because HIV cannot pass through intact latex or polyurethane. Similarly, there is no possibility of HIV transmission when a person has an undetectable viral load.2.42.4.1HIV cannot be transmitted via contact with an environmental surface such as a chair, bench or toilet; from food or drink; or from casual human contact such as hugging, sharing household objects or eating together.HIV cannot survive long in air and is unable to penetrate intact skin. No case of HIV infection from contact with an environmental surface, food or drink or through casual human contact has ever been identified despite many scientific studies considering this possibility 2.4.2▫There is no possibility of HIV transmission via contact with the saliva of an HIV‐positive person, including through kissing, biting or spiting.Numerous studies have considered the possibility of HIV transmission via saliva but none has found any evidence, including a 1997 study of 34,000 cases in the UK ▫There is no possibility of HIV transmission from biting or spitting where the HIV‐positive person's saliva contains no, or a small quantity of, blood.Current evidence suggests HIV cannot be transmitted even when saliva contains small quantities of blood. Despite early research suggesting a theoretical risk of transmission if saliva‐containing blood enters a person's body through contact with mucosal tissue , no cases of HIV transmission resulting from the spitting of blood have been reported ▫and their blood comes into contact with a mucous membrane or open wound, and their viral load is not low or undetectable varies from none to negligible.The possibility of HIV transmission from biting where the HIV‐positive person's saliva contains a significant quantity of blood, Many studies have detailed a large number of cases where bites have not resulted in HIV transmission For transmission to be plausible in the case of biting, the HIV‐positive person must have blood in their mouth at the time of the bite, a sufficient amount of HIV must be present in the blood of the HIV‐positive person, and the bite must be deep enough to penetrate the HIV‐negative person's skin causing trauma and tissue damage 2.5The second section of this Consensus Statement considers the harms of HIV because persistent misconceptions exaggerating the harms of HIV infection appear to influence application of the criminal law The natural course of untreated HIV infection varies widely from person to person Antiretroviral therapies dramatically reduce HIV‐associated disease progression. Globally, treatment guidelines have been revised to recommend initiation of antiretroviral treatment immediately following diagnosis of HIV infection because most people on treatment will achieve an undetectable viral load and maintain a healthy immune system, will remain in good health, and will avoid the complications of long‐term HIV infection Studies from many countries have consistently shown that antiretroviral therapies have radically increased life expectancy, that life expectancy has continued to improve over time, and that the long‐term health and quality of life of people living with HIV has drastically improved Although HIV causes an infection that requires continuous treatment with antiretroviral therapy, people living with HIV can live long, productive lives including working, studying, travelling, having relationships, having and raising children, and contributing to society in various other ways.2.6The final section of this Consensus Statement recognizes the importance of the correct use of scientific and medical evidence in HIV‐related prosecutions where proof of actual transmission from one person to another is at issue.International guidance on HIV in the context of the criminal law recommends that “proof of causation, in relation to HIV transmission, should always be based on evidence derived from a number of relevant sources, including medical records, rigorous scientific methods and sexual history” ▫Medical records can provide contextual information but cannot establish transmission between a complainant and a defendant.The circumstances of the nature and timing of a sexual relationship or other potential sources of a person's HIV infection must be central to any case where sexual transmission of HIV is alleged. When available and lawfully obtained, medical records are valuable for identifying the last HIV‐negative and first HIV‐positive test of the complainant and the defendant. Considering the diagnostic window period of each test, this information can be used to establish the period during which the complainant acquired HIV and whether the defendant was HIV‐positive during this time. Importantly, whether the complainant or defendant was infected first cannot be based on who tested HIV‐positive first or which person brought charges against the other.Information related to HIV viral load and CD4 counts included in medical records has sometimes been presented as evidence establishing the timing of HIV infection. However, viral loads and CD4 counts show considerable inter‐ and intra‐individual variation and therefore cannot be used to determine exactly when someone acquired HIV ▫Phylogenetic analysis can be used as a forensic tool. The results can be compatible with, but cannot conclusively prove, the claim that a defendant has infected a complainant. Importantly, phylogenetic results can exonerate a defendant when the results are not compatible with the allegation that the defendant infected the complainant.Phylogenetic analysis compares the evolutionary relationship between different persons’ HIV, but results must be interpreted cautiously alongside other factual and medical evidence when used in criminal cases Phylogenetic analysis of HIV involves estimating the evolutionary relationships of HIV variants, for example, to investigate HIV transmission networks for public health purposes. In criminal cases, phylogenetic analysis involves investigating whether the complainant(s) and the defendant(s) are part of the same transmission network. The network is represented as a phylogenetic “tree.” Notably, the phylogenetic tree must be understood as an HIV gene tree, which may differ from the transmission history, because HIV variants may predate transmission or disappear after transmission can exonerate a defendant accused of transmitting HIV to a complainant because if the virus strains detected in the defendant and complainant are unrelated, the phylogenetic evidence conclusively contradicts the claim that the defendant was the source of the complainant's virus. HIV phylogenetics is very different from profiling of human DNA as, given the ongoing evolution of each person's HIV variants, phylogenetics cannot obtain an “exact match.” When there appears to be a “phylogenetic match” between two individuals’ HIV it means two or more variants are epidemiologically “linked”, not that they are the same Recent advances in DNA sequencing and phylogenetics allow some consideration of direction and timing of transmission Phylogenetic analysis is complex, and consequently it is important that HIV phylogenetics for forensic purposes is performed and interpreted by experts who fully understand the limitations of the technique and explicitly state these limitations in written reports and oral testimony. Interpretation of phylogenetic results for forensic purposes requires expertise about phylogenetics and the distinction between virus evolutionary trees and transmission histories. This is not straightforward and methodologies have not yet been standardized 3Given the evidence presented in this document, we strongly recommend that more caution be exercised when considering criminal prosecution, including careful appraisal of current scientific evidence on HIV‐related risks and harms. This is instrumental to reduce stigma and discrimination and to avoid miscarriages of justice.In this context, we hope this Consensus Statement will encourage governments and those working in the legal and judicial system to pay close attention to the significant advances in HIV science that have occurred over the last three decades, and make all efforts to ensure that a correct and complete understanding of current scientific knowledge informs any application of the criminal law in cases related to HIV.The authors have no competing interests to declare.All authors participated in numerous rounds of discussions, writing and editing of this Consensus Statement.Supplementary Material S1. Endorsers of the Expert Consensus Statement.Click here for additional data file.Supplementary Material S2. Executive Summary Expert Consensus Statement.Click here for additional data file.Supplementary Material S3. Expert Consensus Statement FRENCH Translation.Click here for additional data file.Supplementary Material S4. Expert Consensus Statement RUSSIAN Translation.Click here for additional data file.Supplementary Material S5. Expert Consensus Statement SPANISH Translation.Click here for additional data file.


In 2012, the Centers for Disease Control and Prevention (CDC) began to de‐emphasize and de‐implement multiple evidence‐based HIV prevention practices that had been around for 20 years, thus changing the scope of implementation across the globe. The authors provide evidence how existing interventions  may influence implementation of interventions that came after the program was discontinued. De‐implementation is an ecological event that influences, and is influenced by, many parts of a system, for instance, implementation of one type of intervention may influence the implementation of other interventions  after a long‐running program is discontinued. Researchers and policy makers ought to consider how de‐implementation of behavioral interventions is influenced by biomedical interventions mass‐produced by companies with lobbying power. The scientific study of de‐implementation will be inadequate without consideration of the political climate that surrounds de‐implementation of certain types of interventions and the promotion of more‐profitable ones. Implementation of one type of intervention  influences implementation of other types  interventions.The surge of biomedical interventions influenced de‐implementation of behavioral interventions.Political climate and community exclusion from implementation decision‐making may lead to early de‐implementation. In “Letting Go: Conceptualizing Intervention De‐implementation in Public Health and Social Service Settings,” the authors offer three criteria for identifying interventions appropriate for de‐implementation: they must be (a) harmful or not effective, (b) not the most effective or efficient to provide, or (c) no longer necessary  acknowledge that “it is important to understand the outcomes of de‐implementation…. It will also be valuable to understand some of the ancillary benefits that may have occurred because of implementing interventions” (p. 10).providers of social and public health services in agencies providing EBIs) and the frequency with which these providers made links to high‐impact services. We found that providers who are exposed to any HIV‐prevention interventions more frequently link clients to HIV, HEP‐C, and STI testing, to primary care, and to drug‐treatment and mental health services, all of which are evidence‐based practices shown to help decrease the spread of HIV. In other words, exposure to many of the interventions, now de‐implemented, may have contributed to the relative success of newly implemented high‐impact interventions. Our findings also suggest a dose effect, with exposure to more EBIs resulting in more links to high‐impact interventions. If exposure to EBIs increased the likelihood of links being made to HIV primary care and other “higher‐value” programs, there exists the potential for de‐implementation to have a long‐term inadvertent effect, potentially negative, on the implementation of future innovations.In our published study  under a subsequent program . “Exposure” suggests the potential for a staged approach to implementation of HIV‐prevention innovations, and perhaps to innovations in other areas of social and public health. Providers were initially exposed to DEBI and were required to offer multiple EBIs; then, after the emergence of HIP, they were required to offer high‐impact innovations. But this historical development was not without unwanted consequences. For instance, de‐implementation/de‐adoption of RESPECT, an individual‐level EBI for reducing HIV risks, has weakened collaborative interagency relationships and caused frustration among staff members , a daily oral dose of antiretroviral therapy prescribed to individuals at risk for HIV infection, emerged as a major HIV‐prevention tool. PrEP was shown to reduce the risk of HIV acquisition by 73% among adult men who have sex with men (MSM) and transgender women, with greater efficacy (up to 99%) for individuals with higher rates of adherence  such cost‐effectiveness may be gained. While the CDC de‐implementation may have taken place for cost‐efficiency or program‐value reasons, we may have lost the relative benefits of continued implementation of behavioral interventions to reach marginalized groups who are at risk for new HIV infections and who may not be accessing important HIV‐prevention tools such as PrEP. We do not yet know the long‐term effects, and agree that de‐implementation must be monitored for potential harmful effects.Community exclusion from decision‐making processes related to scientific research and implementation of its products has been helpful in explaining why community members (who can most benefit from research innovations) and the practitioners who deliver those innovations are wary of scientific knowledge and thus may reject its products . Community exclusion also helps explain why communities, including consumers, providers, and agency administrators, are often suspicious of scientific knowledge and find its products inadequate and sometimes ineffective in the effort to address specific complexities of local populations  and services after a long‐running program is discontinued. Researchers and policy makers have much to gain by considering how de‐implementation of behavioral interventions is influenced by the surge of biomedical interventions that are developed and mass‐produced by companies with lobbying power that greatly surpasses that of behavioral researchers. Finally, the scientific study of de‐implementation will be inadequate without full consideration of the political climate that surrounds the de‐implementation of certain types of interventions and the promotion of more‐profitable ones. Here, genuine inclusion of community in decision‐making about implementation may imbue de‐implementation criteria with the opinions and lived experiences of those who may benefit the most from more‐effective interventions.


GBD 2016 Alcohol Collaborators. Alcohol use and burden for 195 countries and territories, 1990–2016: a systematic analysis for the Global Burden of Disease Study 2016. Lancet 2018; 392: 1015–35—In this Global Health Metrics paper, Stefan Lorkowski has been added to the collaborators list; affiliation details and a declaration of interests statement for Stefan Lorkowski have also been added. These corrections have been made to the online version as of June 20, 2019.


The identification of recycled oceanic crustal signatures in both the trace elements and halogens indicates that subduction and dehydration of altered oceanic crust may exert control on the cycling of volatile elements to the deep mantle.Halogens are primarily located within surface reservoirs of the Earth; as such they have proven to be effective tracers for the identification of subducted volatiles within the mantle. Subducting lithologies exhibit a wide variety of halogen compositions, yet the mantle maintains a fairly uniform signature, suggesting halogens may be homogenized during subduction to the mantle or during eruption. Here we present halogen , K, noble gas, and major and trace element data on olivines from three seamounts along the Hawaiian‐Emperor seamount chain to determine if the deep mantle source has retained evidence of halogen heterogeneities introduced through subduction. High Ni contents indicate that the Hawaiian‐Emperor mantle source contains a recycled oceanic crust component in the form of pyroxenite, which increases from the 46% in the oldest (Detroit) to 70% in the younger seamount (Koko). Detroit seamount retains mid‐ocean ridge basalts (MORB)‐like Br/Cl and I/Cl, while the Br/Cl and I/Cl of Suiko and Koko seamounts are higher than MORB and similar to altered oceanic crust and dehydrated serpentinite. Helium isotopes show a similar evolution, from MORB‐like values at Detroit seamount toward higher values at Suiko and Koko seamounts. The correlation between pyroxenite contributions, Br/Cl, I/Cl, and  Trace elements in Emperor seamount olivines indicate magmatism sampled an increasing proportion of pyroxenite through timeHigher than mantle Br/Cl and I/Cl in the younger seamounts indicate that the pyroxenite contains a subducted volatile componentHelium isotope variations indicate that Hawaiian plume magmatism also sampled primitive material derived from the deep mantle Prior to analysis the background and sensitivity of the microprobe was determined using well‐characterized silicate, oxide, and metal standards  formed during irradiation was monitored using several scapolite and Shallowater meteorite I‐Xe standards  measurements of olivine from each seamount are presented in Table There is a correlation between the Ni, Mn, and Cr concentrations within the olivines and the Fo number with all three seamounts generally following fractional crystallization profiles as shown by the regression lines in Figure The Emperor seamount olivines define two compositional trends shown in Figure The Ca concentration within Suiko and Koko olivines is similar and less sensitive to changing Fo number compared to Detroit. The range of Ca concentrations within Suiko  and Koko  overlaps and is lower than the concentrations within Detroit olivines . Variations in Mn are less pronounced than for Ca but the trend is similar with lower Mn concentrations within Suiko and Koko compared to Detroit at Fo numbers lower than 86   and Koko (28.6 × 10−5) are higher and do not overlap with MORB values. It is also notable that the average K/Cl weight ratio from crushing and step heating of Detroit olivine is 14, within error of the MORB value  or samp20Ne/22Ne ratios within error of air. The 40Ar/36Ar ratios of the olivines from all the seamounts are essentially atmospheric. Only one sample, Detroit 50–65, has an 40Ar/36Ar ratio (566 ± 24) significantly greater than the atmospheric value  decreases with increasing pressure. Therefore, melt formed at higher pressure will be enriched in Ni  exhibits a positive correlation with the Br/Cl, I/Cl, o Figure .3He/4He indicates that subducted material has been incorporated into the primordial undegassed Hawaiian mantle plume source. Dehydrated slab material and mantle metasomatized from the release of slab fluids are envisaged to reach the deep mantle  to melts containing a larger proportion of pyroxenite within the plume. The geochemical signature within Suiko and Koko seamounts therefore has not been diluted to the same degree as that at Detroit seamount, where the incorporation of depleted mantle melts has significantly obscured the geochemical signatures originating from other mantle components within the plume.The evolving geochemical signature of the Emperor seamounts appears directly influenced by the overlying lithospheric thickness during partial melting. Lithostatic pressure ultimately arrests the melting beneath oceanic hot spots, as a result this controls the melting of distinct lithologic components within the ascending mantle if their solidus temperatures differ  end‐member; however, they appear to be dominated by a peridotite mantle source rather than a recycled pyroxenite source as in the Emperor seamounts  at 76 Ma led to a stronger component of melting of depleted peridotite that was accompanied by MORB‐like mantle 3He/4He signatures.The geochemical signature of the Emperor seamount chain has evolved from having a primarily MORB‐like volatile and trace element signature toward signatures similar to the younger Hawaiian Islands. From 65 Ma (Suiko seamount) and younger, olivines in Emperor seamount basalts are more enriched in Ni and more depleted in Ca and Mn, while retaining more primitive Elevated halogen ratios of Suiko and Koko samples are similar to the garnet peridotite products of serpentinite dehydration and may signal the subduction of oceanic crust into the Hawaiian plume mantle source. The mantle is therefore not completely homogenized with respect to subducted halogens and noble gases as previously considered. Evidence for distinct recycled components in the mantle may however be readily overprinted by ambient mantle peridotite in OIB with a smaller recycled crust component or when melting occurs at lower pressures in the mantle such as in the Detroit seamount.Supporting Information S1Click here for additional data file.Data Set S1Click here for additional data file.


Acute myeloid leukaemia (AML) is the commonest indication for allogeneic stem cell transplantation  worldwide. The accumulated experience of allografting in AML over the last four decades has provided critical insights into both the contribution of the conditioning regimen and the graft‐versus‐leukaemia effect to the curative potential of the most common form of immunotherapy utilised in standard clinical practice. Coupled with advances in donor availability and transplant technologies, this has resulted in allo‐SCT becoming an important treatment modality for the majority of adults with high‐risk AML. At the same time, advances in genomic classification, coupled with progress in the accurate quantification of measurable residual disease, have increased the precision with which allo‐mandatory patients can be identified, whilst simultaneously permitting accurate identification of those patients who can be spared the toxicity of an allograft. Despite this progress, disease recurrence still remains a major cause of transplant failure and AML has served as a paradigm for the development of strategies to reduce the risk of relapse ‒ notably the novel concept of post‐transplant maintenance, utilising pharmacological or cellular therapies. The number of adults allografted for acute myeloid leukaemia (AML) has risen sharply in the last decade  unrelated volunteer donor (VUD), compared to those achieved with an MSD . Coupled with increased availability of good quality cryopreserved umbilical cord blood units and evidence that transplantation of haploidentical stem cells can now be achieved with acceptable toxicity, allo‐SCT can now be contemplated in almost all eligible patients. Unrelated adult donors and recipients are routinely typed by high resolution typing at HLA loci A, B, C, The likelihood of identifying a suitably matched unrelated donor has risen to over 75% in Caucasian patients within the last decade   regimen with iv‐Bu/Cy for patients with AML in CR1. Whilst outcomes were similar between the groups, the FluBu4 regimen resulted in a significant reduction in NRM. As a consequence, Flu/iv‐BU is now an increasinly utilised MAC regimen in fit adults with AML in CR  with either total body irradiation (TBI)‐ Cy/TBI‐ or busulfan (Bu)‐ Bu/Cy. Both regimens are still widely used in fit patients with high‐risk disease, transplanted using an MSD or VUD. The majority of studies comparing Cy/TBI with a Bu/Cy regimen  have demonstrated broadly equivalent outcomes     after two courses of induction chemotherapy  (Levis et al., One of the most promising post‐transplant maintenance strategies currently under examination is the administration of + T cell responses to candidate tumour antigens, including MAGE antigens, which are not ordinarily observed post‐transplant, and prospective randomised trials of the impact of such agents as post‐transplant maintenance are ongoing (Craddock et al., An alternative and promising approach is peri‐ or post‐transplant administration of the DNMT inhibitors AZA or decitabine, which are also both well‐tolerated in this clinical setting (Cruijsen et al., The outcome for patients who relapse after allograft remains very poor, with a two‐year OS of 14–25%, although the small number of patients who achieve a second CR after salvage therapy can have a promising outcome after either a second allograft or DLI (Schmid et al., FLT3 ITD, are commonly lost at relapse may be interpreted as supporting the exploration of maintenance strategies which utilise drug or cellular therapies with broader anti‐tumour specificity as opposed to targeted therapies. There is also increased evidence of acquired dysregulation between the donor immune system and recipient haematopoiesis as an important mechanism of disease relapse (Fig It is increasingly recognised that clonal evolution is a fundamental mechanism underlying disease relapse post‐transplant (Quek et al., apse Fig . Acquireapse Fig . A similapse Fig  but appeapse Fig .FLT3 ITD+ disease and appear superior to high dose salvage chemotherapy (Perl et al., A major challenge in the management of relapsed disease is the tolerability of salvage therapies in post‐transplant patients. Azacitidine has recently been shown to be a well‐tolerated and active salvage agent in patients relapsing post‐allograft (Craddock et al., The last decade has seen a remarkable increase in the number of therapeutic opportunities with the potential to reduce the risk of disease relapse in patients allografted for AML. A future priority must be to effectively deliver randomised trials of transplant regimens and post‐transplant maintenance strategies, incorporating both genomic and pre‐transplant MRD studies. This will require far‐reaching improvements in our capability to deliver practice‐informing transplant trials. Much can be learned from the remarkable success of the accelerated trials network developed by the US BMT Clinical Trials Network Network, . AttentiCC has received honoraria from Celgene, Daichi‐Sankyo, Novartis and Pfizer, as well as research funding from Celgene. JL has received travel funding from Novartis and Daichi‐Sankyo.


In recent years, iron sulfur (Fe–S) proteins have been identified as key players in mammalian metabolism, ranging from long-known roles in the respiratory complexes and the citric acid cycle, to more recently recognized roles in RNA and DNA metabolism. Fe–S cofactors have often been missed because of their intrinsic lability and oxygen sensitivity. More Fe–S proteins have now been identified owing to detection of their direct interactions with components of the Fe–S biogenesis machinery, and through use of informatics to detect a motif that binds the co-chaperone responsible for transferring nascent Fe–S clusters to domains of recipient proteins. Dissection of the molecular steps involved in Fe–S transfer to Fe–S proteins has revealed that direct and shielded transfer occurs through highly conserved pathways that operate in parallel in the mitochondrial matrix and in the cytosolic/nuclear compartments of eukaryotic cells. Because Fe–S clusters have the unusual ability to accept or donate single electrons in chemical reactions, their presence renders complex chemical reactions possible. In addition, Fe–S clusters may function as sensors that interconnect activity of metabolic pathways with cellular redox status. Presence in pathways that control growth and division may enable cells to regulate their growth according to sufficiency of energy stores represented by redox capacity, and oxidation of such proteins could diminish anabolic activities to give cells an opportunity to restore energy supplies. This review will discuss mechanisms of Fe–S biogenesis and delivery, and methods that will likely reveal important roles of Fe–S proteins in proteins not yet recognized as Fe–S proteins. Fe–S proteins were not discovered and characterized until the late 1950s and early 1960s when methods were developed that detected their intrinsic magnetic properties, including electron paramagnetic imaging and Mossbauer spectroscopy methods  bind cytosolic iron-sensing proteins now known as iron regulatory proteins 1 and 2 (IRP1 and IRP2) in iron-depleted cells , rather than at room air concentrations of 21%, which constitutes hyperoxia and may adversely affect generation of meaningful experimental results in many settings , a scaffold protein upon which nascent clusters assemble (NifU or IscU), a ferredoxin which likely provides electrons for Fe–S cluster formation, and a chaperone-co-chaperone pair (HscA and B) that likely aid folding or cluster transfer , whereupon specialized proteins in the cytosol, named the cytosolic iron sulfur assembly machinery or CIA machinery completed synthesis and delivery of Fe–S cofactors to numerous cytosolic and nuclear proteins  was postulated because it was alleged to be crucial for Fe–S acquisition of enzymatic function in a critical Fe–S enzyme of the cytosolic leucine biosynthetic pathway of yeast  was discovered to bind directly to the cochaperone, HSC20, the mammalian orthologue of bacterial HscB in the Fe–S synthesis machinery. The yeast homologue of HSC20, Jac1, was already known to bind the yeast orthologues of the scaffold protein IscU, known as Isu1, and the orthologue of the mammalian chaperone HSPA9 (HscA in bacteria), Ssq1, from yeast studies (Andrew et al. S. cerevisiae, although the human cysteine desulfurase is clearly present and functional (Marelja et al. In mammalian cells, a full complement of Fe–S biogenesis proteins is synthesized and targeted to the mitochondrial matrix, but almost all of these proteins have also been observed in the cytosolic/nuclear compartments of cells. Though they are often transcribed from the same gene, cytosolic forms are generated by alternative splicing (Tong and Rouault To better understand the process of Fe–S assembly, native gels and mass spectrometry have been helpful in defining stages in the assembly process. The validity of the approach was established by discovering that SDHB acquired its Fe–S clusters in stages, beginning with binding to HSC20 and ISCU, and progressing to ejection of the HSC20 transfer complex, transfer of the cluster to SDHB, binding of holo-SDHB to SDHA in the multiprotein SDHB complex, and finally to the membrane bound subunits, SDHC and SDHD (Maio et al. Native gels and mass spectrometry also greatly enhanced studies of Fe–S biogenesis in the cytosol, where individual assembly steps have been defined (Kim et al. Studies showed that a branch point was reached in Fe–S cytosolic delivery to Fe–S proteins. In some examples, such as NUBP1, no other steps were required to facilitate transfer from the nascent apparatus, composed of the cysteine desulfurase, primary scaffold, and a chaperone co-chaperone pair. Interestingly, frataxin, which plays a critical function in the mitochondrial Fe–S biogenesis complex Rouault , was cleDiscovery of the important role for LYR motifs opens the possibility that informatics can be used to identify prospective Fe–S proteins, and feasible follow-up steps including analysis by ICP-MS followed by scale up for analysis by definitive techniques that require large amounts of protein such as EPR and Mossbauer could revolutionize Fe–S protein discovery and lead to revision of many metabolic pathways Rouault , b. The In a fundamental process such as Fe–S biogenesis, it is not surprising that mutations to the proteins involved in the process cause human diseases, particularly in cases when loss of function is not complete. The most well-known of these diseases is Friedreich’s ataxia, which causes ataxia and cerebellar dysfunction due to a loss of expression of frataxin, which is caused by by expansion of a trinucleotide repeat in the first intron that represses expression by an unknown mechanism. These diseases may offer insights into the mechanisms that account for tissue-specific pathology in disease. The subject has been extensively reviewed (Rouault and Tong It is likely that the role of Fe–S proteins will become increasingly recognized as key to metabolism and regulation of cellular responses as more key Fe–S proteins are identified and their roles are revealed. It is quite possible that Fe–S proteins represent a repository of energy stored in the form of reducing power, and that cells rely on this energy to maintain homeostasis. One could imagine that many pathways involved in cellular growth and regulation could be disrupted if a Fe–S cofactor becomes oxidized, leading to its dysfunction through changed conformation and/or charge interactions.Iron sulfur cofactors evolved early in evolution, and the iron sulfur origin of life theory is a viable and attractive hypothesis (Boyd et al.


Drosophila melanogaster) eclose from their pupae mainly around dawn. The timing of eclosion is thought to confer adaptive benefits to the organisms and thus shows remarkable accuracy. However, it is not clear what factors are involved in the evolution of such accuracy in natural populations. In this study, we examined the relative contributions of gating of eclosion by the circadian clock versus clock-independent developmental rates and light-induced responses in the eclosion phenotype exhibited by fly populations that have evolved greater accuracy in eclosion rhythms compared to controls. We compared variation in timing of transitions between early developmental stages (pupariation and pigmentation), overall development time under constant light conditions – where circadian clocks are dysfunctional – and eclosion profiles when developmental rate was manipulated using different larval densities in selected and control stocks. Our results showed that stocks that have evolved greater accuracy of eclosion rhythms due to artificial selection do not show reduced individual variation in pupariation and pigmentation time compared to controls, though they do exhibit lower variation in eclosion time. Selected stocks also did not show lower variation in eclosion time under constant light conditions in contrast to the greater accuracy seen under light-dark cycles. Moreover, manipulations of developmental rate by varying larval density and inducing eclosion by changing onset of light phase did not alter the eclosion profile of selected stocks as much as it did controls, since selected stocks largely restricted eclosion to the daytime. These results suggest that fly populations selected for greater accuracy have evolved accurate eclosion rhythms primarily by strengthening circadian gating of eclosion rather than due to fine-tuning of clock-independent developmental processes.Fruit flies (This article has an associated First Person interview with the first author of the paper. Summary: Fruit fly populations that have evolved greater accuracy of eclosion rhythms show enhanced gating of their eclosion by circadian clocks but not reduced variation in clock-independent developmental processes. Drosophila melanogaster populations is noted to be highly accurate . Moreover, we also did not find significant differences in pupariation time between the stocks when this experiment was repeated . These results suggest that pupariation profiles of selected and control stocks are largely similar and the mean pupariation time and variation between individuals are not significantly different between selected and control stocks  occurring on the 8th day and no eclosion after the 9th day. Hence, the figure depicts only eclosion on the 9th day A. The ecP<0.05; P<0.05; P<0.05; P<0.05; P<0.05; We also examined profiles of eclosion rhythms under different larval densities, where developmental rates increase with decreasing densities, to evaluate the role of clock-independent developmental rate on the eclosion rhythm of selected and control stocks. Drosophila clock is rendered arrhythmic . Since development time is extremely susceptible to variation due to changes in environmental conditions, we repeated this experiment to verify these results and found similar trends of delayed pupariation time in selected stocks that were not significantly different from controls . However, we did not find a trend of early pigmentation in selected stocks  as observed in While it is known that fruit flies do not show circadian rhythms of pupariation and pigmentation , reducedWhile selection for early and late timing of eclosion had yielded correlated responses in pre-adult development time , we obseAlthough early development (pupariation and pigmentation) does not appear different between selected and control stocks, variation in proximate mechanisms leading up to eclosion independent of circadian clock control could have evolved, resulting in reduced variation in eclosion time. This role of clock-independent processes could be detected under LL conditions where the circadian clock is rendered dysfunctional  and ecloTribolium freeman , constant temperature (25°C) and constant humidity (∼80%) on banana-jaggery-based food medium solidified with agar  and control (CP) stocks of 1–4) populations and control (CP1–4) populations will be collectively referred to as selected and control stocks.Both sets of populations were maintained as large, outbred populations with about 1200 breeding adults in plexiglass cages (25 cm×20 cm×15 cm) with roughly equal males and females on a 21-day discrete generation cycle. Prior to egg collection, petriplates with banana-jaggery medium coated with yeast paste (yeast plate to induce egg-laying) were provided to the fly populations. Three days later, the yeast plate was withdrawn and replaced by a fresh food plate where vertical cuts were made on the medium and the edges of the food exposed so as to increase area of vertical surfaces available for egg laying (cut-plate). Flies were allowed to lay eggs for 2–3 h before the cut-plate was withdrawn and eggs were collected and transferred into glass vials (20 cm height×2.5 cm diameter) containing ∼10 ml of food. The eggs were dispensed at high densities of roughly 300 eggs per vial to facilitate eclosion over several days. Adult flies eclosing between the 9th and 12th days after egg collection were collected into fresh plexiglass cages to constitute the next generation. While all such flies were collected in CP populations, only those that eclosed in the selection window were chosen to form the breeding population of the next generation in case of PP populations. The selection was carried out on four successive days from eggs that were collected within a 2 h window  in order to avoid indirect selection for development time. The assays described in this chapter have been conducted on populations that have been subject to over 120 generations of selection. The selected (PPAll assays were conducted on standardized flies from selected and control populations which were subjected to a common rearing protocol for one generation where selection pressure was relaxed. The populations which underwent such a generation of common rearing are referred to as standardized populations and the progeny from these populations constituted the sample flies for the assays. All assays were conducted under ambient temperature of 25°C and constant humidity (∼80%) with light intensity ∼100 lux maintained throughout the LL regime and the light phase of LD 12:12.n=300 flies per population, n=1200 each for selected and control stocks) with ∼10 ml of food for each replicate population and maintained under LD 12:12. Low density of eggs  for assay of development time was chosen so as to measure the overall development time of all individuals of a vial in contrast to the maintenance protocol where high density  was used, in order to obtain multiple cycles of eclosion to select for accurate daily timing of eclosion, rather than a particular speed of development. Duration of pupariation and wing pigmentation was estimated by counting the number of pupae formed or pigmented every 2 h. Pupariation was identified by the formation of two spiracles on the head and pigmentation was recorded when the colour of wings on the sides of the pupae changed from white to grayish or black  which was discarded and replaced by another cut-plate after 2 h. The second cut-plate was withdrawn 2 h later and individual eggs were transferred on to agar pieces. Agar pieces with low density of exactly 30 eggs each were then transferred to 10 vials  for a single vial and averaged across 10 vials for each replicate population. These population mean profiles were then averaged across four replicate populations for selected and control stocks and plotted with standard error of mean across the four populations as error bars. Mean pupariation time for each replicate population was also calculated and these population means were used as replicate values for one-way ANOVA with ‘stock’ as fixed factor. Variation in pupariation time was also estimated for each replicate population by calculating the standard deviation in pupariation time across all individuals and was analyzed similar to the mean. Similarly, pigmentation profiles, mean pigmentation time and variation in pigmentation time were estimated for selected and control stocks and compared. Eclosion profiles were calculated across 2 days and plotted similar to pupariation and pigmentation profiles. Mean eclosion time was calculated for all flies eclosing over 2 days and compared using ANOVA with ‘stock’ as a fixed factor. However, variation in eclosion time was calculated only for the second day of eclosion since there were very few numbers of flies eclosing on the first day. Eclosion profiles, mean eclosion time and variation in eclosion time under LL conditions were analyzed similar to pupariation and pigmentation time.For eclosion assays with different larval densities, eclosion profiles were calculated as percentage of eclosion in each 2 h interval normalized by total number of flies emerging in a day for each vial and averaged across days 8–11. Only days in which at least 25 flies eclosed were considered for analysis from each vial and therefore, at lower densities  mostly days 8 and 9 were used, while days 9, 10 and 11 were primarily used for higher densities . After vial-wise averages were calculated across days, the eclosion profiles were then averaged across five vials for each replicate population at each larval density. These population mean profiles were further averaged across four replicate populations to obtain average profiles for selected and control stocks and plotted with SEM across replicate populations as error bars. Further, percentage of eclosion in the 6-h window prior to lights-on (ZT18–ZT0) was calculated as this part of the profile was the most variable across densities. ANOVA was performed on percentage of eclosion before lights-on with ‘density’ and ‘stock’ as fixed factors. The phase of peak of eclosion was also recorded as the time at which maximum eclosion was observed on a given day for a vial. This phase of eclosion peak was averaged across days and across all vials within a population and subsequently averaged across four replicate populations for each stock and plotted. ANOVA with ‘density’ and stock’ as fixed factors was performed on phase of eclosion peak.To study the direct effect of light on eclosion, 0.5 h eclosion profiles were calculated from ZT22–ZT2. The percentage of eclosion in each 0.5 h interval was calculated as a fraction of the total eclosion in a day for a particular vial and averaged across all vials within a population and subsequently, averaged across the four populations and plotted. ANOVA with ‘stock’ and ‘time-point’  as fixed factors was performed separately for each regime.P<0.05.Data were tested for normality using Kolmogorov–Smirnov test. All post-hoc comparisons were done using Tukey's Honest Significant Difference. All statistical analyses were performed on STATISTICA 7.0 and differences were considered statistically significant at