A personal tribute to George L. Wied, M.D., a founder of the medical subspecialty, cytopathology, who died July 25, 2004. I arrived in Chicago last November for the ASC meeting, and reflexively reached for the phone to let Dr. Wied know that I had arrived, just as I had for each of countless trips to Chicago over the past 25 years. Then I remembered that he was no longer here, having passed away in Salzburg, Austria, on July 25th. But as the meeting unfolded, I realized that George's mark was deeply imprinted on the entire scientific program, and that his legacy was carried by most of the people in attendance.George Papanicolaou is attributed with the discovery of the cytologic method for detecting epithelial tumors. His laboratory at Cornell Medical Center in New York was the culture medium for the discipline, instructing such notable cytologists as Leopold Koss and George Wied. The medical specialty itself, however, owes its success to George Wied, for without him, it is doubtful whether it would have survived the skepticism of most other medical specialists.Dr. Wied had faith in the importance of cytology that was unwavering and his conviction was transmitted to all who followed him around the globe. A survivor of Nazi Germany, he was a fierce defender of individual freedoms, and he translated that zeal into inclusion of all peoples in his vision. He was an instinctual teacher and taught those around him to convey the criteria of those strange cellular samples via the Tutorials of Cytology. His attention to detail was impeccable, a trait that he insisted his faculty emulate.The TOC faculty quickly became internationally recognized experts, and the Tutorials were a successful enterprise for over 40 years. Until cytopathology became a mandatory part of the curriculum of U.S. pathology training programs, the Tutorials were among the few opportunities for physicians to become facile with the discipline. Repeat enrollment was common, as the specialty became more complex and its importance became apparent to the medical profession. For many nations TOC remained the sole source of first-hand and first-rate cytology education. Judging by the multinational backgrounds of presenters at the ASC meeting, and the excellence of the papers, Dr. Wied's goal has most assuredly been achieved.Setting and maintaining a level of excellence was a responsibility that Dr. George Wied eagerly assumed, and expected the rest of us to uphold. It was easy to follow his example, for his charisma infused us with commitment to those eager to learn. In order to validate the professionals involved in the practice, he established the examinations of the International Academy of Cytology. He personally traveled to every venue where the exam was held, usually following either a Tutorial or International Congress of the IAC. He savored each opportunity to bring another disciple into the fold of cytology.But where Dr. Wied had the most fun was in the realm of cytology automation. As I listened Sunday to the presentations of experiences with the "new" imaging systems and their integration into the clinical cytology laboratory, I recalled the numerous conferences devoted to development of computerized scanners. As early as 1951, Dr. Wied recognized that computers would become an essential part of our daily lives, for data management and communication. He also realized that the task of screening slides was work intensive, subjective, and fraught with opportunities for error. If Dr. Wied didn't have the knowledge himself, he immediately reached out to others who did, and drafted them to the cause of automating his specialty. One of those recruits was Peter Bartels, a gifted optical scientist and incredibly creative problem solver. Together they built a team of researchers at the University of Chicago that attacked each obstacle to success like an army of dragon slayers.Rather than seek the glory of discovery alone, Dr. Wied's generous spirit inspired him to organize meetings to discuss problems common to automation, bringing scientists from a variety of disciplines and numerous countries. If a potentially important contributor to the meeting did not have funding to attend, Dr. Wied would offer to support them, often personally. He was most supportive of young scientists and mentored them through their careers. These were usually pleasant meetings, as Dr. Wied's gentle and considerate nature didn't allow personal bickering. But he loved scientific controversy and he encouraged us to challenge each other to the next level of achievement. He fully believed that what was good for one research group was good for the entire profession. He was most distressed when the commercialization of automated scanners led to open fighting among the companies trusted to develop the fruits of so many years of collegial research.ACTA Cytologica in 1957 and Analytical and Quantitative Cytology and Histology in 1979.He was a frequent member of NIH study sections that reviewed research proposals in automation. He was also known to drop in unexpectedly on a research group thousands of miles from his home, just to see what was happening. He was free with his advice without being condescending. He would never knowingly offend anyone. His constant encouragement and validation of the importance of scientific discovery for this young specialty was manifested through the two journals he founded and edited, Recognition for his accomplishments came frequently and from various sources, including an Outstanding Investigator Award from the NIH. But if you were to reach into the heart of George Wied, I wager that he felt that his greatest accomplishment was being father to Kazutaka (George) Wied, son of his wife, Kay. An unlikely parent, Dr. Wied soon became a master at constructing dioramas out of shoe boxes, holding flash cards with German vocabulary words, and generally encouraging young George to treasure his education. Being a musician himself, Dr. Wied patiently tolerated the squawks and squeaks of his young son's efforts to be a violinist. As young George became more accomplished, after dinner performances soon became an expected treat whenever the occasion allowed.Dr. Wied and Kay continued to travel the world even though Dr. Wied's health was becoming increasingly fragile. His final trip could have been written by a romantic novelist. He accepted a speaking engagement in Japan, but instead of a simple round trip to Tokyo, he insisted on an around-the-world ticket, "because it's cheaper!" For one of the stops, he chose to go to Vienna, a city that he loved, to once more savor the delicious schnitzel at the Intercontinental Hotel, site of the Vienna Tutorials. Kay realized that the Salzburg Music Festival was nearby and would be a good place to relax and hear the music that Dr. Wied loved so dearly. Young George, having just graduated from Stanford University, was with them, not always the case. They spent the day at numerous concerts in Salzburg, all dedicated to Czech composers, an uncanny coincidence since Dr. Wied was born in what was then Czechoslovakia. After the concerts, Kay, young George and Dr. Wied spent the evening together, remarking on the beauty of the music. Dr. Wied died in the night, with his beloved family nearby, and across the street from the home of his most admired composer, Wolfgang Amadeus Mozart.There is an old adage, that when a loved one dies, you mourn each time for all the roles they played in your life. If it is a parent or sibling, you mourn once. However, if the person has been multiple influences in your life, you will mourn for each role. For Dr. Wied, many of us will mourn multiple times. For me, he was a teacher, mentor, advisor, friend, and role model. I will mourn many times, but will be comforted by having known him. I have already and will continue to repay his influence by transmitting his ideals to those who follow. His is truly a legacy that deserves to be perpetuated. George Wied was a man of rare breadth and depth, the kind of professional and human for which mankind will eternally thirst.


A recent report questioned the identity of tRNP(Ser)Sec as the real SLA antigen. The latter study identified α-enolase as a major anti-SLA target, through proteomic analysis.Antibodies to a cytosolic soluble liver antigen (SLA) are specifically detected in patients with autoimmune hepatitis (AIH). The target of anti-SLA has been identified as a ~50 kDa UGA serine tRNA-associated protein complex (tRNP(Ser)Sec using rat and primate liver homogenate and the recombinant antigens. Thirty-three serum samples, 11 from SLA-positive patients and 22 from SLA negative controls were investigated. SLA antibodies were detected by an inhibition ELISA and confirmed by immunoblot using human liver homogenate. Autoantibody reactivity was further evaluated using preparations of primate and rat liver homogenates. Anti-α-enolase antibody reactivity has been tested by immunoblot using recombinant α-enolase. An affinity purified goat polyclonal anti-α-enolase IgG antibody was used as reference serum sample. Anti-tRNP(Ser)Sec antibody reactivity was detected by ELISA or dot blot using recombinant tRNP(Ser)Sec antigen.In an attempt to explain the observed discrepancy we have investigated reactivity of SLA positive sera against α-enolase and tRNP(Ser)Sec antibody serum gave a single band of ~50 kDa in both liver preparations. All but one anti-SLA antibody positive sera reacted with a ~50 kDa but none immunofixed a 48 kDa band. All anti-SLA antibody positive sera reacted strongly with the recombinant full length tRNP(Ser)Sec protein. None of the anti-SLA negative sera reacted with tRNP(Ser)Sec. Anti-SLA positive, and anti-SLA negative sera reacted equally against recombinant α-enolase by immunoblot. Pre-incubation of anti-SLA positive sera with tRNP(Ser)Sec completely abolished the 50 kDa band. The findings of the present study indicate that α-enolase and tRNP(Ser)Sec are both expressed in primate and rat liver and have a respective MW of 48 and 50 kDa. They also show that anti-tRNP(Ser)Sec – but not anti-α-enolase – correlates with anti-SLA antibody reactivity.The affinity purified IgG antibody directed to human α-enolase gave a band of approximately 48 kDa in both human and rat liver homogenates. A high titre anti-tRNP(Ser)Sec is the most likely target of anti-SLA.Our findings indicate that tRNP In pediibraries -4,7. Anton ELISA .(Ser)Sec is the sole target of anti-SLA antibodies [Using anti-SLA positive sera against rat liver cytosolic fraction in one and two-dimensional immunoblotting analyses and through peptide mass fingerprint analysis, following MALDI-TOF mass spectrometry, Ballot et al.  identifitibodies -8. Crititibodies . Moreovetibodies -18.(Ser)Sec antigen as competitor in inhibition experiments it has been found removal of the 50 kDa band immunofixed by SLA positive sera from immunoblots of primate liver homogenate [(Ser)Sec as a major component of SLA, a view apparently shared by Ballot et al, several questions still remain unanswered:Using recombinant tRNPmogenate . Though 1. Are there any differences in α-enolase expression between rat – used by Ballot et al  – and pr(Ser)Sec is due to its presence in trace amounts in the supernatant of liver homogenate [2. Is it true that failure of proteomic analysis to detect tRNPmogenate ?3. What is the reactivity of SLA positive and negative sera against recombinant α-enolase?(Ser)Sec by the screening of cDNA libraries? Do α-enolase and tRNP(Ser)Sec cross-react?4. How do we explain the apparent paradox of SLA being identified as α-enolase by proteomic analysis and as tRNP(Ser)Sec using rat and primate liver homogenate and the recombinant antigens.In the present study, we have investigated reactivity of SLA positive sera against α-enolase and tRNPThirty-three serum samples, 11 from SLA-positive patients and 22 from SLA negative controls were investigated. SLA-positive patients included 8 paediatric patients with AIH1 [7 female, median age 12, range 5–17 years, all ANA positive, median immunofluorescence (IFL) titre: 1/320, range 1/80–1/1280] and 3 adults with AIH/PBC overlap syndrome, , all but one AMA positive (1/5120), and ANA positive (median tire 1/320, range 1/80–1/640). Eleven case-matched SLA negative patients were tested as pathological controls including 8 with AIH1 and 3 with AIH/PBC overlap syndrome. Eleven demographically matched healthy subjects including 8 children  and 3 adults  negative for SLA were also tested as controls.All sera have been tested for conventional antibodies by indirect IFL using rodent liver, kidney, stomach tissues.SLA antibodies were detected by a modified inhibition ELISA  and confAn affinity purified goat polyclonal anti-α-enolase IgG antibody raised against a peptide mapping near the carboxyl-terminus of human α-enolase, which is common to α, β, and γ isoforms of mouse, rat and human enolase  was used as reference serum sample at a dilution of 1:100, according to the manufacturer's instructions.(Ser)Sec antibody reactivity was detected by ELISA or dot blot using recombinant tRNP(Ser)Sec antigen (Euroimmun). A high-titre anti-tRNP(Ser)Sec antibody positive serum was used as a positive control.Anti-tRNP(Ser)Sec, inhibition experiments were performed using 3 anti-SLA positive serum samples, diluted at 1/1000, and pre-incubated with solid phase recombinant tRNP(Ser)Sec (Euroimmun, UK), as previously described [To investigate whether the 50 kDa band immunofixed by anti-SLA is tRNPescribed .(Ser)Sec antibody serum gave a single band of ~50 kDa in both liver preparations Sec antibody serum in rat liver preparations Sec protein both in ELISA (mean titre 87 ± 23 RU/ml, cut off: 20 RU/ml) and dot blot. None of the anti-SLA negative sera reacted with tRNP(Ser)Sec. Anti-SLA positive, and anti-SLA negative sera reacted equally against recombinant α-enolase by immunoblot with 5/9 cases in each group giving a strong band Sec completely abolished the 50 kDa band immunofixed in either rat or human liver preparation. In contrast, a parallel experiment where the anti-α-enolase antiserum was pre-incubated with recombinant tRNP(Ser)Sec left unaltered the reactivity to the 48 kDa band.Pre-incubation of anti-SLA positive sera with tRNP(Ser)Sec are both expressed in primate and rat liver and have a respective MW of 48 and 50 kDa. They also show that anti-tRNP(Ser)Sec – but not anti-α-enolase – correlates with anti-SLA antibody reactivity suggesting that the target of anti-SLA antibody is tRNP(Ser)Sec and not α-enolase [The findings of the present study indicate that α-enolase and tRNP-enolase -9,19.(Ser)Sec. Ballot et al [(Ser)Sec.However, Ballot et al  state thot et al , however(Ser)Sec is the most likely target of anti-SLA.All the above observations indicate that tRNP(Ser)Sec, tRNA-associated antigenic proteinAIH, autoimmune hepatitis; SLA, soluble liver antigen; tRNPThe author(s) declare that they have no competing interests.DPB designed the study, performed the ELISA and inhibition experiments and contributed in writing the report. DG performed the immunoblot testing of anti-enolase antibodies. IB, SL & YM performed the immunoblot experiments involving liver preparations. RRM helped with the artwork. GMV provided clinical material and contributed in writing the report. DV supervised the study and wrote the report.


Virtual reality (VR) is an innovative tool for sensorimotor rehabilitation increasingly being employed in clinical and community settings. Despite the growing interest in VR, few studies have determined the validity of movements made in VR environments with respect to real physical environments. The goal of this study was to compare movements done in physical and virtual environments in adults with motor deficits to those in healthy individuals.The participants were 8 healthy adults and 7 adults with mild left hemiparesis due to stroke. Kinematics of functional arm movements involving reaching, grasping and releasing made in physical and virtual environments were analyzed in two phases: 1) reaching and grasping the ball and 2) ball transport and release. The virtual environment included interaction with an object on a 2D computer screen and haptic force feedback from a virtual ball. Temporal and spatial parameters of reaching and grasping were determined for each phase.Individuals in both groups were able to reach, grasp, transport, place and release the virtual and real ball using similar movement strategies. In healthy subjects, reaching and grasping movements in both environments were similar but these subjects used less wrist extension and more elbow extension to place the ball on the virtual vertical surface. Participants with hemiparesis made slower movements in both environments compared to healthy subjects and during transport and placing of the ball, trajectories were more curved and interjoint coordination was altered. Despite these differences, patients with hemiparesis also tended to use less wrist extension during the whole movement and more elbow extension at the end of the placing phase.Differences in movements made by healthy subjects in the two environments may be explained by the use of a 2D instead of a 3D virtual environment and the absence of haptic feedback from the VR target. Despite these differences, our findings suggest that both healthy subjects and individuals with motor deficits used similar movement strategies when grasping and placing a ball in the two reality conditions. This suggests that training of arm movements in VR environments may be a valid approach to the rehabilitation of patients with motor disorders. Virtual reality (VR) is a computer-based, multisensory interactive simulation occurring at the same speed and time as events in the physical world. Different levels of immersion can be achieved ranging from complete 3D (cave, head-mounted display) to partial 2D (computer display, TV screen) with different hardware configurations. Interface devices (computer mouse, joystick, force sensor, cyberglove) allow the user to move in and interact with objects in the virtual environment. Of crucial relevance to rehabilitation is the potential for increasing the user's level of interaction with their real physical environment so as to maximize their return to community life . The effNeurophysiologists and rehabilitation specialists like physical and occupational therapists are beginning to be interested in VR as a tool to study motor control and to evaluate and treat motor deficits secondary to central nervous system lesions such as stroke . The useThis study focused on the possibility of using virtual environments for the retraining of arm motor function in individuals with hemiparesis due to stroke. Major barriers to arm motor recovery after stroke are coordination deficits and the use of maladaptive movement strategies for reaching and grasping. Patient motivation and movement repetition are key factors in motor recovery -12. CurrDespite the growing interest in the use of VR for motor retraining, it is not known if movements involving reaching and grasping objects in VR environments are performed in a manner similar to those done in the physical world. Thus, the goal of this study was to validate VR as a tool for studying reaching and grasping in healthy subjects and in individuals with hemiparesis by comparing movement kinematics of identical tasks made in a physical and a virtual environment. Since reaching and grasping deficits have been well characterized in individuals with hemiparesis -17, the Eight healthy subjects  and 7 adults with hemiparesis  with no prior experience with VR participated in the study. Potential participants were identified from discharge lists of Montreal area rehabilitation centres. Out of 17 medical charts screened, 12 patients met eligibility requirements according to study inclusion and exclusion criteria. Patients were included if they were under 60 years old, had sustained a single, non-traumatic unilateral stroke in the territory of the left middle cerebral artery and had arm paresis  on a computer screen placed 75 cm in front of subject's manubrium Figure . The virPrior to data collection, all participants practised the tasks in physical and virtual conditions (20 – 40 min). To better compare the participants' performance in the two environments, the glove and grasp devices were worn on the hand in both conditions Figure .Kinematic data from the right arm were recorded with 6 infrared-emitting diodes (IREDs) placed on the distal phalanx of the index and thumb, the distal head of the first metacarpal, the radial styloid process, the lateral epicondyle of the humerus and the acromion .Each trial was divided in two phases: 1) reaching and grasping the ball and 2) ball transport and release. For the first movement phase, 4 temporal and 4 spatial parameters of reaching and grasping were determined. Temporal parameters were movement time, time to peak wrist velocity (RPV), time to maximal hand aperture (RMGA), and the delay between them (RPV-RMGA). Spatial parameters were endpoint path curvature, maximal grip aperture, angular ranges of joint motion and elbow-shoulder interjoint coordination . For theMovement onsets and offsets of each phase were defined as the times at which the tangential velocity of the IRED on the index finger surpassed and remained above or fell and remained below 10% of the maximal peak velocity respectively. The temporal parameters  were normalized to movement time and the delay between them was calculated. For the spatial parameters, the curvature of the trajectory of the IRED on the index finger was estimated as the ratio between the actual trajectory length and a straight line segment between the initial and final positions . Joint ap < 0.05 was used, adjusted for multiple comparisons by type using the Bonferroni correction.Both parametric and non-parametric statistics were used. For within-group comparisons between the two conditions of reality, Student t-tests were used. However, since variances were not homogeneous (Levene's test) for healthy subjects and participants with hemiparesis, non-parametric tests were used for between-group comparisons . A significance level of All healthy subjects and participants with mild upper limb motor deficits were able to reach, grasp, transport, place and release the virtual ball using movement strategies that were similar to those used for the physical ball Tables  and 3. AIn healthy subjects, the temporal and spatial aspects of the two phases of the task were almost identical between the physical and virtual conditions Tables , 3. HoweMovements made by individuals with hemiparesis in the physical environment differed from those made by healthy subjects in three ways. In both phases, movements were significantly slower and in the second phase, trajectories were more curved and interjoint coordination was altered Tables  and 3. IThe similarity in movement kinematics between physical and virtual reaching and grasping suggests that virtual reality may be an effective environment for rehabilitation. Interest in training in virtual environments is increasing amongst rehabilitation professionals in light of recent evidence suggesting that neuronal recovery after stroke-related brain damage critically depends on the motivation of the individual and the intensity of training ,11. VirtOur results show that subjects tended to decrease wrist extension and increase elbow extension in the virtual compared to the physical condition. Two principal factors may explain those differences: the absence of depth perception in the VR condition and the absence of tactile feedback at the end of the reach.Binocular vision enables humans to perceive depth in a 3 dimensional (3D) environment. This faculty is called stereopsis (for a review see ). Since The difference in depth perception in the 2D virtual environment may also be responsible for the tendency to increase elbow extension in both groups. Previous studies have shown that fine motor corrections are produced by distal joints . The 2D The absence of depth perception cannot explain the decrease of wrist extension at the end of the second phase of the movement in VR compared to the physical condition. A more likely explanation involves the type of haptic feedback provided to the subject. In the physical condition, subjects had to extend the wrist so that the ball and not their fingers would make contact with the target. In the VR condition, wrist extension was not necessary because subjects only had to place the ball at the coordinates of the virtual wall without encountering a physical barrier. To avoid such differences between physical and VR environments, relevant haptic feedback is necessary to indicate contact of the hand with the object or target. Another way is to integrate physical objects into the VR environment such as the manipulation of a real paper envelope in real-time in a VR environment , or the Overall, the finding that both healthy subjects and individuals with motor deficits used similar movement strategies in a physical and a limited virtual environment suggests that VR technology is a valuable tool for studying and retraining reaching, grasping and placing movements. Whether movement kinematics may be improved with the use of interfaces providing stereopsis and more relevant haptic feedback should be investigated by comparing movements made in immersive to those made in non-immersive virtual environments.


Public Library of Science has grown from a grassroots movement to a nonprofit publisher, in order to catalyze change towards open-access publishing of the scientific literature Communication among scientists has undergone a revolution in the last decade, with the movement of scientific publication to a digital medium and the emergence of the Internet as the primary means for distributing information. Millions of articles are, in principle, just a mouse click away from our computers. For many of us, PDFs have replaced printed journals as the primary form in which we read about the work of our colleagues.Yet we have barely begun to realize the potential of this technological change. For practicing scientists, it provides myriad opportunities to expand and improve the ways we can use the scientific literature. Equally important, it is now possible to make our treasury of scientific information available to a much wider audience, including millions of students, teachers, physicians, scientists, and other potential readers, who do not have access to a research library that can afford to pay for journal subscriptions.PLoS Biology, we take on a new role as publishers, to demonstrate that high-quality journals can flourish without charging for access.We founded the Public Library of Science three years ago to work toward realizing these opportunities. We began as a grassroots organization of scientists, advocating the establishment and growth of online public libraries of science, such as the National Institutes of Health's PubMed Central, to provide free and unrestricted access to the scientific literature. Today, with the launch of PLoS Biology, and every PLoS journal to follow, will be an open-access publication–everything we publish will immediately be freely available to anyone, anywhere, to download, print, distribute, read, and use without charge or other restrictions, as long as proper attribution of authorship is maintained. Our open-access journals will retain all of the qualities we value in scientific journals—high standards of quality and integrity, rigorous and fair peer-review, expert editorial oversight, high production standards, a distinctive identity, and independence. Although most readers will be satisfied with the free and unrestricted use of the online edition (including the right to print their own copies), a printed edition of PLoS Biology will be made available, for the cost of printing and distribution, to readers who prefer the convenience and browseability of the traditional paper format. And the full contents of every issue will immediately be placed in the National Library of Medicine's public online archive, PubMed Central, guaranteeing their permanent preservation and free accessibility.Our aim is to catalyze a revolution in scientific publishing by providing a compelling demonstration of the value and feasibility of open-access publication. If we succeed, everyone who has access to a computer and an Internet connection will be a keystroke away from our living treasury of scientific and medical knowledge. This online public library of science will form a valuable resource for science education, lead to more informed healthcare decisions by doctors and patients, level the playing field for scientists in smaller or less wealthy institutions, and ensure that no one will be unable to read an important paper just because his or her institution does not subscribe to a particular journal.Open access will also enable scientists to begin transforming the scientific literature into something far more useful than the electronic equivalent of millions of individual articles in rows of journals on library shelves. The ability to search, in an instant, an entire scientific library for particular terms or concepts, for methods, data, and images—and instantly retrieve the results—is only the beginning. Freeing the information in the scientific literature from the fixed sequence of pages and the arbitrary boundaries drawn by journals or publishers— the electronic vestiges of paper publication—opens up myriad new possibilities for navigating, integrating, “mining,” annotating, and mapping connections in the high-dimensional space of scientific knowledge.Consider how the open availability and freedom to use the complete archive of published DNA sequences in the GenBank, EMBL, and DDBJ databases inspired and enabled scientists to transform a collection of individual sequences into something incomparably richer. With great foresight, it was decided in the early 1980s that published DNA sequences should be deposited in a central repository, in a common format, where they could be freely accessed and used by anyone. Simply giving scientists free and unrestricted access to the raw sequences led them to develop the powerful methods, tools, and resources that have made the whole much greater than the sum of the individual sequences. Just one of the resulting software tools—BLAST—performs 500 trillion sequence comparisons annually! Imagine how impoverished biology and medicine would be today if published DNA sequences were treated like virtually every other kind of research publication—with no comprehensive database searches and no ability to freely download, reorganize, and reanalyze sequences. Now imagine the possibilities if the same creative explosion that was fueled by open access to DNA sequences were to occur for the much larger body of published scientific results.The benefits of open access are incontestable. The questions and concerns that remain focus on finances. As everyone acknowledges, publishing a scientific journal costs money—the more rigorous the peer review, the more efficient and expert the editorial oversight, the more added features and the higher the production standards, the greater the cost to publishers. Most journals today depend on subscriptions and site-licensing fees for most of their revenue. Since these access tolls are incompatible with open access, how will newly formed open-access journals pay their bills, and how will the traditional journals that have served the scientific community for many years survive in an open-access world?http://dx.doi.org/10.1371/journal.pbio.0000036.sd001)—we would retain a robust and competitive publishing industry and gain the benefit of universal open access.Because publishing is an integral part of the research process, a natural alternative to the subscription model is to consider the significant but relatively small costs of open-access publication as one of the fundamental costs of doing research. The institutions that sponsor research intend for the results to be made available to the scientific community and the public. If these research sponsors also paid the essential costs of publication—amounting, by most estimates, to less than 1% of the total spent on sponsored research . Because scientists are so dependent on ready access to previously published work, publishers are able to set their prices with little fear of subscription cancellations. Indeed, journal prices have been rising at a rate well in excess of inflation, straining the budgets of universities, hospitals, and research institutions. Open access would eliminate monopolies over essential published results, diminishing profit margins and creating a more efficient market for scientific publishing—a market in which publishers would compete to provide the best value to authors  at the best price.In recent months, we have witnessed a remarkable surge of awareness and support for open-access publication, both within the scientific community and in the public at large, exemplified by recent newspaper articles and editorials supporting PLoS and open access; by the recent introduction of the Public Access to Science Act in the United States Congress; by the Bethesda Workshop on Open Access; and by public statements of support from organizations as diverse as the NIH Council of Public Representatives, the Association of Research Libraries, and the Susan G. Komen Breast Cancer Foundation. Achieving universal open access will require action from funding agencies and institutions.PLoS Biology. Other major institutional sponsors of biomedical research are actively considering similar policies.The Howard Hughes Medical Institute, the largest private sponsor of biomedical research in the United States, has already taken a leading role in promoting open access. They will provide each of their investigators with supplemental funds to cover the costs of publishing in open-access journals like Private foundations with a commitment to science and education have contributed generously to this cause. Like any new business, PLoS needed to raise funds to cover our startup costs. A generous grant from the Gordon and Betty Moore Foundation enabled PLoS to launch our nonprofit publishing venture. Other individuals and organizations, notably the Irving A. Hansen Foundation, also provided generous and welcome support. These start-up funds made it possible for us to assemble an outstanding editorial board and staff, who have today accomplished the extraordinary feat of launching a new publisher and a premiere journal from scratch in less than nine months.PLoS Biology and BioMed Central's Journal of Biology to lead the way. These new journals face a double challenge. First, we are introducing an unfamiliar model—open-access publication. Second, any new journal, even one with the stringent standards and the extraordinary editorial team of PLoS Biology, must begin without the established “brand name” of the older journals, which, like a designer logo, elevates the perceived status of the articles that bear it. With all that is at stake in the choice of a journal in which to publish—career advancement, grant support, attracting good students and fellows—scientists who believe in the principle of open access and wish to support it are confronted with a difficult dilemma. We applaud the courage and pioneering spirit of the authors who have chosen to send to a fledgling journal the outstanding articles you will read in the premiere issues of PLoS Biology. In the end, it's the contributions of these authors that will make PLoS Biology a success.The opposition of most established journals to open access has left it to new journals like


Emerging Themes in Epidemiology is a new, online Open Access journal. This editorial – which coincides with the Journal's launch – describes its unique review and publication model. The editorial board and review process of ETE will be managed by research degree students and will therefore be a training ground for students . With our mix of Open Access publishing and the strong involvement of students in editing and reviewing, we believe that Emerging Themes in Epidemiology will be a progressive medium for promoting new ideas in epidemiology. Emerging Themes in Epidemiology (ETE) was conceived late on a Friday afternoon. As the rest of the academic and professional work week was coming to an end, the creators of this journal were just beginning to think straight. Clearly the work of students. However, it was not just clandestine meetings at unsociable hours that brought this diverse group of epidemiology PhD students together to launch and direct a new journal.The idea that has given birth to ETE, which will be a training ground for PhD students.Through training and various research projects PhD students become versed in the technical, methodological and practical aspects of epidemiology. While some will stay in academia and some will use their skills in other sectors of public health, our time as PhD students is seen as a time to learn and a time to be a part of the academic community. However, aside from the occasional correspondence with editorial boards, research students receive little training or involvement in the process of scientific publication. We think that this is an important process for us to learn about since, as researchers, our careers are dependent on publication. So we came together to form Emerging Themes in Epidemiology, the philosophical underpinnings of the Journal have been illustrated. Now, some explanation is required of how we will achieve our vision of ETE.This Open Access journal will also be our contribution to the field of epidemiology. Students will manage the editorial board and they will be a part of the review committee for each paper. Through this process we hope to learn. But, we also hope to facilitate the publication of new ideas and to raise issues that concern us as students of epidemiology. In the other editorial in this launch 'issue' of ETE is made up of doctoral students and faculty. The research students are from various academic and public health institutes in London (though we are open to the prospect of overseas expansion). The student members are responsible for the running of the Journal, for managing the review process and communicating with authors and readers. The board is headed by our Editor-in-Chief, Professor Peter Smith, and is overseen by an international panel of Associate Editors. The role of these senior faculty is to consider the major decisions of the Board and to preside over final publication judgements. And, because PhD studentship is a transient state (of varying length), these Associate Editors will maintain the continuity of ETE by recruiting new student editors as the current board graduates.The editorial board of At present, there is a lively debate as to whether peer-review should be 'open' or 'closed'. Recognizing the forceful arguments on both sides, we believe that the scientific community is best served by a system that encourages a constructive dialogue between colleagues. Authors and reviewers will know each others' name and affiliation. However, we recognize that some highly specialized fields of research are small and competitive. In reality, this means that there are often repercussions for giving both criticism and praise to a colleague's work. So at the reviewers' request, we will withhold their names from the authors.ETE, it is assigned to one of our two Deputy Editors. A committee of five research students then screens the manuscript to decide whether it fits within the scope of the Journal. If they decide it does, the paper is sent for external peer review. All papers are seen by a minimum of two reviewers. As part of our dedication to involving research students in the editorial process and facilitating the publication of new ideas, at least one PhD student and one expert in the appropriate field will be asked to review each paper. How will PhD students perform as reviewers? There is evidence that young reviewers (under 40 years old) trained in epidemiology or statistics, and those who can dedicate about 3 hours provide better quality reviews [When a paper is submitted to  reviews . ClearlyETE. Authors can therefore be confident that their papers will have the highest visibility and will be available in PubMed's archives.Final decisions to accept or reject will always be done with the endorsement of one of the Associate Editors, who are senior researchers in various epidemiologic fields. The involvement of students in the review process is admittedly novel, but we believe there are sufficient checks and balances in our review system to ensure quality. Testament to this is the decision by PubMed and PubMed Central to index ETE will encourage openness from authors as well as reviewers. An individual's financial or professional associations do not necessarily constitute conflicts of interest, but failing to declare them can be highly misleading to the reader and the scientific community [The issue of conflict of interest is anathema to many scientists. We are trained to pursue objectivity; the suggestion that one's methods, results or conclusions are in any way affected by funding sources or other affiliations can be seen as a direct challenge to credibility. It shouldn't be. In the complex funding and political environments in which we work, we can no longer deny that these issues must be faced. Evidence has mounted that funding is associated with research outcomes (in tobacco and food) and, recently, high-profile cases related to vaccine safety and nutritional supplements have furthered the case for explicit statements of potential conflicts -5. Once ommunity .ETE is a journal dedicated to active discourse in epidemiology. We will ask contributors to disclose their source(s) of funding – as it could be seen to influence opinions or research agenda. We will also ask authors to disclose links to other associations that are not explicit from their institutional affiliation.Perhaps there is no discipline more appropriate for open-access publication than epidemiologic research and the allied population health sciences. Our work has the aim of revealing the determinants of disease in human populations, with the ultimate goal, through knowledge empowerment, intervention and policy, to reduce suffering from ill health. With such an ethos, how can we justify keeping research findings locked away in expensive medical journals, inaccessible to those who may need it most?The open-access publishing model has the potential to revolutionize the way authors use scientific literature and the manner in which it is received by other scientists and the community at large. The advent of open-access publishing means "it is now possible to make our treasury of scientific information available to a much wider audience, including millions of students, teachers, physicians, scientists, and other potential readers, who do not have access to a research library that can afford to pay for journal subscriptions" .Currently, the most common economic model for sustaining Open Access journals is one where the author pays for the costs incurred in the editorial and publishing process. BioMed Central, our publisher, charges authors per accepted publication (currently $525), but fees are waived for authors from institutions with membership to BioMed Central or authors with financial hardship. Our hope is that a long-term solution will in future be developed by the scientific community as a whole, perhaps through the provision of grant schemes for Open Access publication. Nonetheless, recognizing the difficulties with the current 'author pays' model, we aim to cover the article processing fees for at least the next few years for all submissions not already covered under institutional memberships or other grants or subsidies.ETE will call for papers on a particular subject pertinent to the theory and practice of epidemiology. The series of articles will be built around a framework, designed by the editorial board and a guest editor. The series of articles will be published in tandem, and will be highlighted on a special page on the website. The flexibility of online publishing will allow for articles submitted after the initial publication of the issue and for commentary on the original articles to also be included. Guest editors for special issues will work with the Editorial Board to develop an issue, commission appropriate articles and write a leading editorial. 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A mixed-effects model repeated measures approach (MMRM) was specified as the primary analysis in the Phase III clinical trials of duloxetine for the treatment of major depressive disorder (MDD). Analysis of covariance using the last observation carried forward approach to impute missing values (LOCF_ANCOVA) was specified as a secondary analysis. Previous research has shown that MMRM and LOCF_ANCOVA yield identical endpoint results when no data are missing, while MMRM is more robust to biases from missing data and thereby provides superior control of Type I and Type II error compared with LOCF_ANCOVA. We compared results from MMRM and LOCF_ANCOVA analyses across eight clinical trials of duloxetine in order to investigate how the choice of primary analysis may influence interpretations of efficacy.a priori in the various protocols were included in the comparisons.Results were obtained from the eight acute-phase clinical trials that formed the basis of duloxetine's New Drug Application for the treatment of MDD. All 202 mean change analyses from the 20 rating scale total scores and subscales specified rd decimal place . For the HAMD17 total score, the primary outcome in all studies, MMRM yielded 9/12 (75%) significant contrasts, compared with 6/12 (50%) for LOCF_ANCOVA. The expected success rate was 80%.In 166/202 comparisons (82.2%), MMRM and LOCF_ANCOVA agreed with regard to the statistical significance of the differences between duloxetine and placebo. In 25/202 cases (12.4%), MMRM yielded a significant difference when LOCF_ANCOVA did not, while in 11/202 cases (5.4%), LOCF_ANCOVA produced a significant difference when MMRM did not. In 110/202 comparisons (54.4%) the p-value from MMRM was lower than that from LOCF_ANCOVA, while in 69/202 comparisons (34.2%), the p-value from LOCF_ANCOVA was lower than that from MMRM. In the remaining 23 comparisons (11.4%), the p-values from LOCF_ANCOVA and MMRM were equal when rounded to the 3Important differences exist between MMRM and LOCF_ANCOVA. Empirical research has clearly demonstrated the theoretical advantages of MMRM over LOCF_ANCOVA. However, interpretations regarding the efficacy of duloxetine in MDD were unaffected by the choice of analytical technique. Treatment effects are often evaluated by comparing change over time in outcome measures. However, valid analyses of longitudinal data can be problematic, particularly if some data are missing for reasons related to the outcome measure ,2. SinceA common method of analyzing clinical trial data is to use analysis of variance or analysis of covariance (ANOVA or ANCOVA) with missing data imputed by the last observation carried forward approach (LOCF_ANCOVA). The popularity of LOCF_ANCOVA may be due to its simplicity, and also the belief that violations of the restrictive assumptions inherent to LOCF_ANCOVA lead to a conservative analysis . ConsideOne such method, termed MMRM (Mixed Model Repeated Measures ), has beThe MMRM approach was specified as the primary analysis in the Phase III clinical trials of duloxetine for the treatment of major depressive disorder (MDD), while LOCF_ANCOVA was specified as a secondary analysis. In the present investigation, we provide a comprehensive summary of results from MMRM and LOCF_ANCOVA in the eight acute-phase clinical trials that formed the basis for duloxetine's New Drug Application (NDA) for MDD. The primary objective of this investigation was to determine whether differences in results between MMRM and LOCF_ANCOVA influenced conclusions regarding the efficacy of duloxetine.The data source for this investigation was the eight acute-phase clinical trials in which duloxetine was compared with placebo in the treatment of MDD. Relevant details of these studies are highlighted in Table a priori in the various protocols to be analyzed for mean change from baseline to endpoint, and were collected at more than one postbaseline time point  With regard to statistical significance of the difference between duloxetine and placebo, the proportion of outcomes showing agreement between MMRM and LOCF_ANCOVA was compared with the proportion of outcomes for which MMRM and LOCF_ANCOVA yielded disparate results; 2) The proportion of outcomes for which MMRM yielded the lowest p-value was compared with the corresponding proportion for LOCF_ANCOVA; 3) The number of outcomes for which "substantial evidence of efficacy" was demonstrated. In regulatory settings, the criterion for substantial evidence of efficacy is frequently the demonstration of a statistically significant advantage over placebo in two or more studies. This criterion was utilized here to define substantial evidence of efficacy for a particular outcome.The frequency of lower p-values provides a "fine-tuned" measure of sensitivity of the two analytic methods. However, in certain cases such an assessment may actually be misleading. For example, to distinguish between p= .800 and p = .810, or between p =. 002 and p =. 003, implies that the methods yielded different results when in fact the similarities far outweigh the differences. Hence, it is equally appropriate to simply categorize based upon the presence or absence of a significant difference. Furthermore, given the large number of outcomes assessed across the eight studies, it would not be surprising to see the two methods disagree with regard to statistical significance on at least a small number of outcomes. Therefore, perhaps the most clinically meaningful summary measure is the number of outcomes for which substantial evidence of efficacy was demonstrated.17) total score, the HAMD17 Maier subscale, and the Visual Analog Scale (VAS) for overall pain severity. The HAMD17 total score was an obvious choice as it was the primary outcome in all studies. The other outcomes were selected since they are frequently focal points in manuscripts and presentations regarding duloxetine's efficacy. Finally, we provide case studies to help explain how and why results from MMRM and LOCF_ANCOVA may differ.Three outcomes were selected for more detailed presentation of results: the 17-item Hamilton Rating Scale for Depression , MMRM and LOCF_ANCOVA agreed with regard to the statistical significance of the difference between duloxetine and placebo. In 25 cases (12.4%) MMRM yielded a significant difference whereas LOCF_ANCOVA did not, while in 11 cases (5.4%) LOCF_ANCOVA yielded a significant difference when MMRM did not.17 total scores, leading to a three-fold greater standard error.Both methods tended to yield significance more frequently in depression rating scales and subscales than in outcomes related to somatic and painful physical symptoms. The studies were generally underpowered for these secondary somatic and pain outcomes owing to the greater variance in changes score for these outcomes. For example, the variance in VAS overall pain severity was approximately nine-fold greater than the variance in HAMDrd decimal place .In 110 of the 202 outcomes (54.4%) the p-value from MMRM was lower than that from LOCF_ANCOVA, while in 69 cases (34.2%) the p-value from LOCF_ANCOVA was lower than that from MMRM. In the remaining 23 cases (11.4%) the p-values from LOCF_ANCOVA and MMRM were equal when rounded to the 317 total score, HAMD17 Maier subscale, and VAS overall pain) are presented in Table 17 total score, the advantage of duloxetine over placebo in mean change from baseline to endpoint from MMRM analyses was greater than the corresponding advantage from LOCF_ANCOVA in 9/12 comparisons  are used to further illustrate MMRM and LOCF_ANCOVA  results. Results from these studies were originally reported by Detke et al [17 total score tended to increase over time whereas duloxetine's advantage in VAS overall pain was greatest at intermediate visits  from MMRM in Studies 1 and 2 were 4.86 (p < .001) and 2.17 (p = .024), respectively. The corresponding advantages from LOCF_ANCOVA were 3.80 (p < .001) and 1.73 (p = .048). Although the differences were significant for both methods in both studies, MMRM yielded treatment contrasts that were approximately 25% greater than LOCF_ANCOVA.In the case of the HAMDFor VAS overall pain, the advantage of duloxetine over placebo at endpoint from MMRM in Studies 1 and 2 were 5.88 (p = .055) and 4.40 (p = .135), respectively. The corresponding advantages from LOCF_ANCOVA were 6.91 (p = .019) and 5.17 (p = .037). In both studies, the endpoint differences were significant from LOCF_ANCOVA, but not from MMRM. The LOCF_ANCOVA treatment contrasts were approximately 15% greater than those from MMRM.17 total score and VAS overall pain.Standard errors from LOCF_ANCOVA were approximately 5% smaller than the Week 9 standard errors from MMRM for both the HAMDIn many areas of clinical research, the impact of missing data can be profound ,12-14. TConsiderable advances in statistical methodology, and in our ability to implement these methods, have been made in recent years. Methods such as MMRM, which require less restrictive assumptions regarding missing data, may now be easily implemented with standard software ,5,18,19.No universally superior approach to analysis of longitudinal data exists. However, a series of studies -9 demonsetc. to blind analysts to the treatment names, Molenberghs et al [Similar results regarding control of Type I and Type II error for LOCF_ANCOVA and mixed-effects model analyses have been obtained independently ,20-22. Fhs et al  concludeThe theoretical differences between MMRM and LOCF_ANCOVA have been summarized ,18, estaThe VAS pain results highlight a limitation of endpoint analyses of any type, namely that they provide only a snapshot view of the response profile. From LOCF_ANCOVA analysis, one can only conclude that drug was superior to placebo at endpoint. However, MMRM analysis reveals that drug had a significant effect early in the trials, but that advantage was somewhat transitory as the placebo group tended to "catch up" over time. In order to understand the response profile of a drug, the entire longitudinal profile should be considered . From MMIn the duloxetine database, results from MMRM and LOCF_ANCOVA were in general agreement regarding substantial evidence of efficacy and frequency of significant differences. However, MMRM tended to be more sensitive to drug-placebo differences for outcomes related to overall depressive symptoms and core emotional symptoms of depression, with mean advantages over placebo that were 10% to 20% greater than LOCF_ANCOVA. However, MMRM did not universally increase duloxetine's advantage over placebo in comparison to results from LOCF_ANCOVA. For example, in somatic and painful physical symptom outcomes, results from LOCF_ANCOVA showed mean advantages over placebo that were approximately 40% greater than that from MMRM.Therefore, while the overall conclusions regarding the efficacy of duloxetine were unaffected by the choice of analytic method, this should not mask the important differences between MMRM and LOCF_ANCOVA. The advantages of MMRM and similar methods over LOCF_ANCOVA have been conclusively demonstrated in many studies and are evident in the duloxetine data.17 total score), while LOCF_ANCOVA produced a 50% success rate, in comparison to the expected rate of 80%. While many factors may reduce the success rate in Phase III clinical trials, the use of a statistical method with known inflation of Type II error  is an obvious suspect.Khan et al  compiled17 total score) when LOCF did not was in the Phase II study (Study 3).An unduly high rate of false negative results could be especially problematic in early phases of drug development where only one or two chances exist to make the correct decision regarding the efficacy of a drug. It is noteworthy that one of the instances when MMMRM yielded a significant difference on the primary outcome  is an obvious suspect. Thus, the unexpectedly low success rate in Phase III is consistent with the conclusion that LOCF_ANCOVA inflates Type I error .Hence, results from the duloxetine NDA are consistent with research suggesting a move away from LOCF_ANCOVA and other simple analytic techniques to methods such as MMRM that are more robust to the biases from missing data.Important differences exist between MMRM and LOCF_ANCOVA. Research has clearly demonstrated the advantages of MMRM over LOCF_ANCOVA. However, interpretations regarding the efficacy of duloxetine in MDD were unaffected by the choice of analytical technique.Drs. Mallinckrodt, Raskin, Wohlreich, Watkin, and Detke are employees of Eli Lilly and Company.CHM designed the statistical analyses, and participated in interpretation of data and drafting of the manuscript. JR, MMW, JGW, and MJD participated in interpretation of data and drafting of the manuscript. All authors read and approved the final manuscript.The pre-publication history for this paper can be accessed here:


The exclusion of individuals with elevated acute phase proteins has been advocated in order to improve prevalence estimates of vitamin A deficiency in surveys, but it is unclear whether this will lead to sampling bias. The purpose of the study was to determine whether the exclusion of individuals with elevated acute phase proteins is associated with sampling bias and to characterize inflammation in children with night blindness.1-acid glycoprotein (AGP) were measured.In a survey in the Republic of the Marshall Islands involving 281 children, aged 1–5 years, serum retinol, C-reactive protein (CRP), and αP = 0.03) and with anemia was 58.3% and 35.7% (P = 0.029), respectively. The proportion of children with serum retinol <0.70 μmol/L was 52.0% after excluding children with elevated acute phase proteins. Among children with and without elevated acute phase proteins, mean age was 2.8 vs 3.2 years (P = 0.016), the proportion of boys was 43.1% vs. 54.3% (P = 0.075), with no hospitalizations in the last year was 11.0% vs 23.6% (P = 0.024), and with anemia was 43.8% vs 31.7% (P = 0.05), respectively.Of 281 children, 24 (8.5%) had night blindness and 165 (58.7%) had serum retinol <0.70 μmol/L. Of 248 children with AGP and CRP measurements, 123 (49.6%) had elevated acute phase proteins (CRP >5 mg/L and/or AGP >1000 mg/L). Among children with and without night blindness, the proportion with serum retinol <0.70 μmol/L was 79.2% and 56.8% (Exclusion of children with inflammation in this survey of vitamin A deficiency does not improve prevalence estimates for vitamin A deficiency and instead leads to sampling bias for variables such as age, gender, anemia, and hospitalization history. Vitamin A is essential for normal immune function, hematopoiesis, growth, and vision [Vitamin A deficiency is a major cause of morbidity and mortality among preschool children in developing countries . Vitamind vision . Among pd vision ,3, recend vision , and lowd vision . The synd vision ,6,7.The identification of populations at risk for vitamin A deficiency is important in order to target groups that would benefit from interventions to improve vitamin A status. Xerophthalmia, such as night blindness and Bitot spots, is used to determine the prevalence of clinical vitamin A deficiency in populations . Other sWe hypothesized that exclusion of individuals with elevated acute phase proteins, and selection of only apparently healthy children would lead to lead to sampling bias in a survey of vitamin A deficiency. A secondary hypothesis was that children with night blindness would have elevated acute phase proteins. Vitamin A deficiency has been recognized as a major public health problem in many areas of the South Pacific region , includi2. The sampling strategy for the study was based on the 1988 census of the Republic of the Marshall Islands, which provided data on the average number of children of the target age group within each household, determined by dividing the number of children in a locality by the number of households in the same location. This number was then divided into the number of children to be sampled to obtain the number of households to be visited. Households to be visited were chosen by systematic sampling of every fifth household. When available, the birth dates of the children were ascertained from the children's health cards; otherwise, the birth dates were obtained by asking the parent or guardian. The survey teams consisted of at least one Marshallese-speaking health care worker, a phlebotomist, and a medical doctor.A population-based survey, the Republic of the Marshall Islands Vitamin A Deficiency Study, was conducted between November 1994 and March 1995 to assess the prevalence of vitamin A deficiency among children, aged 1–5, in the Republic of the Marshall Islands. The Republic of the Marshall Islands (population 68,000) is a complex of 28 coral atolls and 5 small (non-atoll) islands, with a total of 1136 islands spreading over a very small land area of 134 kmOf the children in the survey, a systematic subsample of every fifth child was obtained in which a standardized questionnaire was used to collect information on various risk factors such as night blindness, demographics, breastfeeding, visits to the hospital, presence of a vegetable garden, and food consumption. Parents were asked if the child currently had night blindness. A history of night blindness has been shown to be a reliable indicator of vitamin A deficiency . Oral ing × 10 min, room temperature). Aliquots of serum were made, kept in liquid nitrogen or at -70°C, and shipped by overnight air express from the Republic of the Marshall Islands to New York City. Retinol remains stable at -70°C for 15 y or more [1-acid glycoprotein (AGP) was measured using radial immunodiffusion assay (Bindarid, The Binding Site, Birmingham, UK). Interleukin-6 (IL-6) was analyzed using a high sensitivity commercial enzyme-linked immunosorbent assay (R&D systems, Inc, Minneapolis, MN). Serum retinol, CRP, AGP, and IL-6 values were available for 281, 250, 248, and 176 of 281 pre-school children, respectively. Missing values were due to inadequate volume of sera. Controls provided by the manufacturer were used to measure intra- and inter-assay coefficients of variation (CV) in laboratory analysis. For serum retinol, the within-assay and between-assay CVs were 3% and 8%, respectively. For serum CRP, AGP, and IL-6, the within-assay and between-assay CVs were 4.1 and 5.0, 3.9 and 1.6, and 3.2 and 2.9, respectively. The study protocol was approved by the institutional review board of the Pacific Health Research Institute of Hawaii and the Ministry of Health and Environment of the Republic of the Marshall Islands. The study was conducted in accordance with the Helsinki Declaration.Blood samples were obtained by venipuncture. Hemoglobin was measured using a HemoCue instrument (HemoCue, Inc., Mission Viejo, CA). Venous blood samples were collected in red-top serum separator tubes and immediately wrapped in aluminum foil, continuously shielded from light, and stored at 4°C until centrifugation : Ailuk (16), Arno (26), Enejelar (3), Enewetak (14), Kwajalein (73), Majuro (77), Namu (32), Utrik (25), and Wotje (15). There were 141 boys and 140 girls, and the mean (±standard deviation) age of the children was 2.93 ± 1.38 years. Due to limited sample volume, serum CRP, AGP, and IL-6 were not measured in 31, 33, and 105 pre-school children, respectively. Of the 281 children, there were 24 (8.5%) with night blindness and 165 (58.7%) with serum retinol <0.70 μmol/L. Of 248 children who had both AGP and CRP measured, there were 123 (49.6)% with inflammation (CRP >5 mg/L and/or AGP >1000 mg/L).P = 0.016), were more likely to have been hospitalized in the last year (P = 0.024), and were more likely to be anemic (P = 0.05) compared with children without inflammation. Mean retinol concentration was lower (P = 0.0005) and the proportion of children with serum retinol <0.70 μmol/L was higher (P = 0.013) among children with inflammation compared to children without inflammation. The findings suggest that children with inflammation were more likely to be girls (P = 0.075) compared to children without inflammation. There were no significant differences in the proportion with night blindness, the mean number of people living in the house with the child, current breastfeeding status, and the presence of a vegetable garden at home between children with and without inflammation.The characteristics of children with and without inflammation are shown in Table P = 0.08).Of the 281 children 58.7% had serum retinol <0.70 μmol/L. In contrast, if children with inflammation are excluded as proposed by Thurnham and colleagues , the proP = 0.03), had lower serum retinol (P = 0.007), were more likely to have serum retinol <0.70 μmol/L (P = 0.03), and to be anemic (P = 0.029) compared to children without night blindness. The findings were suggestive that there may be a higher proportion of boys than girls who are night blind (P = 0.09), and with lower mean hemoglobin concentrations (P = 0.09). There were no significant differences in geometric mean AGP, CRP, IL-6, or inflammation between children with and without night blindness.The characteristics of children with and without night blindness are shown in Table P < 0.0001 for both). Serum AGP and CRP individually were inversely correlated with serum hemoglobin (P < 0.0001) and retinol (P = 0.0004). IL-6 was positively associated with CRP (P < 0.0001), and inversely associated with retinol (P < 0.0001). Hemoglobin had low correlation with retinol that was of borderline significance (P = 0.09) and low inverse correlation with IL-6 that was also of borderline significance (P = 0.08).Spearman correlations of serum AGP, CRP, IL-6, retinol, and hemoglobin are shown in Table In this population of children from the Republic of the Marshall Islands, more than half had serum retinol concentrations <0.70 μmol/L. According to criteria of the World Health Organization, vitamin A deficiency is considered a public health problem if more than 15% of the population has serum or plasma retinol concentrations <0.70 μmol/L . VitaminNearly half of the children in this study had inflammation, as indicated by elevated AGP and/or CRP. These findings suggest that the prevalence of subclinical infection is high in this population that has a high prevalence of vitamin A deficiency. These findings are consistent with the concept that the syndrome of vitamin A deficiency is associated with depressed immunity and increased infections ,7. ChildAlthough the exclusion of individuals with elevated acute phase proteins has been advocated to improve prevalence of vitamin A deficiency in surveys that rely upon plasma or serum retinol concentrations , this stThe alternative method of having serum retinol concentrations "adjusted using acute phase proteins"  involvesChildren with night blindness had significantly lower serum retinol concentrations compared with children without night blindness, a finding that is consistent with previous studies ,7,15. ThThe relationship between IL-6 and elevated acute phase proteins has not well been characterized in epidemiological studies of vitamin A deficiency. IL-6 is a proinflammatory cytokine that plays a role in the upregulation of CRP  and AGP In summary, the method of excluding individuals with elevated acute phase proteins from this survey of vitamin A deficiency resulted in sampling bias and a prevalence estimate that was based upon about half of the original sample. The remaining sample was healthier, older, less likely to have been hospitalized, and with a higher proportion of boys and a lower proportion with anemia. The method of excluding individuals with elevated acute phase proteins may lead to underestimation of the prevalence of vitamin A deficiency. Vitamin A deficiency remains a major problem in many developing countries worldwide, and further studies are needed to develop unbiased epidemiological methods for the estimation of the prevalence of vitamin A deficiency in populations.


The high rate of antibiotic prescriptions general practitioners (GPs) make for respiratory tract infections (RTI) are often explained by non-medical reasons e.g. an effort to meet patient expectations. Additionally, it is known that GPs to some extent believe in the necessity of antibiotic treatment in patients with assumed bacterial infections and therefore attempt to distinguish between viral and bacterial infections by history taking and physical examination. The influence of patient complaints and physical examination findings on GPs' prescribing behaviour was mostly investigated by indirect methods such as questionnaires.Direct, structured observation during a winter "cough an cold period" in 30 (single handed) general practices. All 273 patients with symptoms of RTI  were included.2 = 0,696). Efforts to reduce antibiotic prescribing, e.g. recommendations for self-medication, counselling on home remedies or delayed antibiotic prescribing were rare.The most frequent diagnoses were 'uncomplicated upper RTI/common cold' (43%) followed by 'bronchitis' (26%). On average, 1.8 : 1.7–2.0) medicines per patient were prescribed (cough-and-cold preparations in 88% of the patients, antibiotics in 49%). Medical predictors of antibiotic prescribing were pathological findings in physical examination such as coated tonsils (odds ratio (OR) 15.4, 95%-CI: 3.6–66.2) and unspecific symptoms like fatigue (OR 3.1, 95%-CI 1.4–6.7), fever (OR 2.2, 95%-CI: 1.1–4.5) and yellow sputum (OR 2.1, 95%-CI: 1.1–4.1). Analysed predictors explained 70% of the variance of antibiotic prescribing  predictors. The method of direct observation was shown to be accepted both by patients and GPs and offered detailed insights into the GP-patient-interaction. Over-prescribing in respiratory tract infections (RTI) has been the topic of numerous studies in general practice. Explanations for the overuse of antibiotics despite weak scientific evidence are multifaceted and vary from efforts to protect patients from "complicated" courses of disease to assumed patient expectations and to inadequate knowledge of physicians -4. RecenWe performed a structured, direct observation-based study. This concept is borrowed from social and cultural anthropology developed in the early 1960s . StartinData collection focused on patient complaints, results of physical examinations, further diagnostic procedures and diagnoses. Physical examination findings were coded according to the level of precision obtained . The registration of medication included prescriptions as well as recommendations for over-the-counter medicines (OTC) and the dispensing of drug samples . Data were mostly acquired through observation during the consultation.Potentially participating GPs were randomly selected from a GP register and addressed with a form letter. Of 62 GPs addressed, 30 participated this study . To avoid biasing GPs' behaviour no specific hypotheses were shared with the participants.GPs were visited for one day by S. F.  during a "cough and cold" period in winter; and all consecutive patients (age above 14 years) with symptoms consistent with RTI were included. All patients were informed that a medical student wanted to participate in the consultation and all of them accepted her attendance.SAS software (Version 8.1) was used to analyse the data . RespiraGPs' median age was 48 years, their median experience in general practice was 12 years and 17% of the GPs were female. A total of 273 patients (51% women) were included . The median number of included patients per GP was 9. No patient refused participation. Patients' median age was 37 years (range 14 to 88).Upper respiratory tract infections/common cold (URTI), bronchitis, tonsillitis and sinusitis were the dominating diagnoses and 88% of all patients got at least one of these diagnoses Table . In tota49 patients (18%) were asked by their GPs about self-medication and 83% of them acknowledged the previous use of OTC- drugs (predominantly symptomatic cough and cold drugs, particularly mucolytics). In 40% of these patients, GPs prescribed an OTC-preparation of the same ATC-classification. The use of household remedies was addressed in 12% of the patient encounters and three-quarter of the patients confirmed that they tried them before the consultation .On average 1.8 (95%-CI: 1.68–1.95) drugs were prescribed per patient Table . Only 12As shown in table antibiotics (Table 2 = 0,695). Patients' age, smoking status and symptoms such as cough, sore throat, hoarseness, sputum, sneezing, headache and earache had no influence. Also, GPs' characteristics influenced the prescription rate: Although patients' age was uniformly distributed between younger and older GPs, younger GPs (<50 years) prescribed fewer antibiotics than older ones did (37.3 versus 54.4% of the patients, p < 0.05). Neither the number of treated patients  nor the GPs' experience in general practice (measured as time of practicing) had influence on antibiotic prescription rates.Multiple logistic regression analyses demonstrated associations between patient complaints, physical examination results and the prescription of cs Table . The calCough and cold preparations were associated with symptoms such as cough (OR 5.09, 95%-CI: 2.43–10.69), headache (OR 2.43, 95%-CI: 1.02–5.83), sneezing (OR 2.39, 95%-CI: 1.2–4.76), and abnormal findings in throat examination (OR 2.16, 95%-CI: 1.11–4.2). The calculated model explained 19% of the variance of cough and cold preparation prescriptions (R2 = 0.186).Recommendations for non-pharmaceutical treatment were given to 35% patients . The advice of increased fluid intake was given to 27% of patients with the prescription of acetylcysteine (which is recommended in the package insert). One quarter of smokers were advised to stop smoking.A sickness certification was issued to 57% of the professionally active patients for a median duration of 4 days. 18% declined the proposal of a sick note.43% of the patients were asked to return for a "control visit" and 21% were informed to revisit if they became worse. Patients with the diagnoses 'tonsillitis' (in 63%) and 'bronchitis' (61%) were more often asked for a "control visit" than patients with the diagnoses 'URTI/ common cold' (23%).Our observational study confirmed the overuse of antibiotics and cough and cold preparations despite the lack of scientific evidence. The analysis of predictive indicators for the prescription showed that antibiotic prescribing was associated with specific patient symptoms and physical examination results whereas the prescription of cough and cold preparations was mostly performed indiscriminately. Efforts to reduce antibiotic prescribing, e.g. recommendations for self-medication, counselling on home remedies or delayed antibiotic prescribing were rare.RTI consultation rates of up to one fifth of all patients seen a day may appear high when compared internationally . HoweverIn our study, the demonstrated antibiotic prescription rate was high in comparison to other European investigations and rather approaches US-American levels ,8. ParalIn our observation, patient complaints and physical findings such as 'pathologically altered tonsils' or 'wheezing/rales' in lung auscultation have been shown to be strong predictors of GPs prescribing behaviour comparable to recent investigations ,6. BesidDiagnoses made by GPs were strong predictors of prescribing behaviour. Patients with the diagnosis 'URTI/ common cold' received significantly fewer antibiotics than those with 'tonsillitis', 'sinusitis' or 'bronchitis'. Despite the controversial scientific evidence for antibiotic therapy in those conditions, the majority of participating GPs seemed to be convinced that antibiotics were necessary or likely to be helpful ,5,22. FuComplementary to quantitative analysis direct observation offered interesting insights in the GP-patient-consultation. Only one patient asked for an antibiotic prescription, which is in contrast to the common belief about patient expectations ,23. SurpThe impact of non-medical reasons of prescribing should not be disregarded . Our obsPrescription of cough and cold preparations in patients with acute RTI is not supported by scientific evidence -29, in pInternational comparisons of OTC-prescription rates are restricted because of different health insurance systems (and refunding policies) in different countries. Recent data from Belgium – a country with an at least partially comparable health system – showed high prescription rates for cough and cold preparations, too . InteresThe fact that only 19% of the variance of cough and cold preparation prescriptions was explained by clinical signs and patient symptoms supports the assumption that non-medical reasons dominated the decision to prescribe. Frequently, the term "pseudo-placebo" is used in this context to describe the function of those preparations  and assuEighty-three percent of patients asked confirmed the use of OTC-preparations before the consultation, which were often identical with the subsequently prescribed preparations. GPs did not appreciate the opportunity to encourage this self-medication and cost reducing patient behaviour, perhaps because they were afraid that patients would not consult them with similar complaints at another time . In thisThe method of direct observation allowed for detailed and sensitive insights in GP-patient-consultations. Nevertheless, investigators must be aware of the specific disadvantages of the method (e.g. the "observer effect").Furthermore, we could demonstrate that patient complaints and physical examination results had a strong impact on GPs prescribing behaviour, especially on antibiotic prescription. Although this GP behaviour is not in accordance with actual scientific evidence, GPs' understanding of pathogenesis and the value of clinical signs should be strongly considered when efforts are made to reduce the overuse of antibiotics in primary care.ATC code = "Anatomic, Therapeutic, Chemical Classification" codeCI = Confidence intervalGP = General practitionerOR = Odds ratioRTI = Respiratory tract infectionURTI = Upper respiratory tract infectionThe author(s) declare that they have no competing interests.TF participated in the study design, analysis, interpretation of data and drafted the manuscript. SF participated in the study design, analysis, interpretation of data, draft of manuscript and performed the practice observations. MMK participated in the study design and draft of manuscript. EHP participated in the study design, analysis, interpretation of data and draft of the manuscript. All authors read and approved the final manuscript.The pre-publication history for this paper can be accessed here: