Tinnitus is the perception of a sound in the absence of an objective physical source. Up to now, there is no generally accepted view how these phantom sounds come about, and also no efficient treatment. Patients are turning to complementary or alternative medical therapies, such as acupuncture. Based on the theory of traditional Chinese medicine, acupoints located on both the adjacent and distal area of the disease can be needled to treat disease. Furthermore, the way of combining acupoints is for strengthening the curative effect. We aim to evaluate the efficacy of acupuncture at local points in combination with distal points in subjective tinnitus patients.This trial is a randomized, single-blind, controlled study. A total of 112 participants will be randomly assigned to one of four treatment groups receiving acupuncture treatment for 4 weeks. The primary outcome measure is subjective tinnitus loudness and annoyance perception, which is graded using the Visual Analogue Scale (VAS). The assessment is at baseline (before treatment initiation), 4 weeks after the first acupuncture session, and 8 weeks after the first acupuncture session.Completion of this trial will help to identify whether acupuncture at local acupoints in combination with distal acupoints may be more effective than needling points separately.International Standard Randomized Controlled Trial Number Register: ISRCTN29230777 Subjective tinnitus is a frequent auditory sensation  experienced in the absence of an external or internal acoustic stimulus. An estimated prevalence in the adult population across studies is about 10% to 15% , where 1Acupuncture has been used to treat tinnitus for a long time. Neuroscience studies related the effects of acupuncture to neuronal stimulus, activation of endogenous opioid mechanisms and neuropeptides which stimulate specific brain structures . It has The aim of our study is to evaluate the efficacy of acupuncture at local points in combination with distal points in subjective tinnitus patients.We perform the study according to common guidelines for clinical trials /WHO Good Clinical Practice standards (GCP) including certification by an external audit). The trial protocol has been approved by the Research Ethical Committee of Beijing Hospital of Traditional Chinese Medicine Affiliated to Capital Medical University. This trial was registered with ISR CTN at Current Controlled Trials (ISRCTN29230777).Patients will be recruited in acupuncture clinic, Beijing Hospital of Traditional Chinese Medicine affiliated to Capital Medical University with a target sample size of 112 subjects. The trial is executed from February 2012 to December 2014.Patients who require for all of the following conditions will be considered for enrollment:1. Diagnosis of subjective tinnitus, unilateral or bilateral2. Age 18 to 65 years, either sex3. Tinnitus duration of at least 3 months4. Not to receive another treatment during the clinical trial period5. Written and informed consent1. Objective tinnitus 2. Underlying disease or history:2.1. Cerebral vascular events2.2. Neurodegenerative disorders2.3. Otitis media2.4. Acoustic tumor2.5. Prior brain surgery2.6. Inner ear malformation2.7. Head trauma2.8. Ototoxic drug medication3. Women in pregnancy and lactation or without contraception4. Inability to correct use of test equipment: unable to cooperate during audiologic examination5. Patients who cannot communicate reliably with the investigator or who are not likely to cope with the requirements of the trialThis trial is a randomized, single-blind, controlled study. Participants will receive acupuncture for 4 weeks. All candidates go through a standardized interview and receive more information about the study and the treatments. They also undergo audiological testing of hearing thresholds, minimal masking levels, and loudness discomfort levels carried out by an audiologist in Tongren Hospital affiliated to Capital Medical University.Time points are as follows Figure :Visit 1: screeningVisit 2: treatment initiation, participants will receive acupuncture for 4 weeksVisit 3: 4 weeks after first acupuncture, follow-up and treatment finishVisit 4: 8 weeks after first acupuncture, follow-upPatients who meet the inclusion criteria and none of the exclusion criteria are randomized to one of four treatment groups: group A will receive acupuncture at local acupoints in combination with distal acupoints; group B will receive acupuncture at local acupoints in combination with distal non-acupoints; group C will receive acupuncture at local non-acupoints in combination with distal acupoints; group D will receive acupuncture at local non-acupoints in combination with distal non-acupoints. Acupuncture points selected as follows Figure :Distal non-acupoint I: the radial side of forearm, the ulner side of the middle of the line joining Shaohai (HT 3) and Shenmen (HT 7), between heart meridian of hand-shaoyin and small intestine meridian of hand-taiyangDistal non-acupoint II: the radial side of forearm, the junction of the upper 1/4 and lower 3/4 of the line joining HT 3 and HT 7, between heart meridian of hand-shaoyin and small intestine meridian of hand-taiyangDistal non-acupoint III: lateral side of lower leg, 3 cun above the tip of external malleolus, 1.5 cun behind anterior crest of the tibia, between the stomach meridian of foot-yangming and gall bladder meridian of foot-shaoyangDistal non-acupoint IV: lateral to the shank, 3 cun below Yanglingquan (GB34), the midway between gall bladder meridian of foot-shaoyang and bladder meridian of foot-taiyangLocal non-acupoint I: one finger-breadth (middle finger) inferior to Daying (ST5) in the body of mandibleLocal non-acupoint II: midpoint between Yangbai (GB14) and the midline of the head, between governor meridian and gall bladder meridian of foot-shaoyangLocal non-acupoint III: high spot of the angle of mandibleLocal non-aupoint IV: on zygomatic arch, the midway between ST 7 and GB 3Acupuncture will be performed with sterile needles by one therapist with more than 22 years of experience and an acupuncture license from the Chinese medicine practitioner license from the Ministry of Health of the People’s Republic of China. Treatment will be conducted over a period of 4 weeks, at a frequency of two sessions per week. No additional treatment is allowed.A research coordinator screen and enroll participants at clinic. After participants completed a baseline evaluation, another research coordinator who is uninvolved with data collection randomly assigns them to one of four treatment groups by using a computer-generated, blocked random-allocation sequence. The random list is generated with SAS software . This research coordinator informs the acupuncturist of the treatment assignment.The patients, data collection staff, and data analysts are blinded during the study period. The acupuncturist is not blinded to the treatments they delivered because acupuncture manipulation makes it impossible. During the intervention, acupuncturist and the personnel who collect data are segregated immediately after the treatment start and are instructed not to exchange information with each other.Subjective tinnitus loudness and annoyance perception is graded using the VAS. The assessment is at baseline (before treatment initiation), 4 weeks after the first acupuncture session, and 8 weeks after the first acupuncture session.1.1. F: Functional subscale (11 factors)1.2. E: Emotional subscale (9 factors)1.3. C: Catastrophic subscale (5 factors)versus week 8).Each question of the THI can be answered by the patient with either ‘often’ (4 points), ‘sometimes’ (2 points), or ‘never’ (0 points) with a maximum total score of 100 points indicating most severe suffering from tinnitus. The assessment is at baseline (before treatment initiation), 4 weeks after the first acupuncture, and 8 weeks after the first acupuncture.Participants should report the adverse events they experienced, including discomfort or bruising at the sites of needle insertion, nausea, or feeling faint after each treatment.The trial aims to detect a difference in loudness and annoyance perception of tinnitus between the four study groups. This is deemed a clinically significant difference of average 20-point reduction on the VAS scores of loudness in acupuncture group when comparing with the sham-control group based on previously published studies . The staThe following formula was used for a four-group trial :Calculations are performed using 80% power, a 5% significance level (two-side). The required sample size is approximately 24 participants for each group. We plan to enroll a total of 112 participants with 28 in each group, allowing for a 25% withdrawal rate.Data will be analyzed according to the 2×2 randomized factorial study designs. The statistical model includes two fixed factors . Interaction is evaluated using the interaction term in the ANOVA model and by visual assessment of profile plots. As no interaction between local acupoints and distal acupoints is detected, results are presented as a factorial design .Data analysis will be conducted by statisticians who are independent from the research team. Every analysis is conducted using the SPSS software (SPSS 12.0 KO for Windows ©).We have presented the design and the protocol for the randomized controlled trial of acupuncture at local and distant points for subjective tinnitus. Completion of this trial will help to identify whether acupuncture at local acupoints in combination with distal acupoints may be more effective than needling points separately.It is hoped that this study will encourage renewed interest in acupuncture’s potential for treating people with tinnitus. Recent researches have created a climate where acupuncture might be assumed to be ineffective . HoweverIn this study, the side-effects of acupuncture will be reported by patients. Several large surveys have also provided evidence that acupuncture is a relatively safe treatment . The resPatients are informed in a manner suggesting that two different types of acupuncture treatment were compared. One type is traditional, another is new, and the effect of both is uncertain, not mentioning terms such as ‘placebo’ or ‘sham’. Similar strategies of informed consent have been used in most previous acupuncture trials .However, our study has several limitations. Deciding on an appropriate control procedure for clinical studies on acupuncture is a particular challenge . PreviouOne limitation concerns the fact that this is a single-center study. It does not lend the results to great generalizability to more diverse sets of patients in more diverse settings. Our directed acupuncture treatment is uniformly applied, and adjunctive therapies are not used. In addition, acupuncturists in diverse settings with different philosophies, backgrounds, training, and clinical experiences might have chosen different technique for the directed acupuncture intervention. Therefore, the external validity can be somewhat hard to achieve. But we ensure that the risk of contamination between groups will be minimized, while maintaining the integrity of the experimental group comparison. As a result, whether the findings can be generalized to the clinical setting is unclear and more work is needed.Another limitation is that the therapist is not blinded in the present trial. A blinded study of acupuncture is challenging to conduct because it is almost impossible to blind acupuncturists to the treatments they are delivering. This study does not allow us to determine whether the observed effectiveness is due to placebo effects, intensity of provider contact, or a physiologic effect of needling. Therefore, a bias due to unblinding cannot be ruled out. In addition, the sample size in the present study is relatively small.The trial is currently in recruitment phase.The authors declare that they have no competing interests.CZL conceived of the study and prepared the initial protocol. LPW made amendments and participated in design of the trial protocol. GXS drafted the manuscript and participated in design of the study. LLH participated in the development of the acupuncture point protocol. QQL and LYL participated in the design of the study. All authors read and approved the final manuscript.


HIV/AIDS related stigma is a major barrier to uptake of HIV testing and counselling (HTC). We assessed the extent of stigmatising attitudes expressed by participants offered community-based HTC, and their anticipated stigma from others to assess relationship with HIV test uptake. From these data, we constructed a brief stigma scale for use around the time of HIV testing.Adult members of 60 households in urban Blantyre, Malawi, were selected using population-weighted random cluster sampling and offered HTC with the option to self-test before confirmatory HTC. Prior to HTC a 15-item HIV stigma questionnaire was administered. We used association testing and principal components analysis (PCA) to construct a scale measure of stigma. Of 226 adults invited to participate, 216 (95.6%) completed questionnaires and 198/216 (91.7%) opted to undergo HTC . Stigmatising attitudes were uncommon, but anticipated stigma was common, especially fearing verbal abuse (22%) or being abandoned by their partner (11%). Three questions showed little association or consistency with the remaining 12 stigma questions and were not included in the final scale. For the 12-question final scale, Cronbach's alpha was 0.75. Level of stigma was not associated with previously having tested for HIV (p = 0.318) or agreeing to HTC (p = 0.379), but was associated with expressed worry about being or becoming HIV infected (p = 0.003).Anticipated stigma prior to HTC was common among both men and women. However, the high uptake of HTC suggests that this did not translate into reluctance to accept community-based testing. We constructed a brief scale to measure stigma at the time of HIV testing that could rapidly identify individuals requiring additional support following diagnosis and monitor the impact of increasing availability of community-based HTC on prevalence of stigma. In sub-Saharan Africa, where two-thirds of the world's people living with HIV reside In Malawi, the adult HIV prevalence was estimated to be 11.9% in 2007 HIV/AIDS-related stigma is a well-documented barrier towards the uptake of HIV/AIDS related services Research on the consequences and public health implications of stigma for people living in high HIV prevalence countries has focused on two areas We previously undertook a pilot study to assess the feasibility, acceptability and accuracy of supervised self-testing for HIV using oral mouth swab kits in urban communities in Blantyre, Malawi The College of Medicine of Malawi Research Ethic Committee (COMREC) and the London School of Hygiene and Tropical Medicine Ethics Committee granted ethical approval for the study. All participants provided written informed consent to participate.The study was a prevalence study of stigmatising attitudes towards HIV, nested within a cross-sectional community-based random sample survey of feasibility and acceptability of community-based HTC. Community health worker catchment areas  in the three high-density residential suburbs of northwest of Blantyre were delineated by circumferential walks, with global positioning system (GPS) mapping of boundaries. Four of 51 catchment areas were randomly selected with probability proportional to size. Satellite maps were used to randomly select 15 dwellings from each of these four catchment areas. Selected dwellings were visited to identify all households . A single household-per-dwelling was then randomly selected, and all adults aged 16 years and over residing within the household were invited to participate in the study.Individuals who agreed to participate underwent a baseline interviewer-administered questionnaire that collected information on demographic characteristics and experience of previous HIV testing. A stigma questionnaire comprising of 15 questions was undertaken that asked about attitudes towards people living with HIV (“stigmatising attitudes”) and how they felt they would be perceived if they were to be diagnosed with HIV (“anticipated stigma”). Each question was answered on a three-item Likert-type scale.The stigma questionnaire was based upon the UNAIDS People Living with HIV Stigma Index Following completion of questionnaires, participants were offered three HTC options: self-testing plus confirmatory standard HTC , standard HTC alone, or no testing. Pre- and result-based post-test counselling was provided to all participants accepting HTC. Oral self-testing was conducted using OraQuick ADVANCE HIV I/II  followed by confirmatory finger-prick testing using Determine  and Unigold , with a third test  in the event of discordant results. Data describing test performance and acceptability have been published elsewhere Participants who were found to be HIV positive were given a referral form with instructions to attend their nearest primary health care centre for comprehensive HIV care, including assessment for ART eligibility.Demographic characteristics and responses to stigma questions were tabulated. Chi-squared tests were used to examine associations between each question, and gender differences in question responses.Principal components analysis of the 15 stigma questions was undertaken to examine patterns of dependence, to assess whether any questions did not fit within the construct that was being measured by the other questions, and to identify whether different constructs were being measured by the “stigmatising attitudes” and “anticipated stigma” questions. Cronbach's alpha coefficient and correlations between each item and the scale formed by the remaining questions were calculated to measure the internal consistency of the questions Variables were coded in an ordinal fashion from 0 to 2, with 0 representing the lowest degree of stigma exhibited in a response, and 2 the highest. A stigma score was calculated by taking the sum of the scores for each of the items included in the final scale. After attempting a number of transformations to normalise the data, we recoded the final stigma scale into four approximately equally sized groups corresponding to none, low, medium and high levels of stigma. We used logistic regression to assess the impact of stigma on uptake of HIV self-testing and on worry about being diagnosed with HIV. We used ordered logistic regression to assess the following potential predictors of stigma: gender, age, marital status, education, poverty (as measured by problems getting food in the last month), previous HIV testing and personal knowledge of someone who has died of HIV. Multivariable regression was used to adjust for potential confounders.Statistical analysis was undertaken using STATA 11.1 .Between March and July 2010, 226 household members were randomly selected and invited to participate in the study; 216 (95.6%) consented to take part and completed questionnaires. Of these 198/216 (91.7%) opted to undergo supervised oral self-testing followed by standard HTC. Baseline characteristics of study participants are shown in Responses to the 15 stigma questions are given in Similar response patterns of responses to questions were seen between men and women were found. Of interest, a high proportion of both men (66.0%) and women (58.2%) felt that HIV-positive women should be prevented from having babies . Additionally, a similar proportion of men (11.3%) and women (10.0%) feared that their partner would leave them should they be diagnosed HIV-positive . Women (26%) were less likely than men (45%) to want others to know if someone in the family was ill with HIV .Pairwise associations between responses to the 15 stigma questions were calculated and showed that response to question 6 (“Women with HIV should be prevented from having children”) was not associated with response to any other question. Additionally, question 15 (“Would you want others to know if a family member became sick with HIV”) and question 14 (“If a married person goes for HIV testing and counselling then he/she must be unfaithful”) were associated with only one and two other questions respectively. As anticipated, there appeared to be two main blocks of association corresponding to the two categories of questions (“stigmatising attitudes” and “anticipated stigma”).Coefficients of the first two principal components obtained in principal component analysis are shown in Cronbach's alpha for the combination of all 15 questions was 0.69, and was 0.75 when questions 6, 14 and 15 were removed. We also calculated Cronbach's alpha for two stigma subscales. For the stigmatising attitudes questions Cronbach's alpha was 0.66, but was 0.71 when question 6 was removed. For the anticipated stigma questions Cronbach's alpha was 0.61, but was 0.70 when questions 14 and 15 were removed. On the basis of these findings, for the final stigma scale we excluded questions 6, 14 and 15 and retained the remaining 12 questions. On the final 12-item scale, 111/216 (51.4%) of participants respondents responded with a strong degree of stigma to at least one question.The distribution of stigma scale scores calculated by taking the sum of each of the 12 items included in the final scale is shown in Using the stigma scale as the explanatory variable, in univariate logistic regression , we founThe main findings of this study are that a simple stigma scale was developed with good internal consistency, which had the capacity to separate individuals into those with and without stigmatising attitudes and anticipated stigma. Responses to 3 questions were not included in the final scale due to lack of explanation of variation in the data or lack of internal consistency. Our 12-item final scale constructed through principal components analysis demonstrated good internal consistency when questions asking after both stigmatising attitudes and anticipated stigma were included. The good consistency shown for subscales of stigmatising attitudes and anticipated stigma demonstrate that these more targeted scales could be used within specific situations. However, it will be important to validate both the final scale and subscales in larger and different populations.Using the final 12-item scale, a higher level of stigma was independently associated with worrying about being or becoming HIV-infected, but was not associated with past HIV test uptake or testing decision, although power to address the latter question was limited by the high HTC acceptance rate of 92%.Brief stigma scales administered at the time of HIV testing, such as the one constructed, are potentially useful in monitoring changes in stigma over time in sentinel populations  and 95% reporting that teachers with HIV should be allowed to continue work (compared to 93% in our sample) The higher affirmative response to the question “Would you fear getting HIV from caring for a person with HIV or AIDS?” could relate to lack of access to consumables  required for safe and hygienic management of severely ill HIV-infected individuals in impoverished homes. The question “Women with HIV should be prevented from having children” was a newly added question that interestingly had the highest stigmatising response rate (62%). This was not probed further in the current study but may reflect, first, the prominence during post-test counselling given to family planning and the need to avoid unprotected sex to prevent HIV super-infection even within concordant HIV-positive couples and, secondly, widespread awareness about the risk of mother-to-child transmission of HIV resulting from recent scale-up of programs to prevent mother to child transmission of HIV (PMTCT). Previous studies among health workers In contrast to the low prevalence of stigmatising attitudes, we found higher levels of anticipated stigma in certain questions, with participants concerned about being verbally abused or being left by their partner if they found to be HIV positive, and not wanting others to know if a family member was sick with HIV. This suggests that, whilst knowledge and awareness of HIV in Malawi has become widespread , although guided by reported utility in other settings Scales that incorporate questions on anticipated stigma, such as the one developed here, are important to capture all domains of stigma. A central criticism of previous studies that have developed scales to measure HIV stigma is that they have included only one stigma domain within their construct Our data suggest that the availability of community-based HTC could overcome some of these stigma-related barriers to HIV testing. The scale described here could be used to assess the impact of anticipated stigma on newly diagnosed HIV positive individuals' uptake of ART, a major current programmatic limitation within ART programmes in numerous high HIV prevalence countries We describe the construction of a simple scale measure of stigma encompassing both stigmatising attitudes and anticipated stigma that holds promise for a practical approach to quantifying stigma around the time of HIV testing. If generalisable, this scale could be used to monitor the impact of increasing availability of community-based HIV testing on prevalence of stigma over time in high prevalence settings and identify individuals at high risk of adverse experiences during the testing process. Additionally, it could allow investigation of the acceptability and feasibility of different HTC approaches (e.g. community-based versus facility-based) in the same population.


Video encoding for multimedia services over communication networks has significantly advanced in recent years with the development of the highly efficient and flexible H.264/AVC video coding standard and its SVC extension. The emerging H.265/HEVC video coding standard as well as 3D video coding further advance video coding for multimedia communications. This paper first gives an overview of these new video coding standards and then examines their implications for multimedia communications by studying the traffic characteristics of long videos encoded with the new coding standards. We review video coding advances from MPEG-2 and MPEG-4 Part 2 to H.264/AVC and its SVC and MVC extensions as well as H.265/HEVC. For single-layer  video, we compare H.265/HEVC and H.264/AVC in terms of video traffic and statistical multiplexing characteristics. Our study is the first to examine the H.265/HEVC traffic variability for long videos. We also illustrate the video traffic characteristics and statistical multiplexing of scalable video encoded with the SVC extension of H.264/AVC as well as 3D video encoded with the MVC extension of H.264/AVC. Network traffic forecasts, such as the Cisco Visual Networking Index , predictThe encoders that are used to compress video before network transport have significantly advanced in recent years. These video encoding advances have important implications for the network transport of encoded video. The purpose of this paper is to first give an overview of the recent developments in video coding. Then, we examine the traffic characteristics of long videos encoded with the recently developed video coding standards so as to illustrate their main implications for the transport of encoded video in communication networks.This paper covers three main areas of video coding advances: (i) efficient encoding of conventional two-dimensional 2D) video into a nonscalable video bitstream, that is, a video bitstream that is not explicitly designed to be scaled  during network transport, (ii) scalable video coding, that is, video coding that is explicitly designed to permit for scaling  so as to make the video traffic adaptive (elastic) during network transport, and (iii) efficient nonscalable encoding of three-dimensional 3D) video. For conventional 2D video, we start from the video coding standards MPEG-2 and MPEG-4 Part 2 and outline the advances in video coding that have led to the H.264/MPEG-4 Advanced Video Coding (H.264/AVC) standard   − 1 frame periods compared to the classical B frame prediction structure.If the dyadic hierarchical B frame structure of H.264/AVC and H.265/HEVC is employed, it imposes additional constraints on the timing of the frame transmissions compared to the classical B frame prediction employed in the preceding MPEG-2 and MPEG-4 Part 2 standards. Generally, a given frame can only be encoded MPEG-2 and MPEG-4 provide scalable video coding into a base layer giving a basic version of the video and one or several enhancement layers that improve the video quality. The quality layering can be done in the dimensions of temporal resolution (video frame frequency), spatial resolution , or SNR video quality. The layering in the SNR quality dimension employs coarse quantization (with high QP) for the base layer, and successively finer quantization  for the enhancement layers that successively improve the SNR video quality. These layered scalability modes permit scaling of the encoded video stream at the granularity of complete enhancement layers; for example, a network node can drop an enhancement layer if there is congestion downstream. MPEG-4 has a form of sublayer SNR quality scalability referred to as fine grained scalability (FGS). With FGS there is one enhancement layer that can be scaled at the granularity of individual Bytes of video encoding information. With both MPEG-2 and MPEG-4, the flexibility of scaling the encoded video stream comes at the expense of a relatively high encoding overhead that significantly reduces the RD efficiency of the encoding and results in very limited adoption of these scalability modes in practice.Similar to the preceding MPEG standards, the Scalable Video Coding (SVC) extension of H.264/AVC  providesH.264 SVC has a novel Medium Grain Scalability (MGS) that splits a given SNR quality enhancement layer of a given video frame into up to 16 MGS layers that facilitate highly flexible and RD efficient stream adaption during network transport.E = 25. When encoding this enhancement layer, the 16 coefficients resulting from the discrete cosine transform of a 4 × 4 block are split into MGS layers according to a weight vector (a similar splitting strategy is employed for larger blocks). For instance, for the weight vector W = , the 16 coefficients are split into six MGS layers as follows. The lowest frequency coefficient is assigned to the first MGS layer m = 1, the next two higher frequency coefficients are assigned to the second MGS layer m = 2, and so on, until the four highest frequency coefficients are assigned to the sixth MGS layer m = 6, the highest MGS layer in this example. For network transport, the base layer and each MGS layer of a given frame are encapsulated into a so-called network adaptation layer unit (NALU).As for all SNR quality scalable encodings, the base layer of an MGS encoding provides a coarse quantization with a relatively high QP; for example, B = 35 or 40. MGS encodings have typically one enhancement layer providing a fine quantization with a relatively small QP; for example, 8 in 1–B7 and B9–B15. It is therefore recommended to drop MGS layers first from the B frames without any dependent frames, that is, the odd-indexed B frames in the highest layer in When scaling down an MGS video stream at a network node, dropping MGS layers uniformly across the frame sequence results in low RD efficiency of the downscaled stream. This is due to the dependencies in the B frame hierarchy. Specifically, dropping an MGS layer from a B frame that other B frames depend on, for example, frame BAlternatively, MGS encodings can be conducted so that each NALU is assigned a priority ID between 0 indicating lowest priority and 63 indicating highest priority for RD efficiency. These priority IDs can be assigned by the video encoder based on RD optimization. For downscaling an MGS stream with priority IDs, a network node first drops MGS layers (NALUs) with priority ID 0, then priority ID 1, and so on.Sony HD video for the priority ID stream scaling. We compare the RD curves of the MGS streams with cascaded QPs (C) with the RD curve from single-layer encoding, whereby all encodings have a GoP structure with one I frame and β = 15 B frames with the prediction structure illustrated in E = 25, in In E = 25) gives somewhat lower RD efficiency and substantially higher traffic variability at the encoder output than the corresponding B = 35, E = 25 encoding. That is, the increased adaptation flexibility of the B = 40, E = 25 encoding comes at the expense of reduced RD efficiency and very high traffic variability reaching CoV values above 3.5 at the encoder output.Similar to the single-layer encoding, we observe from the comparison of MGS streams encoded without and with cascaded QPs in Figures E = 25 MGS, C encoding, the CoV values of the MGS encoding are close or slightly below the single-layer CoV values.We observe from Cmin plots in The 3D video employs views from two slightly shifted perspectives, commonly referred to as the left view and the right view, of a given scene. Displaying these two slightly different views gives viewers the perception of depth, that is, a three-dimensional (3D) video experience. Since two views are involved, 3D video is also sometimes referred to as stereoscopic video. The concept of employing multiple views from different perspectives can be extended to more than two views and is generally referred to as multiview video.3D video streaming requires the transport of the two sequences of video frames resulting from the two slightly different viewing perspectives over the network to the viewer. Since the two views capture the same scene, their video frame content is highly correlated. That is, there is a high level of redundant information in the two views that can be removed through encoding (compression). The Multiview Video Coding (MVC) standard builds on the inter-coding techniques that are applied across a temporal sequence of frames in single-layer video coding to extract the redundancy between the two views of 3D video. More specifically, MVC typically first encodes the left view and then predictively encodes the right view with respect to the left view.One approach to streaming the MVC encoded 3D video is to transmit the encoded left and right views as a frame sequence with twice the frame rate of the original video, that is, left view of first video frame (from first capture instant), right view of first video frame, left view of second video frame, right view of second video frame, and so on. Since the right view is encoded with respect to the left view, it is typically significantly smaller (in Bytes) and the sequence of alternating left and right views result in high traffic variability, as illustrated in the next section.multiview frame for transmission. The sequence of multiview frames has then the same frame rate as the original video.Another MVC streaming approach is to aggregate the left and right views from a given video frame (capture instant) into one An alternative encoding approach for 3D video is to sequence the left and right views to form a video stream with doubled frame frequency and feed this stream into a single-view video encoder, such as H.264 SVC or H.265/HEVC. This approach essentially translates the interview redundancies into redundancies among subsequent frames. Similar to MVC encoding, the two encoded views for a given capture instant can be transmitted sequentially or aggregated.Yet another encoding alternative is to downsample (subsample) the left and right views to fit into one frame of the original video resolution. For instance, the 1920 × 1080 pixels of left and right views are horizontally subsampled to 960 × 1080 pixels so that they fit side-by-side into one 1920 × 1080 frame. This side by side approach permits the use of conventional 2D video coding and transmission systems but requires interpolation at the receiver to obtain the left and right views at the original 960 × 1080 pixel resolution.β = 7 B frames between successive I and P frames and 16 frames  per GoP. We observe from Alice video compared to the IMAX video and (ii) that the QP cascading improves the RD efficiency by up to about 0.5 dB for the Alice video and almost 1 dB for the IMAX video. These different RD efficiency levels and RD efficiency increases with QP cascading are due to the different content of the two videos. The IMAX video is richer in texture and motion and thus “more difficult” to compress and higher RD efficiency gains can be achieved with improved coding strategies.In IMAX video gives higher frame size variability and that (ii) QP cascading increases the frame size variability, mirroring the above observations for single-view (2D) video. We also observe from We observe from Recent studies  indicateWe have given an overview of modern video coding standards for multimedia networking. We have outlined the advances in the main video coding standards for nonscalable (single-layer) video and scalable video. For single-layer video, we gave an overview of H.264/AVC and H.265/HEVC. We compared their rate-distortion (RD) and rate variability-distortion (VD) characteristics before and after smoothing as well as link bitrate requirements. This comparison included the first study of the traffic variability (before and after smoothing) and statistical multiplexing characteristics of H.265/HEVC encoding for long videos as well as an original study of the effects of cascading of quantization parameters (QPs) for the different levels of the hierarchical dyadic B frame prediction structure of H.264/AVC. We found that the advances in the video coding standards have led to increased RD efficiency, that is, higher video quality for a prescribed mean video bitrate, but also substantially increased traffic variability at the encoder output (which is effectively mitigated through smoothing). We also found that elementary smoothing with moderately sized buffers and statistical multiplexing during network transport translate the RD improvements of H.265/HEVC into commensurate reductions of the required network link bitrate .For scalable video coding, we gave a brief overview of H.264 SVC Medium Grain Scalability (MGS) and the scaling of an encoded H.264 SVC MGS video bitstream in a network node. We compared the RD and VD characteristics (before and after smoothing) as well as the link bitrate requirements of the scaled H.264 MGS stream with corresponding single-layer encodings. We illustrated that H.264 SVC MGS streams can be flexibly scaled in the network in the lower region of the quality adaptation range while maintaining RD efficiency and link bitrate requirements very close to the unscalable single-layer encodings. For 3D video, we outlined the encoding and streaming of the two views and examined the RD and VD characteristics of the streams.The H.264/AVC video coding standard formed the basis for the development of the highly efficient scalable video coding (SVC) extension as well as extensions for stereo and multiview video coding suitable for 3D video . Similar


Global health equity strategists have previously focused much on differences across countries. At first glance, the global health gap appears to result primarily from disparities between the developing and developed regions. We examine how much of this disparity could be attributed to within-country disparities in developing nations.We used data from Demographic and Health Surveys conducted between 1995 and 2010 in 67 developing countries. Using a population attributable risk approach, we computed the proportion of global under-five mortality gap and the absolute number of under-five deaths that would be reduced if the under-five mortality rate in each of these 67 countries was lowered to the level of the top 10% economic group in each country. As a sensitivity check, we also conducted comparable calculations using top 5% and the top 20% economic group.In 2007, approximately 6.6 million under-five deaths were observed in the 67 countries used in the analysis. This could be reduced to only 600,000 deaths if these countries had the same under-five mortality rate as developed countries. If the under-five mortality rate was lowered to the rate among the top 10% economic group in each of these countries, under-five deaths would be reduced to 3.7 million. This corresponds to a 48% reduction in the global mortality gap and 2.9 million under-five deaths averted. Using cutoff points of top 5% and top 20% economic groups showed reduction of 37% and 56% respectively in the global mortality gap. With these cutoff points, respectively 2.3 and 3.4 million under-five deaths would be averted.Under-five mortality disparities within developing countries account for roughly half of the global gap between developed and developing countries. Thus, within-country inequities deserve as much consideration as do inequalities between the world’s developing and developed regions. Increased attention has recently been given to economic disparities in health status, health service coverage, and their impact on health improvements in developing countries -5. In thOn the surface, calls for such a focus on health conditions among the poor within developing countries in order to reduce the global health gap might seem strange. By some standard measures, inequalities between the world’s developed and developing regions are far larger than intra-country disparities. Take the case of under-five mortality rates (U5MR), or the number of children dying between birth and age five per 1000 live births. U5MR is the health status indicator for which the greatest amount of relevant evidence is available and a key indicator for the fourth Millennium Development Goal of reducing U5MR by two-thirds between 1990 and 2015. A recent survey of 56 low- and middle-income countries (LMIC) found that the U5MR within the poorest 20% of a national population during the 1990s was, on average, almost twice as high as that in the population’s best-off 20% . Seen inWe argue in this paper that such conclusion would be misguided without careful analysis of the magnitude of intra- and inter-country disparities. Such an analysis would examine each kind of disparity’s contribution to the gap between developed and developing countries by applying a variant of the population attributable risk approach, widely used to estimate the reduction in mortality brought about by eliminating a particular cause of death.The purpose of our study is to determine how much of the under-five mortality gap between developed and developing countries can be reduced by improving mortality rates of developing countries to the level of their highest income groups. Although several studies, such as the “Global Health 2035” Lancet Commission report,  have recOur findings highlight the magnitude of mortality burden attributable to within-country inequalities and corroborate the need for focused priority policies toward reducing socioeconomic inequalities in health in developing countries.To estimate U5MR for different economic strata of developing country populations, we required data that 1) include both information about mortality and socio-economic status, and 2) are large enough to contain information on under-five deaths that provide reliable estimates on subgroups of the population. We selected most recent Demographic and Health Surveys (DHS), which meet the aforementioned criteria. We used the wealth index as indicator of economic status. The wealth index, a composite measure of a household economic status, is derived from household assets and characteristics using principal component analysis ,14. The Data on under-five mortality in the highest economic groups needed for the estimation of the intra-country mortality gap was derived by pooling all country datasets on interviewed women’s birth histories. Prior to pooling those datasets, the wealth index already available in each DHS dataset, was used to split the household population into groups of equal size. The principal focus was on the top 10% (decile) of the sample population; as sensitivity checks, we also selected the top 5% (ventile) and 20% (quintile) of the sampled population. The pooled dataset was used to compute U5MR for the top decile, ventile, and quintile of the total population of the 67 countries, using appropriate adjustment weights. The wealth quintile variable  was already available in each country dataset. The pooled U5MR was weighted such that the mortality rate obtained is equivalent to the weighted average of the 67 country-specific U5MRs, with weights representing the proportion of country births in the total number of births. The final weights used in the pooled dataset combined these latter weights with sampling weights provided in each dataset. We computed the U5MR using life table approach with a synthetic cohort approach .Application of the population attributable risk approach to determine the proportion of the total gap that is due to within-country disparities in U5MR involved three steps. The first is estimating the size of the global gap. The second consists of preparing an alternative estimate, to show how large the gap would be if mortality inequalities within developing countries were eliminated in the manner described below – that is, by improving health among low- and middle-income groups to the levels already enjoyed by the highest ones within the countries concerned. The third and final step involves comparing the estimates prepared in steps one and two.The estimate prepared in the first step will be called the “actual mortality gap” between developed and developing countries. It consists of the observed number of under-five deaths in developing countries, minus the number of such deaths were all economic groups in those countries to enjoy the same U5MR as the average found in developed countries. We calculated the observed number of under-five deaths in developing countries by using country population, crude birth rate, and U5MR in 2007 for all 67 countries included in the analysis. The country population, crude birth rate, and U5MR were taken from the World Development Indicators  using appropriate weights as described above.We then applied the U5MR of the top economic group to the total number of births in 2007 in the 67 countries to estimate the number of under-five deaths. The “reduced mortality gap” is the difference between the observed number of under-five deaths in these 67 developing countries in 2007 and the estimated number of deaths using the U5MR in the top economic group in the same 67 developing countries.In the third step, the estimates produced in the first and second steps are compared. The outcome expressed in relative terms represents the percentage decrease in the actual mortality gap that would result from achieving the reduced gap just described. We used Stata version 12.0 for the analysis.The list of 67 DHS datasets used and summary of the data can be found in Additional file The gap analysis results using the top 10% (decile) of the population based on the wealth index as the top economic group are presented in Table Lines 1 and 2 of Table In Section B, the same analysis is repeated, only now using the U5MR of 44.1 deaths per 1000 live births, the mortality rate experienced in the top economic decile of the included 67 developing countries. Line 7 represents the number of under-five deaths that would have occurred in the 67 countries had their average U5MR been 44.1 deaths per 1000 live births.  by the actual gap . The result is 48%, indicating that the actual global mortality gap could be reduced by 48% if the U5MR in all economic groups in developing country populations could be lowered to that of the top 10% in those populations. As can be seen from line 8 of the table, this would result in around 2.9 million fewer under-five deaths.The analyses using mortality rates from the top wealth quintile and ventile are available in Additional file Using a population attributable risk approach, we demonstrated that about half of the under-five mortality gap between developed and developing countries can be reduced by lowering U5MR in each developing country to the level enjoyed by the top economic stratum of these countries. For our purposes, these developing countries were represented by the 67 countries with available DHS data. An accurate appreciation of the findings’ implications requires a clear awareness of the many nuances in the approach taken and the data used. In the present case, three of the nuances are especially noteworthy.First, the size of the intra-country mortality gap inevitably depends upon how the best-off group is defined, and there is no known conceptually-satisfying definition for such a group. For example, it is likely that there exists within even the poorest countries some very small but highly privileged groups with mortality rates close to or even better than those of the industrialized world. Were this small group to be taken as the best-off for the purposes of the calculations performed here, then almost all of the global mortality gap would be attributable to differences within countries. Using lower economic cut-offs (e.g. everyone above the poverty line) would produce a higher U5MR among the reference population we are using as “best off,” and a correspondingly lower portion of the global gap resulting from intra-country disparities.While the top wealth decile that we used in our analysis has served as the indicator of choice whenever the available datasets are adequately large, it is largely arbitrary. Plausible alternatives exist, and their use could produce notably different results. For example, using the top 5% of the population as the reference group would increase the relative difference between the two gap measures from 48% to 57%, and the absolute difference from 2.9 million to 3.4 million under-five deaths. Had the reference group been the population’s top 20%, the gap reported would have fallen to 38% and 2.3 million under-five deaths. Such variability argues for considering the results produced based on a 10% figure as no more than a general sense of the magnitudes involved. It would be safer to say, for example, that eliminating U5MR disparities within developing countries by reducing U5MR among developing countries could reasonably be expected to reduce the actual global mortality gap by roughly a half, and avert two to three million under-five deaths.Second, while the population attributable risk approach used here is quite standard, it also represents no more than a very schematic approximation of reality. In actuality, it is obviously unrealistic to expect a sudden decline in under-five mortality that would leave other important parameters unchanged. Far more likely would be an extended process of change, involving a wide range of broad social and economic developments, as well as health service modifications, shifting the relative economic positions of different groups, and influencing fertility as well as mortality. Whether such a process might lead to results similar to those reported here is difficult to say. The analysis presented does not however make any assumption or claims about the determinants or causes of mortality change.Finally, the DHS data used were collected between 1994 and 2011 and the mortality rates computed referred to a period ten years preceding each survey. The range in survey years introduces variability in the data. It should be noted that our analysis utilizes estimates that are averages over this period.Findings in our study suggest that the global under-five mortality gap could be reduced by roughly half by eliminating intra-country U5MR disparities by economic status, and doing so could avert around two to three million deaths annually. The findings presented cannot plausibly be viewed as providing more than general orders of magnitude. However, our findings support two principal points: that economic inequalities with respect to health within developing countries contribute much more to the global health gap than might appear to be the case at first glance and that these inequalities deserve at least as much attention as does the gap between those countries and developed countries.Over six million more under-five deaths occur in developing countries than in developed countries every year. Under-five mortality disparities within developing countries account for roughly half of that mortality gap. Within-country inequities require as much consideration as do inequities between the world’s developing and developed regions.The authors report no competing interests.AA and DRG designed the study, AA, NK, and DRG analyzed the data, AA, NK, and DRG contributed to the writing of the manuscript. All authors read and approved the final manuscript.The pre-publication history for this paper can be accessed here:http://www.biomedcentral.com/1471-2458/14/216/prepubTable S2. Demographic and Health Survey and population data from 67 low- and middle-income countries contributing to the analysis. Table S3a-c. Pooled U5MR by wealth quintiles, deciles, and ventiles for 67 developing countries included in the analysis. Table S4a. Impact of eliminating mortality disparities within developing countries, by lowering mortality rates to those enjoyed by the highest wealth quintile within those countries: 67 Low- and Middle-Income Countries, 2007. Table S4b. Impact of eliminating mortality disparities within developing countries, by lowering mortality rates to those enjoyed by the highest wealth ventile within those countries: 67 Low- and Middle-Income Countries, 2007.List of countries by World Bank income categories. Click here for file


E. coli genome sequences were made publicly available. The suitability of BG7 for genome annotation has been proved for Illumina, 454, Ion Torrent, and PacBio sequencing technologies. Besides, thanks to its plasticity, our system could be very easily adapted to work with new technologies in the future.BG7 is a new system for de novo bacterial, archaeal and viral genome annotation based on a new approach specifically designed for annotating genomes sequenced with next generation sequencing technologies. The system is versatile and able to annotate genes even in the step of preliminary assembly of the genome. It is especially efficient detecting unexpected genes horizontally acquired from bacterial or archaeal distant genomes, phages, plasmids, and mobile elements. From the initial phases of the gene annotation process, BG7 exploits the massive availability of annotated protein sequences in databases. BG7 predicts ORFs and infers their function based on protein similarity with a wide set of reference proteins, integrating ORF prediction and functional annotation phases in just one step. BG7 is especially tolerant to sequencing errors in start and stop codons, to frameshifts, and to assembly or scaffolding errors. The system is also tolerant to the high level of gene fragmentation which is frequently found in not fully assembled genomes. BG7 current version – which is developed in Java, takes advantage of Amazon Web Services (AWS) cloud computing features, but it can also be run locally in any operating system. BG7 is a fast, automated and scalable system that can cope with the challenge of analyzing the huge amount of genomes that are being sequenced with NGS technologies. Its capabilities and efficiency were demonstrated in the 2011 EHEC Germany outbreak in which BG7 was used to get the first annotations right the next day after the first entero-hemorrhagic  The massive production of bacterial genome sequences using NGS technologies is demanding new automated systems capable of getting an accurate annotation of a complete genome in a short enough time. Classical annotation systems are based on two completely separate phases: 1) ORF prediction and 2) Functional annotation. Classical ORF prediction methods are totally dependent on the detection of start and stop codons. This is an efficient strategy whenever the sequencing technology has minimal sequence errors since only predicted ORFs have to be annotated - avoiding an important amount of unnecessary comparisons. However, all NGS technologies generate sequences with substitution, deletion and insertion errors. Each technology is prone to generate different types of errors: 454 technology generates deletions and insertions at homopolymeric regions This scenario demands new approaches better fitted to the new sequencing technologies framework.After analyzing the publications about bacterial annotation pipelines we found that the use of the classical genome annotation paradigm - composed by a first phase of ORF prediction and a subsequent phase of functional annotation, continues being the standard also for NGS annotation.DOE-JGI MAP E. coli. The first assembly had 3057 contigs and a high error rate and the last assembly had 4 contigs and a very low error rate. The annotation used for determining the correct genes was the BROAD annotation of the last assembly that included 5164 genes (The BROAD reference genes used in the test are in File S1). For the first assembly (named BV1) BG7 yielded 6190 genes with 197 false negatives and 186 false positives and RAST 8253 genes with 247 false negatives and 455 false positives. For the last assembly (named BV4) the data for BG7 was 5210 genes, 163 false negatives and 271 false positives while the number of genes obtained with RAST was 5446 with 116 false negatives and 321 false positives. (See S2). BAsys RAST Using the classical paradigm - ORF prediction first and then gene annotation, any gene lost in the first phase cannot be recovered in later phases of the annotation.Specifically, when using Glimmer, any minimal error affecting start or stop codons can cause unrecoverable gene loss or false gene prediction. BG7 has been designed for NGS based on a new paradigm that integrates gene finding and annotation in a single step centered on massive analysis of similarity with reference proteins. For each annotation project a large set of proteins from Uniprot E. coli outbreak https://github.com/ehec-outbreak-crowdsourced/BGI-data-analysis/tree/master/strains.The efficacy, speed and specificity of BG7 for NGS projects were proved in a real case, the 2011 Germany A schematic overview of BG7 is displayed in Once the compatible HSPs are joined, the region of similarity is extended upstream and downstream searching for start and stop codons taking into account the orientation of the gene based on the alignment with the reference protein.In the case where stop or start codons are not found within a fixed distance, the gene is considered as a gene with “non-canonical” start or/and stop. This strategy prevents gene loss when there is a lack of either stop or start codons caused by sequencing errors. If a sufficient similarity supports the existence of a gene, BG7 maintains this region as a gene even though the start and end of the genes are not well defined. This strategy also solves the problem of detection of not complete genes located at the ends of contigs.In the case where the same contig region is similar to several different reference proteins, the best hit  is chosen. Once all similarity-supported putative genes are defined, the system solves the overlapping between them, selecting the better gene for each sequence fragment. So each final gene is supported by the similarity with a specific and unique Uniprot protein. A threshold for gene overlapping can be set so gene overlaps beyond the fixed threshold are solved based on the E value of each putative gene and on the provenance of the annotating protein. When the overlapping genes have different E values, the gene with the lowest E value is chosen.Otherwise, when they have the same E-value, the one from the closest organism isselected. The closest organism is inferred by analyzing the tBLASTn results in a preprocessing step. The system evaluates which organism has more ortholog proteins in the genome under analysis, assuming then the closest organism as the preferential one for the annotation process. Preference is given to the closest genome providing a more uniform annotation probably more fitted to the real evolutionary origin of the genes. With this strategy the final predicted genes can only overlap at their ends with a length below the fixed threshold. This parameter can be adjusted depending on the features of the genome to be annotated.RNAs are also predicted by similarity using BLASTn. Contigs of the genome under analysis are compared against the reference RNAs, obtaining in the end a complete annotation with protein coding and RNA genes.In order to ensure high quality annotations, the size of the reference protein set should be maximized. Normally around 200,000 reference proteins are used to annotate a 3 Mb bacterial genome but there is no algorithmic limitation on the size of the protein and RNAs reference sets. The limit is only imposed by the memory available on the machine.Project_prefix , Virus_flag (boolean indicating whether this genome corresponds to viruses or bacteria/archaea), Extension_threshold (when searching for start and stop codons this parameter fixes the length in bp that the ends of the preliminary protein-similar segments will be extended) and Overlapping_threshold  should be fixed for each project. The specific input for each program is detailed in Table S3.The input data for BG7 is: 1. the sequences to be annotated, 2. the set of reference proteins, 3. the tBLASTn results from the comparison of the set of reference proteins against the contigs in xml format, and 4. the BLASTN results from the comparison of the contigs against the set of reference RNAs. The values for the parameters Table S3). A prototype of BG7 result files from E.coli K12 annotation is available at: https://s3-eu-west-1.amazonaws.com/era7bioinformatics-project-data/BG7_Deliverables_Prototype.zip. The availability of intergenic regions could be very useful for the analysis of regulatory signals. Besides, intergenic regions in which genes have not been detected can be used as the input for a second round of annotation. Using a not restrictive E-value  in the initial tBLASTn, very short genes or genes fragmented at the end of the contigs could be detected. In addition, a totally different set of reference proteins could be used in this second round of annotation that could be focused on specific functionalities of special interest such as metabolic pathways, virulence, pathogenicity, or antibiotic resistance. Thus, the re-annotation of the non-coding space of sequences provides a higher confidence annotation.Using cloud computing Running BG7 on virtual machines on top of aws infrastructure we get a system highly scalable and parallelizable, making possible to annotate hundreds of genomes simultaneously with a time cost similar to the time needed for just one genome annotation. This could be a great advantage in public health emergency situations since hundreds of genomes could be annotated in just two hours using a specific parallelized architecture that can be easily designed for each project.Escherichia coli K12 E. coli K12 checking functional annotations with experimental work. The selection of E. coli K12 to test BG7 was aimed to evaluate our automated annotation in the least favorable scenario. In this case our system would be an automated system that would not be taking advantage of the inherent accuracy of Sanger sequencing. The results of the comparative analysis between NCBI and BG7 annotations are summarized in Table S4). These results demonstrate that BG7 annotations are quite similar to NCBI annotations. This is a remarkable fact since BG7 is an automated system and the annotation was done in roughly 2 hours. Not only that, E. coli K12 annotations were done by a large community of researchers being E. coli K12 the quintessential bacterial model organism. It is important to note that in BG7 annotation all proteins from E. coli K12 were excluded from the reference protein set.BG7 was tested in two real-world scenarios. The first scenario was the genome of E. coli outbreak. BG7 was used for annotating the sequences of the Shiga toxin-producing E. coli O104 (STEC O104:H4) strain, which is able to cause hemolytic, uremic syndrome and enterohemorrhagic diarrhea syndrome E. coli outbreak that were made available  were obtained using Ion Torrent technology. BG7 demonstrated its speed, efficacy, and error tolerance even when using technologies that introduce many errors in the sequence. The genome assembly available on 2-Jun 2011 was fragmented in 3057 contigs with an N50 of 3.56 Kb. The first BG7 annotation done by Era7 Bioinformatics was available to the public on 3-Jun 2011 (doi:10.1038/npre.2011.6001.1). All results from the analysis of the E. coli strain that caused the outbreak in Germany are gathered in a public repository available at: https://github.com/ehec-outbreak-crowdsourced/BGI-data-analysis. The last version of the same E. coli genome was sequenced with paired-end Illumina technology and the assembly was improved until obtaining a closed genome with one chromosome and three plasmids. We analyzed in detail the performance of BG7 in these two extreme stages of genome assembly. To detect BG7 false negative and false positive genes we used BLASTN taking as reference the BROAD Institute annotation for this last assembly  thus it is cross-platform. It uses the following open source libraries: 1. BioinfoXML which includes XML wrappers for bioinformatics entities plus some utilities, 2. BioinfoUtil which consists of a set of bioinformatics-related utility classes, and 3. Apache commons HttpClient 3.1 which is used for connecting with Uniprot Web Services. Both libraries BioinfoXML and BioinfoUtil have been developed at Era7.Table S9 shows how BG7 performs with different instance types. The structure of our system is modular and the pipeline is composed by several Java programs that run sequentially. The pipeline is expressed in an XML file that determines the sequential execution of the different modules as well as setting the parameters for each module. The exchange of data among programs is also based in XML files, which maintain partial results in this structured and interoperable format.BG7 is designed to easily run in virtual machines using cloud computing (Amazon Web Services) The system can be parallelized by means of using cloud computing. A set of machines could be launched, - one for each genome annotation project, and the annotation for all genomes would be obtained in roughly the same time than one genome. Even though BG7 does not include any parallelization system itself, it is very simple to include it in parallelized architectures.BG7 is an annotation system based on a new approach especially designed for NGS that in some aspects can outperform the current microbial genome annotation methods. BG7 is an open, flexible, fast and tunable tool with new error dealing capabilities that allows the annotation of microbial genomes in very preliminary stages. BG7 only depends on Uniprot database and it is not needed to know previously the taxonomic affiliation of the genome to be annotated. BG7 can perfectly run in the cloud and is easy to be incorporated to parallelized architectures. The coexistence of different systems for microbial genome annotation is highly enriching since each system outperforms the others in different aspects of the complex genome annotation process.https://github.com/bg7/ released under the license AGPLv3.BG7 code is available at GitHub: We are working on improving the annotation with a special focus on finding alternative annotations in the cases where the most similar protein is poor in functional annotation data. We plan to provide access to all proteins with significant similarity to any gene region. This new possibility could facilitate the characterization of genes annotated only as hypothetical or uncharacterized proteins.We also plan to include a new module in order to get a more direct characterization of the predicted proteins using InterproScan – detecting all the InterPro motifs and domains in the predicted protein.Another research line the BG7 team is focused on is the generation of rich comparative genomics results based on the initial tBLASTn step. In the case of bacteria with complete proteomes available in Uniprot, it would be possible to do an orthology study with the proteins predicted for the genome under analysis. This would provide additional results very useful for the interpretation of the functional capabilities and phylogenetic relationships of the annotated bacterium.We are also working on the development of new tools for the visualization of both annotation data and comparative genomics results obtained.http://bg7.ohnosequences.com/All improvements will be reported in the BG7 website: File S1BROAD Institute reference genes used in the test. The annotation used for determining the correct genes was the BROAD Institute annotation of the last assembly that included 5164 genes. The gene sequences were downloaded on 20-Aug-2012 from: http://www.broadinstitute.org/annotation/genome/Ecoli_O104_H4/FeatureSearch.html. The name of the file downloaded was: Escherichia_coli_o104_h4_str._ty-2482_1_genes.fasta.(FASTA)Click here for additional data file.Table S1General BG7 and RAST comparison. BG7 and RAST were compared in terms of 1. Input requirements, 2. File formats of the annotation output files provided, 3. Additional output provided files, 4. Availability of the code, 5. Possibility of installing the tools in-house, 6. License and 7. Dependences.(DOCX)Click here for additional data file.Table S2BG7 and RAST comparison in the annotation of two very different stages of completion and error rate of E.coli O104:H4 genome. For the first assembly (named BV1) BG7 yielded 6190 genes with 197 false negatives and 186 false positives and RAST 8253 genes with 247 false negatives and 455 false positives. For the last assembly (named BV4) the data for BG7 was 5210 genes, 163 false negatives and 271 false positives while the number of genes obtained with RAST was 5446 with 116 false negatives and 321 false positives. The BROAD reference genes used in the test are in File S1.(XLSX)Click here for additional data file.Table S3Input, output, and parameters of the BG7 annotation programs. This table summarizes the names of the main java programs that integrate BG7, their input and output files and the parameters to set for each of them. BG7 improvements and updated versions will be available at GitHub: https://github.com/bg7/(DOCX)Click here for additional data file.Table S4Gene-by-gene comparative analysis between NCBI and BG7 annotations for E. coli K12 genome. Detailed results of the gene-by-gene comparison are available in excel format. E. coli K12 NCBI annotation are in blue rows and BG7 annotations are in white rows. In the column corresponding to manual check 0 (in pink) means that the BG7 annotation does not detected that gene, 1 (in yellow) means there are slight differences in the protein name or in start or stop positions, and 2 (in green) means identical annotation in NCBI and in BG7.(XLSX)Click here for additional data file.Table S5BG7 annotation for the first assembly of the German E. coli outbreak obtained with Ion Torrent technology.(XLSX)Click here for additional data file.Table S6BG7 annotation for the assembly of the German E. coli outbreak obtained with PacBio technology.(XLSX)Click here for additional data file.Table S7BG7 annotation for the assembly of the German E. coli outbreak obtained with 454 technology.(XLSX)Click here for additional data file.Table S8BG7 annotation for the assembly of the German E. coli outbreak obtained with illumina technology.(XLSX)Click here for additional data file.Table S9BG7 computation time in different Amazon instances.(DOCX)Click here for additional data file.


In order to facilitate and improve the use of antiretroviral therapy (ART), international recommendations are released and updated regularly. We aimed to study if adherence to the recommendations is associated with better treatment outcomes in the Swiss HIV Cohort Study (SHCS).Initial ART regimens prescribed to participants between 1998 and 2007 were classified according to IAS-USA recommendations. Baseline characteristics of patients who received regimens in violation with these recommendations (violation ART) were compared to other patients. Multivariable logistic and linear regression analyses were performed to identify associations between violation ART and (i) virological suppression and (ii) CD4 cell count increase, after one year.Between 1998 and 2007, 4189 SHCS participants started 241 different ART regimens. A violation ART was started in 5% of patients. Female patients , those with a high education level  or a high CD4 count  were more likely to receive violation ART. The proportion of patients with an undetectable viral load (<400 copies/mL) after one year was significantly lower with violation ART than with recommended regimens  whereas CD4 count increase after one year of treatment was similar in both groups.Although more than 240 different initial regimens were prescribed, violations of the IAS-USA recommendations were uncommon. Patients receiving these regimens were less likely to have an undetectable viral load after one year, which strengthens the validity of these recommendations. After 1996, the use of combined antiretroviral therapy (ART) transformed HIV infection from a progressive, fatal disease to a treatable chronic condition and dramatically reduced its related morbidity and mortality In 2001, the Swiss Federal Office of Public Health stopped issuing recommendations Even though comprehensive treatment recommendations are now widely available, choosing an initial antiretroviral combination often remains a challenging task. In the industrialized world, the availability of more than twenty different antiretroviral drugs has led to highly personalized ART prescriptions, according to patient characteristics and wishes, co-morbidities and resistance testing. This contrasts to the public health approach to ART, as shown by Keiser et al., who compared initial ART regimens prescribed in South Africa and the SHCS CD4 cell count, viral load and socio-demographic characteristics such as age and ethnicity have been shown to be associated with the type of ART prescribed and the probability of being treated with an ART in violation of current guidelines The main goal of official treatment guidelines is to improve the choice of ART. A publication from a small US cohort showed that patients receiving ART according to the IAS recommendations had clear virological and immunological benefits The aims of this study were to (1) describe first-line ART regimens in the SHCS from 1998 to 2007 and compare them with the corresponding IAS-USA recommendations, (2) describe the socio-demographic and clinical characteristics of the HIV-infected sub-population that did not receive regimens consistent with the IAS-USA recommendations, and (3) compare the efficacy of regimens prescribed according to these recommendations with those in violation with them.Local ethical committees of all seven participating study sites , Ethikkommission des Kantons St. Gallen, Kantonale Ethikkommission Zürich, Comitato etico cantonale del Ticino, Commission d'éthique de la recherche clinique de la faculté de biologie et de medicine de l'université de Lausanne, Comité d'éthique du départment de médicine des hôpitaux universitaires de Genève) have approved the study and written consent has been obtained from all participants.www.shcs.ch) is a prospective cohort study with the ongoing enrolment of HIV-positive adults in seven outpatient clinics and their affiliated hospitals and private practice physicians in Switzerland. It is estimated to cover about 45% of the cumulative number of HIV infections declared to the Swiss public health authorities and 69% of all patients living with AIDS in Switzerland Set up in 1988, the Swiss HIV Cohort Study  who started ART between 01.08.1998 and 31.12.2007, 4189 were included in the analysis. Of the 115 patients that were excluded, 8 received unspecified ART and the remaining 107 patients were women who started treatment because of pregnancy while having a CD4 count above 200 cells/µl. Unlike patients with severe immune deficiency, many of these women started treatment because of the pregnancy and stopped it after having given birth. These women could therefore not be compared with the other patients.Initial ART regimens were compared with the newest IAS recommendations available at treatment start date. Updated recommendations were taken into account from the first day of the month following the publication. ART regimens were classified by two experienced physicians as follows: 1) recommended regimen; 2) alternative regimen; 3) active but not yet recommended regimen; 4) drug combination suggesting primary resistance ; 5) prescription within a study protocol; 6) violation of recommendations. Any regimen consisting of one or two active drugs only were considered as violations of the recommendations. In 1998, only PI-based regimens were recommended. In 2000, NNRTI-based regimens were added to the recommended treatments list and triple-NRTI regimens were only accepted as first-line treatments in the 2002 recommendations. We encountered several difficulties in the classification process. There were inconsistencies between the presentations of the different recommendations from one year to the other. For instance, the recommendations elaborated in 2000 did not mention any alternative treatments, and the ones released in 2002 did not specify which combinations of NRTI were to be used or avoided. Importantly, new drugs and combinations are approved all year long but are only included in the subsequent guidelines. To avoid classifying such new, active and safe regimens as violations of recommendations, we created a separate category for them (category 3: active but not yet recommended regimen).All statistical analyses were performed using Stata 10 . Baseline characteristics of patients receiving violation treatments were compared to all other patients (categories 1–5). Crude associations between baseline characteristics and initial regimen type were evaluated using chi-square tests. Predictors of receiving a violation regimen were then assessed in a multivariable logistic regression analysis. Explanatory variables included sex, age category at treatment start , region of origin , risk group according to mode of HIV acquisition (Men who have Sex with Men (MSM), heterosexual, Injection Drug Users (IDU) or other), source of follow-up (SHCS center or other), CD4 count at treatment initiation , hepatitis B antigenemia at baseline (defined as having had at least once a positive HBsAg), chronic hepatitis C virus (HCV) infection (defined as positive HCV serology and HCV RNA), plasma HIV load at baseline , education level (low or high education) and start of ART more than three months before being included in the SHCS.The proportion of patients achieving undetectable viral load after 12 months of ART , as well as the CD4 increase during this period, were compared between patients receiving violation ART and the others, using multivariable logistic and linear regression analyses respectively. An undetectable viral load was defined as being less than 400 copies/ml. In a sensitivity analysis, a threshold of 50 copies/ml was used. Time to switching of ART was evaluated using a Cox proportional hazards model.Multiple imputation was used to impute missing values at baseline. Imputations were based on logistic and linear regression models with six dependent variables  and six independent variables . Analyses were run on each of 25 datasets and results were combined with Rubin's rules. In a sensitivity analysis we also compared these results with a complete case analysis.Baseline socio-demographic and clinical characteristics of the 4,189 HIV-infected patients who started ART between August 1998 and December 2007 are listed in The classification of initial ART regimens according to the IAS-USA recommendations in force at time of ART start is listed in online supporting information: Table S1). This was also true for patients older than 40 years, compared to those under 30. Patients originating from Sub-Saharan Africa were less likely to achieve an undetectable viral load compared to those from Western Europe, as were IDU's compared to MSM. Finally, participants starting ART more than 3 months before enrolment in the SHCS or with a CD4 count above 350 cells/µl were least likely to have an undetectable viral load after one year.Patients who initiated ART in the SHCS between 1998 and 2007 had a median CD4 count increase of 165 cells/µl (IQR 76-273) within one year, and 85% of them had a viral load below 400 copies/ml one year after treatment start. Patients given violation regimens were less likely to achieve an undetectable viral load  one year after treatment start Table 4.online supporting information: Table S2).Immunological recovery one year after ART start was similar in both treatment groups Table 4.Within the first year of treatment, 34% of patients on violation regimens were switched to a second regimen, as compared to 25% in the other groups. The corresponding adjusted hazard ratio in the Cox regression model was 1.66 .When the threshold of HIV viral load was set at 50 copies/ml instead of 400, the difference in the proportion of patients having a favourable outcome at one year between the two treatment groups was not statistically significant. With this second definition, participants with initial treatment in violation of the recommendations were 25% less likely to reach an undetectable viral load compared to those on recommended regimens, after adjustment for all co-factors . When analyzed separately, patients on dual-NRTI regimen were also less likely to reach an undetectable viral load (<400copies/mL) after 1 year than patients on recommended regimens .Since 1998 and the first version of the IAS-USA recommendations, more than four thousand treatment-naïve HIV-infected patients started ART in the SHCS until the end of 2007, being prescribed one of more than two hundred different regimens. The majority of these participants received initial treatment according to IAS-USA recommendations in force at the time ART was started, as well as according to IAS-USA recommendations on antiretroviral drug resistance testing if primary antiretroviral resistance was shown The baseline characteristics of the patients receiving violation combinations were of special interest, since it was suspected that these regimens might lead to a higher proportion of viral failures. In our study, these patients were more often female and highly educated. Even though it is unknown why these sub-populations were more likely to receive initial ART in violation of recommendations, physicians should be aware of these associations in order to avoid the prescription of these inferior regimens.. Highly educated patients may also have been more active in the negotiations regarding the choice of the initial treatment regimen with the physician, favouring sometimes regimens known to be of low virological efficacy. In their small study of self-reported initial treatment in a US-cohort of HIV infected women, Cocohoba et al. reported a significant association between the prescription of violation regimens in patients with a higher CD4 count and lower viral load at baseline In the SHCS, patients treated according to the IAS-USA recommendations between 1998 and 2007 were significantly more likely to achieve an undetectable viral load (<400 cp/mL) after one year, compared with those on a violation regimen. Importantly, patients on violation regimens were more likely to change their treatment to a different regimen during the first six months of ART. As a consequence, changes from a violation regimen to a recommended one might have had a positive impact on clinical outcomes in this sub-group of patients. This might have decreased the difference in virological outcome between patients on violation ART and those on recommended regimens. When the definition of an undetectable viral load was restricted to less than 50 copies/ml, the association between treatment group and virological outcome was not statistically significant. Even though this second definition is widely used in the clinical settings, it may not be appropriate in this context, as some patients might experience single episodes of low-level viremia, so-called blips, which often do not have any clinical consequences. Notably, IDU's and patients from Sub-Saharan Africa were less likely to have an undetectable viral load one year after treatment start compared to the other risk groups, even though they were not prescribed violation regimens more often. These associations have both been described in previous studies and might be explained by lower treatment adherence, as described in the SHCS by Glass et al. Another relevant finding of this study relates to the important role of the SHCS in the daily clinical management of HIV infected patients in Switzerland. Participants who started ART more than 3 months before being included in the SHCS were more often prescribed regimens in violation with the recommendations and were less likely to have an undetectable viral load after one year. Although this seems to support the management of HIV infected patients by physicians experienced in the field of HIV medicine, this finding could also be explained by differences in access to care or other socio-economic factors.Finally, immunological response to ART in patients on violation regimens showed a less favorable trend compared to CD4 recovery in patients on recommended initial ART, although the difference was not statistically significant in the multivariable model. As expected, younger patients and those with a low CD4 count at baseline had a better immunologic recovery than the others.There are several limitations to this study. First, it has to be noted that our classification of the different initial ART regimens in the SHCS was based on expert judgement and not on pre-specified objective criteria. Second, detailed description of antiretroviral combinations is not available in every set of recommendations thus allowing for a wider range of treatments accepted in the different treatment categories depending on the year of release. Third, our statistical analyses rely on observational data, which leads to different types of bias. For example, data on patient history is only collected twice a year, and is therefore subject to recall bias. CD4 counts and viral load values were not always obtained exactly 12 months after ART initiation, the ones measured closest to this time period had to be used. Finally, the logistic regression models cannot be adjusted for every single existing confounder, which means that residual confounding has to be taken into account when interpreting the results. Finally, given the large amount of ART-related issues discussed in the IAS-USA treatment recommendations, we could not consider all determinants of ART prescription for our analyses. As a consequence, even though possible adverse events and pill count, for instance, should be considered when choosing the appropriate initial ART regimen, we decided to limit our analyses to the description of individual predictors for receiving violation regimens and the evaluation of the virological and immunological response to the different ART regimens.In summary, our results show that in the SHCS, 5% of patients received initial regimens in violation with the IAS-USA recommendations. These patients were less likely to achieve viral load suppression after one year. Our results suggest that, in the context of constant increase in the number of therapeutic options and knowledge on specific drug-related side-effects and interactions, the release of updated treatment recommendations as well as the promotion of their use are important to guarantee the best possible care of HIV infected patients.Table S1Univariable and multivariable regression models estimating the proportion of patients having an undetectable viral load (<400 copies/mL) one year after ART start.(DOCX)Click here for additional data file.Table S2Univariable and multivariable regression models estimating the average change in CD4 cell count one year after initial ART start.(DOCX)Click here for additional data file.