Prenatal chorionicity assessment relies on ultrasound, which can be confounded by many factors. Noninvasive assessment of zygosity is possible using single nucleotide polymorphism (SNP)‐based cell‐free DNA testing. Our objective was to determine the relationship between provider‐reported chorionicity and SNP‐cfDNA assignment of twin zygosity.All twin pregnancy blood samples received by a reference laboratory between September 27, 2017 and September 8, 2021 were included. Chorionicity assignment was requested on the requisition, recorded as; monochorionic (MC), dichorionic, or “don't know”. SNP‐cfDNA zygosity results, monozygotic (MZ) or dizygotic (DZ), were correlated with chorionicity assignment.59,471 twin samples  were received and analyzed; 55,344 (93.1%) received zygosity assignment. SNP‐cfDNA reported 16,673 (30.1%) MZ and 38,671 (69.9%) as DZ. Provider‐reported chorionicity was compared to the zygosity assignment for each case. Of 6283 provider‐reported MC twins, 318 (5.1%) were reported as DZ using SNP‐cfDNA.One in 20 suspected MC twin pregnancies were reported as DZ using SNP‐cfDNA. Approximately 30% of 55,344 twin pregnancies were found to be MZ, including cases where chorionicity was unknown. SNP‐cfDNA zygosity assessment is a useful adjunct assessment for twin pregnancies, particularly those reported as MC or without determined chorionicity. What's already known about the topic?The assignment of chorionicity early in pregnancy can improve perinatal outcomes of twin pregnancies. Prenatal assessment of chorionicity relies on ultrasound (US), and the accuracy of US can be confounded by many factors, including gestational age (GA) and operator experience. Noninvasive assessment of zygosity is now possible using single nucleotide polymorphism (SNP)‐based cell‐free DNA (cfDNA).What does this study add?This is the first population‐based study describing the correlation between cfDNA assessment of zygosity and chorionicity. In 55,344 twin pregnancies, approximately 30% were found to be monozygotic (MZ), including cases where chorionicity was unknown. We found that one in 20 suspected monochorionic (MC) twin pregnancies were reported as dizygotic (DZ) twins (DZ) by SNP‐cfDNA testing. SNP‐cfDNA zygosity assessment is a useful adjunct assessment for twin pregnancies, particularly those reported as MC or without determined chorionicity. Egg‐donor or surrogate pregnancies, samples that failed quality control, samples with a known vanishing twin and those not receiving zygosity assignment were excluded. All samples were analyzed for zygosity and aneuploidy risk using an SNP‐based cfDNA methodology and algorithms that have been validated previously.2.2Maternal demographics, including GA at the time of blood draw, maternal weight, age, ethnicity, and use of in vitro fertilization (IVF) were reported by providers.2.3Information regarding the assignment of chorionicity was obtained from the requisition, which included a query using a check box . If the algorithm identifies an MZ pregnancy, fetal sex is only reported if confidence is high that twin fetal sexes are either female/female or male/male. For this analysis, we assumed that all MZ pregnancies would have same‐sex fetuses, 50% female and 50% male. Conversely, for DZ pregnancies, we assumed a sex distribution in which 25% are both male, 25% are both female, and 50% have one male and one female.2.5z‐tests for independent samples  were used to examine differences in demographic characteristics between males and females. p‐values are not adjusted for multiple comparisons and are intended to be hypothesis‐generating only.Pearson chi‐square tests for independence  or This is a retrospective cohort study of cfDNA screening tests from twin gestations received by a Clinical Laboratory Improvement Act (CLIA)‐certified and College of American Pathologists (CAP)‐accredited laboratory . This study was designed in compliance with an investigational review board approved protocol .3A total of 59,471 twin samples were received, of which 55,344 (93.1%) received a result for zygosity. Zygosity could not be assigned in 6.9% of samples, primarily due to suboptimal sample quality, lab processing errors, and low FF.p < 0.001).Cohort demographics and clinical characteristics are summarized in Table A total of 9389/55,344 (17.0%) samples provided an ICD‐10 code that specified information regarding chorionicity Table . As descGiven the identified discrepancies between provider‐reported chorionicity and SNP‐cfDNA reported zygosity within our cohort, we performed an exploratory analysis comparing the observed to expected fetal sex distribution for provider‐assigned chorionicity and SNP‐cfDNA assigned zygosity. Only cases where both chorionicity and zygosity were reported were included in this analysis. Fetal sex was requested by the ordering physician in 51,258/55,344 (92.6%) of samples. Fetal sex was reported in 15,409 samples identified as MZ by SNP‐cfDNA (98.8%) and 29,732 samples identified as DZ (83.3%). The primary reason for nonreporting of fetal sex in DZ pregnancies was low FF.p < 0.001 compared to expected ratio of 50%/50%). Conversely, the observed fetal sex distribution was not significantly different from the expected distribution for DZ twins . By contrast, for the total cohort, fetal sex distribution was similar to expected in pregnancies assigned as MZ or DZ using SNP‐cfDNA  and reported in 231. The observed fetal sex distribution for this group was significantly different from the expected distribution for MZ twins  upon pregnancy outcome. Our study lays a foundation for such research, to better determine the degree to which these two factors contribute independently to complicated and normal outcomes.Andrei Rebarber is a speaker for Natera, Inc. and Hologic. Kimberly A. Martin is a consultant to Natera, Inc. with stock/options to own stock in the company. Russ Jelsema, Allyson Koyen Malashevich, Katelyn Hashimoto, Sheetal Parmar, Zachary Demko, and Paul Billings, are employees of Natera, Inc. with stock/options to own stock in the company.This study was designed in compliance with an investigational review board approved protocol . Written informed consent was obtained from all study participants.The authors confirm that this manuscript represents original research that has not been previously published nor being considered for publication elsewhere.Supporting Information S1Click here for additional data file.


The effect of long‐standing prediabetes or its transition on incident cardiovascular disease (CVD) is unclear. This study aimed to evaluate the association of changes in fasting blood glucose (FBG) status with the risk of developing CVD.This research included 12 145 Chinese adults aged 35–74 years and free from diabetes mellitus (DM) at baseline. Study participants were cross‐classified into six categories according to glucose at the first (1998–2001) and the second visit after 8 years: normal fasting glucose , impaired FBG , and DM. Cox proportional hazard regression model was used to estimate the hazard ratio (HR) and 95% confidence interval (CI) for CVD associated with transition of glucose status.During a median follow‐up of 5.5 years, 373 incident CVD cases occurred. Compared with participants remaining persistent NFG, a higher risk of developing CVD was identified among those remaining persistent IFG, progressing to DM from NFG or from IFG, with the multivariate‐adjusted HR (95% CI) of 1.792 , 1.723  and 1.946 , respectively. Furthermore, when stratified by glucose status at baseline, persistent IFG and progression from IFG to DM still increased CVD risk in comparison with reversion from IFG to NFG, with the multivariate‐adjusted HR (95% CI) of 1.594  and 1.913 .Participants with long‐standing IFG and progressing to DM had a higher risk of developing CVD. Further well‐designed studies are warranted to assess the association of other phenotypes or prediabetes duration with CVD. HighlightsAdjusted for age, gender, BMI, FBG at the first visit, smoking status , drinking status (yes vs no), physical activity, hypertension status (yes vs no), dyslipidemia (yes vs no), region (south vs north), area , education, income and family history of CVD.BMI, body mass index; FBG, fasting blood glucose; NFG, normal fasting glucose (50‐99 mg/dL); IFG, impaired fasting glucose (100‐125 mg/dL); DM, diabetes mellitus ; CVD, cardiovascular disease. It may provide evidence of long‐term blood glucose control for the primary prevention of CVD in the population without diabetes.22.1Participants were from two cohorts of the China‐PAR project, the China Multi‐Center Collaborative Study of Cardiovascular Epidemiology (China MUCA‐1998), and the International Collaborative Study of Cardiovascular Disease in Asia (InterASIA). These two cohorts were established in 1998 and 2000–2001, respectively. Detailed description of study design was previously published.n = 11 480 from China MUCA‐1998; n = 15 540 from InterASIA). Participants were excluded due to (1) absence of the second visit (n = 7518), of whom 1045 died; (2) loss to follow‐up (n = 3015); (3) the history of CVD  before the second visit (n = 705); (4) the diagnosis of diabetes at the first visit (n = 723); (5) hypoglycemia ; and (6) missing records on glucose‐related information at both visits (n = 2823). Finally, altogether 12 145 subjects were included in the analysis . Detailed information on blood specimen measurement is elaborated in Appendix Study participants were defined as follows based on blood glucose level: normal fasting glucose , IFG 100–125 mg/dl), and DM .5 mg/dl, 2.32.Hypertension was defined as currently using antihypertensive medicine and/or an average of three sitting systolic blood pressure (SBP) measurements ≥140 mmHg and/or an average of three sitting diastolic blood pressure measurements ≥90 mmHg after at least a 5‐min rest and with a 30‐s interval. Total cholesterol (TC), triglyceride (TG), and high‐density lipoprotein cholesterol (HDL‐C) were assessed on a Hitachi 7060 Clinical Analyzer  with cholesterol oxidase method, glycerophosphate oxidase method, and direct chemically modified enzyme method, respectively.Other known or suspected risk factors for CVD, including demographic characteristics, lifestyle, medical history of chronic diseases, and family history of CVD, were collected using structured questionnaires face to face at each visit by trained data investigators. Age (in years), smoking status , drinking status (current drinker or not), educational level , monthly household income per capita , and physical activity level  were assessed with standardized questionnaires. The definition of ideal physical activity level was at least 150 min of moderate‐intensity physical activity or at least 75 min of vigorous‐intensity physical activity per week, which was recommended by World Health Organization.2.4International Classification of Diseases, 10th Revision, I00 to I99, I20 to I25, I21, and I60 to I69 were codes for CVD death, ischemic heart disease, acute MI, and stroke, respectively. In this study, CVD was defined as presence of acute MI, unstable angina, nonfatal stroke, heart failure, and death from any CVD causes.Medical and death records were collected from hospital records and local public health department to obtain information on disease status and vital outcome events during the follow‐up visit. Information from electrocardiogram, 2D echocardiography, computed tomography scan, coronary arteriography, or cardiovascular biomarkers, etc. was used to ascertain the final diagnosis. A study‐wide end point assessment committee at Fuwai Hospital reviewed and verified the diagnosis. CVD death included any death from CVD during the follow‐up period. According to the CHD was defined as a diagnosis of acute MI, unstable angina, or death due to CHD. Acute MI was identified as a change in biochemical markers of myocardial necrosis accompanied by any of the following four characteristics: ischemic symptoms, pathological Q waves, ST‐segment elevation or depression, or coronary intervention.2.5t test to compare values of normal distributions, Wilcoxon's rank‐sum test to compare data of abnormal distributions, and the chi‐square test to compare the frequencies of categorical variables between groups.Continuous variables were expressed as mean ± SD or median values with the interquartile range. Categorical variables were expressed as numbers (percentages). Person‐years of follow‐up were calculated as the interval from the date of the second visit to the date of the final follow‐up interview, death, or the occurrence of incident CVD, whichever occurred first. We applied Cox proportional hazards regression model was used to estimate the association of FBG with CVD outcome as hazard ratios (HR) and 95% confidence interval (CI). Model 1 adjusted for age and gender, and model 2 additionally adjusted for BMI, smoking and drinking status, physical activity and educational level, hypertension status, dyslipidemia, FBG at the first visit, region, urbanicity, income, and family history of CVD.p value <.05.Sensitivity analysis was conducted to explore the influence of excluding CVD incidence in the first year of the follow‐up period, excluding development of diabetes ahead of the onset of CVD during follow‐up period, including vegetable and fruit intake at the second visit into the multivariate‐adjusted model, taking cohort source as a stratum in the model to address potential effect variation between two cohorts. All analyses were performed using SAS, version 9.4 , and the statistical significance was set at a two‐tailed 33.1n = 2475) were more likely to be older and more obese, with high blood pressure, TC, TG, and LDL‐C levels and low HDL‐C levels, and they were less likely to have ideal physical activity and more likely to live in an urban area  were identified. The incidence density (per 1000 person‐years) of CVD was 4.87 in participants remaining NFG, 5.21 in those progressing from NFG to IFG, 11.99 in those progressing from NFG to DM, 5.22 in those reverting from IFG to NFG, 9.28 in those remaining IFG, and 10.28 in those progressing from IFG to DM  was 1.828 , and 2.135 , respectively. Persistent IFG had a higher risk of CHD , whereas it was not significantly associated with stroke risk .Ptrend, .015 and .036, respectively). Compared with participants reverting from IFG to NFG, those remaining IFG had a higher risk of CVD , and those progressing from IFG to DM had a higher risk of CVD  and stroke  after adjustment for confounding factors , persistent IFG, progression from NFG to DM, and progression from IFG to DM in comparison with persistent NFG still significantly increased the CVD risk. Multivariate‐adjusted HRs (95% CI) for CVD did not obviously change in those groups of FBG status, which were 1.746 , 1.779 , and 2.012 , respectively (Appendix n = 202), adding vegetable and fruit intake into the covariates, or using the cohort‐stratified Cox regression model to estimate HR and 95% CI.After excluding participants with the diagnosis of CVD within the first year of the follow‐up period (Appendix . Moreove4In this prospective cohort study, long‐standing IFG and progression to DM from NFG and IFG were associated with an elevated risk of CVD. Although several studies indicated that prediabetes significantly increased CVD risk only when it was defined by IGT or combined criteria,Meta‐analysis showed IFG based on single measurement was associated with an increased CVD risk, whereas several studies reported inconsistent results.In line with previous studies involving FBG transition, we drew steady results that newly discovered diabetes increased CVD risk.Potential mechanisms of hyperglycemia in increasing CVD risk are as follows. First, evidence showed that the transition from NFG to prediabetes in subjects was associated with increased weight and insulin resistance and reduced endogenous insulin secretion.Though both IGT and IFG were related to the increased risk of CVD,Several strengths of this study are as follows. We used data from a nationwide, prospective cohort from 14 provinces with long‐time multiple rounds of follow‐up survey.However, the limitations of our research warrant consideration: first, postprandial glucose, 2‐h plasma glucose during 75‐g oral glucose tolerance test, and HbA1c were not measured, which are also important for the development of atherosclerosis and can reflect prediabetes according to different criteria and minimize the possibility of misclassifying prediabetes.5Long‐standing prediabetes status defined by IFG and progressing from NFG/IFG to DM within the interval of 8 years had an increased risk of developing CVD. Emphasis on surveillance and management of FBG for prediabetes is indispensable for the primary prevention of CVD. Further well‐designed studies are needed to examine the effect of different prediabetes phenotypes and prediabetes duration on health outcome.The National Key Research and Development Program of China, Grant/Award Numbers: 2018YFE0115300, 2017YFC0211703; the Chinese Academy of Medical Sciences Innovation Fund for Medical Sciences, Grant/Award Numbers: 2021‐I2M‐1‐010, 2017‐I2M‐1‐004, and 2019‐I2M‐2‐003; the National Natural Science Foundation of China, Grant/Award Numbers: 82030102, 12126602; the Research Unit of Prospective Cohort of Cardiovascular Diseases and Cancers, Chinese Academy of Medical Sciences, Grant/Award Numbers: 2019RU038.The authors have no conflicts of interest to disclose.Appendix S1. Supporting information.Click here for additional data file.


In this placement, (1) the intended relational correspondences are readily apparent, and (2) the influence of potential competing correspondences is minimized. There is evidence for the spatial alignment principle in adults’ visual comparison . Here, we test whether it holds for children. Six‐ and eight‐year‐old children performed a same‐different task over visual pairs. The results indicated that direct placement led to faster and more accurate comparison, both for concrete same‐different matches (matches of both objects and relations) and for purely relational matches—evidence that the same structural alignment process holds for visual comparison in 6‐ and 8‐year‐olds as in adults. These findings have implications for learning and education.Visual comparison is a key process in everyday learning and reasoning. Recent research has discovered the spatial alignment principle, based on the broader framework of structure‐mapping theory in comparison. According to the spatial alignment principle, visual comparison is more efficient when the figures being compared are arranged in  Children were shown two model buildings, one with a diagonal brace  and one with a horizontal crosspiece, lacking a diagonal brace (which was unstable). The buildings were either highly similar and easy to align or dissimilar and harder to align. When children were asked “Which is stronger” and allowed to wiggle them, they discovered that the stable building barely moved, while the unstable one could be bent nearly to the ground. At this point, all children recognized 1.2Given the prevalence and importance of visual comparison both in classroom learning   are more likely to spontaneously engage in comparison between similar items than between dissimilar ones.There is considerable evidence that children are more likely to compare and align two things if they are spatially and temporally juxtaposed. For example, in Christie and Gentner's  study d, geoscie1.3spatial alignment  as well as those varying in color.To test this prediction, Matlen et al.  conductesquare‐triangle‐square has the same relational pattern as red‐blue‐red, so they should press “same” for pairs like this. The results again supported the spatial alignment principle, with faster performance on direct trials than on impeded trials. The results also showed that participants were slower on relation‐only trials  than on object+relation trials  . This is consistent with prior findings that comparison is in general easier when there exist object correspondences as well as relational correspondences  trials was lower for horizontal pairs than for vertical pairs. This lower impedance cost for horizontal pairs, termed the 1.4Our chief goal in this work is to discover to what extent the spatial alignment principle operates in children. An answer to this question will shed light on the development of visual comparison ability and provide educators with a better understanding of how to design instructional materials. Because children are highly reliant on perceptual supports for structural alignment, our expectation is that spatial alignment will be at least as important in children's comparison as in adults’.As a secondary goal, we further explored Matlen et al.’s  speculatChildren start to form print awareness at age 4, including recognizing that print is read from left to right Hiebert, . ReasoniWe adapted the procedures used in Matlen et al.  to be chTo prepare them for the task, children were given extensive training (see study 1 procedure). In addition, we used a fixed order of blocks, such that the cross‐dimension block was always given after the two within‐dimension blocks. This is based on findings that children find purely relational matches harder to process than concrete object+relation matches  comparisons and for cross‐dimension  comparisons. Our second prediction, also following structure‐mapping theory, is that children's performance should be better on within‐dimension (object+relation) trials than on cross‐dimension (relation‐only) trials.Our third prediction was that, as in Matlen et al. , there sBeyond testing the above three predictions, a further goal of this research was to explore whether children would show a horizontal advantage—that is, lower impedance cost for horizontal figures than for vertical figures—and if so, whether this would be related to their reading fluency. If, as Matlen et al. (2020) conjectured, robust horizontal encoding is the result of experience—especially practice in reading—then (1) this pattern should be more pronounced in 8‐year‐olds than in 6‐year‐olds, and (2) if a horizontal advantage does appear, it should be related to reading ability. We measured reading fluency in children through two standard reading assessments, word reading fluency (WRF) and oral reading fluency (ORF) tests, adapted from Dynamic Indicators of Basic Early Literacy Skills  and study 2 (8‐year‐old children).22.12.1.1M = 6.51 years, Range = 6.00–7.06 years, 11 females; among the families who indicated race, 79% were White, 11% were Asian, 7% were multiracial, and 3% indicated other races and ethnicities). For the 28 families who indicated parental educational level, all except one indicated college degree or higher. One additional child was recruited but excluded due to failure to complete the experiment. Children were recruited through a database of families willing to participate in child research from a large Midwestern city. All families had English as the primary language.Twenty‐nine 6‐year‐old children participated . Pairs varied in the structural axes of triplets  and relative placement between the two triplets . Pairs depicted either the same or different patterns (referred to as Concordance). The design was within‐subject and consisted of 2 Dimension (within vs. cross) × 2 Orientation  × 2 Placement (direct vs. impeded) × 2 Concordance (same vs. different). Each within‐dimension block contained 36 test trials and four catch trials, and the last cross‐dimension block contained 72 test trials and eight catch trials. In total, participants were required to complete 160 trials in the test phase in addition to the trials used in training. Table Stimuli were adapted from Matlen et al.’s  experime2.1.2.1color‐color trials, each triplet consisted of star shapes of two colors . In shape‐shape trials, each triplet consisted of three black geometric shapes . This resulted in 18 possible pairings [3×3×2], twice as many as those in each within‐dimension block. A “same” response was called for when triplets within a pair contained the same pattern. Having the same patterns required matching on first‐order relation and, therefore, spatial correspondence for the unique element within each triplet . One‐third of all test trials were “same.”A similar plan was used for cross‐dimension triplets. In color‐shape trials, one triplet consisted of stars of reds and blue and the other of black squares and triangle. Fig. direct trials, the placement of triplets was orthogonal to their structural axes . In impeded trials, triplets were placed consecutively along their structural axes . Together with the number of possible pairings, our manipulation resulted in 36 [9×4] test trials for each within‐dimension block and 72 [18×4] test trials for the cross‐dimension block.We systematically varied the orientation and placement of each pair and created four possible combinations . Triplets within a pair always shared the same orientation of their structural axes, but their placement varied. In Triplets were presented on a 13‘ monitor at 1024×768 resolution. Displays centered at a black fixation cross (16×16 pixels in width and height) on a white background. Each triplet element had a 74×74 bounding box. For within‐dimension triplet, elements were 10 pixels away from each other, resulting in the size of 74×242 pixels for vertical triplets and 242×74 pixels for horizontal triplets. For cross‐dimension triplet, elements were 40 pixels apart. Centers for all triplets were 205 pixels away from the fixation.2.1.3After obtaining parental consent and children's verbal assent, the experimenter brought participants into a quiet room with a 13‐inch MacBook Pro open. There were four experimental phases: key orientation, same‐different training, testing, and reading assessment, with the first three phases run on the software platform of PsychoPy3  if a letter “L” showed up on the left side of the screen and the right‐hand key (“comma”) if a letter “R” showed up on the right side of the screen. The trial began after children press the “Next” key. A fixation cross first appeared for 1000 ms, after which the picture appeared with the fixation cross still at the center. This presentation format was used throughout the experiment. This was repeated for six random trials, and after the third trial, children were prompted to respond as fast as they could. During training, automated feedback appeared on the screen after each response—“Great Job!” for the correct answer and “Whoops!” for the wrong answer.2.1.3.2elephant‐elephant‐rabbit), let children name the pattern on the right, and asked them whether the triplets contained the same or different patterns. Depending on children's response, the experimenter said “Yes/ no. They have different patterns even though they have the same animals.” Additional explanation was provided if children were wrong: “Here (pointing to the left triplet) the rabbit is at the bottom but here (pointing to the right triplet) the rabbit is at the top. The animals are in different order. And that's why they don't have the same patterns, even though they have the same animals.” Children were asked to do the rest on their own.The goal of this phase was to provide a series of training steps so that children would understand the task. Children were told to press “S” if they think the two pictures were the same and “D” if they were different. Automated feedback was provided after each response. There were eight trials in a fixed order, alternating between different and same trials and between vertical and horizontal placement. The first four trials required children to do a same‐different match on two animals . The last four trials showed pairs of animal triplets in direct placement (consisting of two elephants and a rabbit) Fig. . On the 2.1.3.3The testing phase was divided into three blocks as detailed above: The first two were object+relation, within‐dimension  and the last one was always relation‐only, cross‐dimension . Children were instructed to respond as fast as they could while also trying to get all trials correct. Within each block, children first completed randomized practice trials with feedback provided (three for within‐ and six for cross‐dimension blocks). If children made no error, they proceeded to the test trials after the experimenter checked for whether they had any questions. No feedback was provided for the test trials. If children erred on any practice trials, they were asked to redo the practice trials for that block up to three times to ensure understanding of the task instructions. For within‐dimension blocks, no children erred more than three times. All triplet pairs in the practice contained different elements from those in the actual experiment. Shape practice trials consisted of octagons and stars; and color practice trials consisted of black and white stars. Both direct and impeded placements were included in practice trials.blue‐red‐blue and square‐circle‐square, he would say, “here (pointing to the first triplet) the red star is different from the two blue stars and this different thing is in the middle. Here (pointing to the second triplet) the circle is different from the two squares and this different thing is also in the middle. So they have the same patterns.” If children erred on any practice trial, they were asked to redo the practice up to three times. The same explanation was repeated on the first practice trial, but in a more engaging fashion—for example, by checking with children for understanding throughout the explanation. All but one child passed the cross‐dimension practice within three tries. Data of this child for the cross‐dimension block were excluded from further analyses.For cross‐dimension practice trials, the pairs contained circle and squares for the shape triplets and one red and two blue stars for the color triplets. Special instructions were given on the first practice trial. The experimenter paused and explained to children that they would need to match locations of the unique element in each triplet instead of relying on identical patterns. Specifically, the experimenter read the patterns of triplets on the screen. For example, if the pair depicted There were 36 test trials for within‐ and 72 test trials for cross‐dimension blocks, with trial order randomized within each block. Each trial began with an instruction screen that wrote “Ready? Press ‘Next’.” After participants pressed the space bar, a fixation cross appeared for 1000 ms, following which a pair of triplets showed up. Participants indicated whether the triplets shared the same or different patterns by pressing “S” and “D,” respectively. Response was timed after stimulus onset and no feedback was provided. After each response, the instruction screen for the next trial appeared. After completing the first block, the experimenter told children that they were going to play the same game but with new pictures. After completing the second block, the experimenter told children that the same pattern matching game was made harder, and he would explain why it was harder during the practice.blue‐blue‐blue and blue‐blue‐blue) or shape . The cross‐dimension catch trials consisted of eight directly placed pairs of triplets that contained a single color and shape . Catch trials varied in triplet orientation  and in element value (blue and red for colors and square or triangle for shapes), but all elements came from the test trials of the same block. The exclusion criterion was predetermined to be more than one error for each within‐dimension block and more than two errors for the cross‐dimension block.Immediately after all test trials in each block, catch trials were included to detect children who might be responding randomly due to fatigue or failure to conform to the instructions. There were four catch trials for each within‐dimension block. In these trials, the triplets in a pair were in direct placement and contained only a single color . We used the materials intended for first‐grade students because we focused on 6‐year‐olds. Following the instructions and requirement specified by DIBELS  that 6‐year‐olds would be faster and/or more accurate on trials in direct placement than on trials in impeded placement  and (2) that children would perform better on within‐dimension trials than on cross‐dimension trials Figs.  and 8. BDimension: within or cross, referring to the triplet types (color‐color/shape‐shape or color‐shape)Orientation: vertical or horizontal, referring to the structural axes of tripletsPlacement: direct or impeded, referring to the relative placement of the two tripletsConcordance: whether the two triplets share the same or different patternsFor the overall analysis, we conducted 2 (Placement) × 2 (Dimension) × 2 (Orientation) × 2 (Concordance) repeated‐measures ANOVAs for error rates and response times, with subject as the error term. Definitions of the terms are summarized below  = 24.10, p < .001, ηp2 = 0.06. There was no significant effect of dimensionality, F = 3.33, p = .07, ηp2 = 0.01, nor any significant interaction.The repeated‐measures ANOVA on error rates revealed only a significant main effect of placement, t‐testsM = 0.12, SD = 0.15) than on direct trials , t = 5.15, p < .001, d = 0.38, 95% CI = 0.23–0.53. Our second prediction was that children should be faster and/or more accurate on within‐dimension trials than on cross‐dimension trials. In this case, the difference in error rates between cross‐dimension trials  and within‐dimension trials  was not statistically significant, t = 1.93, p = .055, d = 0.16, 95% CI = −0.004 to 0.33.Planned paired 2.2.1.2F = 11.65, p < .001, ηp2 = 0.03, and dimensionality, F = 200.30, p < .001, ηp2 = 0.35. There was no significant interaction effect.The repeated‐measures ANOVA on response times revealed main effects of placement, t‐tests showed that, consistent with Prediction 1, children were faster on direct  than on impeded trials , t = 4.59, p < .001, d = 0.19, 95% CI = 0.11–0.28. Prediction 2 was also borne out: children were faster on within‐dimension trials  than on cross‐dimension trials , t = 11.65, p < .001, d = 0.71, 95% CI = 0.58–0.85.Planned paired Taken together, the results confirm our first hypothesis: children were both faster and more accurate on trials in direct placement than on trials in impeded placement (Prediction 1). The results also bore out our second hypothesis: children were faster, though not more accurate,2.2.2Our third main prediction was that the advantage of direct over impeded placement would be greater for within‐dimension trials than for cross‐dimension trials. The rationale is that the salient object matches should facilitate performance in direct placement, but interfere with performance in impeded placement; this will not apply to cross‐dimension trials, which do not involve the same objects.d) is used instead of simple mean because blocks with different dimensionality might have different means and standard deviations, which is better addressed by an effect size measure. For this analysis, we combine horizontal and vertical triplets, because our third prediction applies to both orientations and because the ANOVA models above did not reveal any effect of orientation. However, for clarity, we present separate graphs for horizontal and vertical triplets  was significantly smaller for horizontal triplets than for vertical triplets , t = 5.55, p < .001, d = 0.99, 95% CI = 0.55–1.43. For the cross‐dimension trials, the magnitude of the direct‐over‐impeded advantage did not differ between horizontal and vertical triplets , p = .98. These results are consistent with there being a horizontal advantage for within‐dimension, but not cross‐dimension, trials.To arrive at the degree of horizontal advantage, for each child, we first subtracted the response times of direct trials from those of impeded trials, for triplets of both horizontal and vertical orientations . A positive score indicates a direct‐over‐impeded advantage; the smaller it is, the lower the impedance cost is. If children mirror the horizontal advantage found in adults, they will show a lower impedance cost for horizontal than for vertical figures. Paired HorizontalM = 0.076, HorizontalSD = 0.122) was nonsignificantly higher than the impedance cost for vertical triplets . Although this difference was not statistically significant in a t‐test, p = .22, we cannot rule out the possibility of a speed‐accuracy tradeoff. Thus, we cannot conclude that 6‐year‐old children showed a horizontal advantage in response time. However, it remains possible that across individuals, there could be a correlation between reading ability and a horizontal advantage in response time.However, before drawing conclusions, we must consider children's error rates on within‐dimension trials. Using the same procedure, we calculated the impedance cost for error rates . The impedance cost for horizontal triplets .SD = 11.55) correct words in the WRF test and 32.96 (SD = 36.15) correct words in the ORF test  for horizontal and vertical triplets for each child, as described above. We then subtracted the impedance cost for horizontal triplets from that for vertical triplets. A positive score means a lower horizontal impedance cost, because it indicates that the direct‐over‐impeded advantage is smaller for horizontal than for vertical triplets. Using the horizontal advantage as the dependent measure, we ran a linear regression with composite reading score as the predictor Fig. . Reading2.3The results from study 1 are consistent with the idea that visual comparison in 6‐year‐olds follows the structural alignment process and is facilitated by spatial alignment. Our first hypothesis, that the spatial alignment principle applies to even 6‐year‐olds, was confirmed. Children were faster and more accurate for direct placement than for impeded placement. Evidence also confirmed our second hypothesis, that structural alignment should be easier when both object correspondences and relational correspondences are present, compared to when correspondences are purely relational. Here, 6‐year‐olds were faster (and no less accurate) on within‐dimension (object+relation) trials than on cross‐dimension (relation‐only) trials.For our third hypothesis, we predicted an interaction between placement and dimensionality. This is because objects are highly salient in structural alignment process. This means that in direct placement, objects should add another basis (besides relations) for a same‐different judgment, but in impeded placement, object mismatches should pose another source of interference in arriving at the correct correspondences. The current results failed to provide statistical support for this hypothesis. For both response time and error rate, the direct‐over‐impeded advantage for the within‐dimension trials was not significantly greater than that for the cross‐dimension trials.We also had two exploratory questions regarding the horizontal advantage. Our first question was whether 6‐year‐olds, like adults, would show a horizontal advantage . The evidence was inconclusive: the direct‐over‐impeded advantage in response time was smaller for horizontal figures on within‐dimension trials ; but because we saw the opposite (nonsignificant) pattern for error rates, we cannot rule out the possibility of a speed‐accuracy tradeoff where children might have sacrificed accuracy in the interest of speed. Nonetheless, we went on to explore our second question—whether the degree of horizontal advantage would correlate with reading ability. A regression analysis failed to show a significant relationship.An important overarching question in this research concerns how these processes develop over age and experience. Do the patterns found among 6‐year‐olds also hold for 8‐year‐olds? And do some patterns change as a result of greater experience? In study 2, we tested the same set of hypotheses and questions in 8‐year‐olds. We hypothesized continuity, in that visual comparison in 8‐year‐olds should also follow the structural alignment process and should be facilitated by spatial alignment. However, we also predicted change: we expected the older group to show a higher horizontal advantage, given they should have more experience in reading. We also asked whether this potentially stronger effect of horizontal advantage would be correlated with children's reading fluency.33.13.1.1M = 8.08 years, Range = 7.51–8.81 years, 15 females; among the families who indicated race, 79% were White, 5% were Asian, 10% were multiracial, and 5% indicated other races and ethnicities). For the 21 families who indicated parental educational level, all except one indicated college degree or higher. Children were recruited through a database of families from a large Midwestern city who had expressed interest in participating in child research. All families had English as the primary language.Twenty‐nine 8‐year‐old children participated  8‐year‐olds were faster and more accurate on direct placement than on impeded placement, and (2) children were faster on within‐dimension trials than on cross‐dimension trials  = 20.83, p < .001, ηp2 = 0.05, and concordance, F = 7.69, p = .01, ηp2 = 0.02. The main effect of dimensionality was not significant, F = 1.99, p = .16, ηp2 = 0.005. There was a significant interaction between placement and dimensionality, F = 4.52, p = .03, ηp2 = 0.01. We report the effect of concordance and the interaction in Supplementary Materials.The repeated‐measures ANOVA on error rates revealed significant main effects of placement, t‐tests were carried out for our first two predictions. First, we found evidence for the advantage of direct placement—children made significantly more errors on impeded trials  than on direct trials , t = 4.60, p < .001, d = 0.40, 95% CI = 0.22–0.58. Our second prediction, that performance would be better on within‐dimension trials than on cross‐dimension trials, was not borne out. There was no significant difference in error rates between cross‐dimension trials  and within‐dimension trials , t = −1.49, p = .10, d = −0.12, 95% CI = −0.28 to 0.04.Two planned paired F = 55.82, p < .001, ηp2 = 0.12, dimensionality, F = 284.03, p < .001, ηp2 = 0.40, and concordance, F = 7.58, p = .01, ηp2 = 0.02. There was also a significant interaction between placement and orientation, F = 15.60, p < .001, ηp2 = 0.04. We report the effect of concordance and the interaction in Supplementary Materials.The repeated‐measures ANOVA on response times revealed main effects of placement, t‐tests showed that, consistent with Prediction 1, children were faster on direct trials  than on impeded trials , t = 8.98, p < .001, d = 0.33, 95% CI = 0.25–0.40. Results were also consistent with Prediction 2: children were faster on within‐dimension trials  than on cross‐dimension trials , t = 15.27, p < .001, d = 0.73, 95% CI = 0.63–0.84. Together with the error rates, the results supported our first hypothesis: 8‐year‐olds, like 6‐year‐olds, were both faster and more accurate on direct placement than on impeded placement (Prediction 1). The results were also consistent with our second hypothesis: children were faster on within‐dimension trials than on cross‐dimension trials (with no evidence of a speed‐accuracy tradeoff) (Prediction 2).Planned paired 3.2.2Our third hypothesis was that the advantage of direct over impeded placement would be greater for within‐dimension trials than for cross‐dimension trials. To test this, we compared the effect sizes of error rates and response times between direct and impeded placement and did this for both within‐ and cross‐dimension trials. As for study 1, we combine horizontal and vertical figures for this analysis, but present separate graphs for the two orientations for clarity. Table ds were not significantly larger for the within‐dimension trials than for the cross‐dimension trials—a pattern that parallels the findings for the 6‐year‐olds. A combined analysis over both age groups revealed significantly larger effect sizes for direct placement over impeded placement for within‐dimension than for cross‐dimension trials. This held for both error rates and response times, providing support for our third hypothesis , t = 3.68, p < .001, d = 0.97, 95% CI = 0.33–1.62, and cross‐dimension trials , t = 3.54, p = .001, d = 0.60, 95% CI = 0.23–0.97. Thus, 8‐year‐olds, like adults and unlike 6‐year‐olds, show a significant horizontal advantage in response time—a lower impedance cost for horizontal figures than for vertical figures. Importantly, we did not see evidence of a speed‐accuracy tradeoff for 8‐year‐olds: t‐tests showed that the impedance cost for error rates did not differ between horizontal and vertical triplets for within‐dimension trials , p = .90, nor for cross‐dimension trials , p = .09. In fact, for cross‐dimension trials, the impedance cost for error rate was nonsignificantly lower for horizontal than for vertical triplets; thus, we can rule out a speed‐accuracy tradeoff for this condition. These findings provide support for the idea that the horizontal advantage increases over age and experience.The horizontal advantage was measured by first subtracting the response times of direct trials from those of impeded trials. A positive score indicates the presence of an impedance cost . This difference score was calculated for triplets of both horizontal and vertical orientations. If 8‐year‐olds mirror the adults’ pattern, they will show a lower impedance cost in response time for horizontal than for vertical figures . Paired 3.2.4Last, we ask whether the horizontal advantage in response time is related to reading fluency. To do so, we regress the degree of horizontal advantage on children's reading ability, as assessed by the four standard reading tests. We report 8‐year‐olds’ performance both using the first‐grade reading materials (so that the results are directly comparable across ages) and the age‐appropriate third‐grade materials.SD = 20.55) correct words in the WRF test and 130.61 (SD = 40.39) correct words in the ORF test (see Appendix B). As expected, 8‐year‐olds outperformed 6‐year‐olds in the first‐grade reading materials for both WRF and ORF, both ps < .001. Performance on the two tests was highly correlated, r = .76, p < .001, so we combined the WRF and ORF scores into a composite score using the weights intended for first‐grade materials [WRF * 0.1351 + ORF * 0.2536]. The resulting reading score had a mean of 42.22 and a standard deviation of 12.47.For the first‐grade materials, on average, 8‐year‐old children read 67.30  correct words in the WRF test and 118.74 (SD = 44.91) correct words in the ORF test (see Appendix C). Performance on the two reading scales was highly correlated, r = .83, p < .001, so the WRF and ORF scores were combined into a composite score using the weights intended for third‐grade materials [WRF * 0.1983 + ORF * 0.3942]  were planned based on our main within‐subject predictions for spatial alignment. But to adequately test the between‐subject relationship between reading ability and performance , a sample size of at least 44 is required. In order to achieve the necessary power for a between‐subject analysis, we combined the two age groups, using the same first‐grade reading assessment for both groups. This gave us a sufficient sample size (48 participants who completed the reading assessments) to test the correlation between reading ability and the degree of horizontal advantage in response time. The regression analyses showed that reading skills failed to predict the degree of horizontal advantage for both within‐dimension trials, β = −4.21, t = −1.34, p = .18, ηp2 = 0.04, and cross‐dimension trials, β = 2.00, t = 0.28, p = .78, ηp2 = 0.002. Fig. As discussed above, our sample sizes for the individual age groups  trials than on cross‐dimension (relation‐only) trials. This prediction too was borne out—both age groups were faster  for the within‐dimension trials than for the cross‐dimension trials. This is consistent with structure‐mapping theory  should be larger in 8‐year‐olds, who have more experience in reading, than in 6‐year‐olds. Second, the degree of horizontal advantage should be related to reading proficiency. Our findings supported the first prediction: We failed to find evidence of a horizontal advantage in 6‐year‐olds, but 8‐year‐olds showed a strong horizontal advantage in response time for both object+relation and relation‐only matches. The second prediction was tested by regressing the degree of horizontal advantage on children's reading ability. The results failed to support the claim that reading contributes to more fluent horizontal encoding. Indeed, we found an opposite pattern in 8‐year‐olds.Given our evidence of an experience‐based horizontal advantage, one possibility is that this advantage arises from more general experience with patterns in the world, such as frequent exposure to horizontally symmetric faces. Alternately, it may be that reading experience does contribute but that fluency takes longer to develop. Future research should continue to explore possible effects of reading proficiency, as well as other pathways to horizontal fluency.4.2These findings bear on the well‐supported generalization that spatiotemporal proximity facilitates visual comparison (Christie & Gentner, One limitation of the current study is that the cross‐dimension block always followed the within‐dimension blocks. This could have affected children's performance on the cross‐dimension trials. Because children had the opportunity to engage in within‐dimension pattern matching before the more difficult cross‐dimension matching task, this could have facilitated children's performance on the later purely relational matches. This is consistent with the idea of progressive alignment (Kotovsky & Gentner, Another limitation is that the current study focused on simple spatial patterns with a clear structural axis. A key issue is how well the spatial alignment principle will scale up to more complex spatial patterns. There is some encouraging research in this direction. Simms et al.  showed s4.3Visualizations are prevalent in education and important for students’ learning (Ainsworth et al. Comparison highlights similarities and also differences that are connected to a shared system of relations (Goldwater & Gentner, Yet, despite the potential advantages of visual comparison, recent research on the presentation of visual pairs in textbooks has found wide variation in the effectiveness of this presentation (Jee, Matlen, Gentner, & Simms, 5We found that the spatial alignment principle held for both 6‐ and 8‐year‐olds, showing continuity in visual comparison processing across development. These results support the idea that visual comparison involves a process of structural alignment. Apart from their implications for basic visual processes, the results are relevant for learning and instruction. Attention to the spatial alignment principle and other principles of visual comparison could have important benefits for the design of instructional materials.Table S1: Effect size estimates for response time in 6‐year‐olds after excluding fast trials.Table S2: Effect size estimates for response time in 6‐ and 8‐year‐olds using MAD.Click here for additional data file.


Noninvasive detection of histological abnormalities remains challenging in patients with HBeAg-negative chronic HBV infection with normal or mildly elevated levels of alanine aminotransferase (ALT). This study aimed to assess the utility of serum quantitative hepatitis B surface antigen (qHBsAg) in identifying significant histological lesions in this population.  This is a single-center study with retrospective analysis of 392 treatment-naive patients of HBeAg-negative chronic HBV infection with normal or mildly elevated levels of ALT. N = 236) had more hepatic inflammation of ≥G2  or fibrosis ≥ S2  compared to those without (N = 156). Serum HBsAg (cutoff point = 1000 IU/mL), aspartate aminotransferase (AST) level (cutoff point = 25 IU/L), age (cutoff point = 40 years), and HBV family history were identified as independent predictors of significant histological abnormalities in multivariate logistic analysis.  In this cohort, significant necroinflammation and fibrosis were found in 69.4% and 61.5% of patients, respectively. Patients with qHBsAg >1000 IU/mL ( A significantly higher proportion of patients with histological abnormalities were found in patients with qHBsAg >1000 IU/mL than those without. The qHBsAg level together with age, AST, and family history of HBV infection could be used as an algorithm to help noninvasive patient selection for antiviral therapy. Chronic hepatitis B virus (HBV) infection is one of the common causes of chronic liver diseases and remains a global public health threat. During the natural course of HBV infection, around 15–40% will develop HBV-related complications including hepatitis B flare-up, liver cirrhosis, and hepatocellular carcinoma (HCC) –4. NoninIn untreated patients, quantitative hepatitis B surface antigen (qHBsAg) declines slowly through the natural course and remains stable for a long time after HBeAg seroconversion. Several studies reported that a qHBsAg level below 1000 IU/ml in HBeAg-negative patients was strongly associated with the inactive carrier state, especially in patients with a low serum level of HBV DNA and normal ALT –7. Thus,The current study is designed to assess the utility of qHBsAg in distinguishing significant histological abnormalities in treatment-naive HBeAg-negative chronic HBV infection patients with normal or mildly elevated ALT levels.This is a single-center, cross-sectional observational study of HBeAg-negative treatment-naive CHB patients with normal or mildly elevated ALT levels. All patients underwent liver biopsy for the assessment of a liver disease between January 2009 and December 2019. Inclusion criteria were defined as follows: (i) aged older than 18 years; (ii) with positive serum hepatitis B surface antigen for at least 6 months; and (iii) with serum HBeAg negative. Exclusion criteria were defined as follows: (i) with HCC or other chronic liver diseases such as other viral hepatitis, autoimmune hepatitis, and nonalcoholic fatty liver disease; (ii) with previous history of any antiviral therapy; (iii) with unqualified liver samples, defined as less than 10 mm or containing less than six portal triads; and (iv) with ALT >80 IU/L. This study complied with the Declaration of Helsinki and was approved by the Institutional Ethics Review Committee at Ruijin hospital.G ≥ 2 (significant inflammation) and/or S ≥ 2 (significant fibrosis) were considered as having significant histological abnormalities. These patients are indicated for antiviral therapy irrespective of the ALT level according to the AASLD [Demographical, laboratory, and histological data were collected within one month before liver biopsy. The upper limit of normal (ULN) for ALT was 40 U/L according to the EASL criterion. Qualitative HBsAg and HBeAg analyses were performed by AxSYM or Architect assays . All patients provided written informed consent for liver biopsy. The indications for liver biopsies are as follows: (1) staging of inflammation and fibrosis to guide antiviral therapy and 2) differential diagnosis of other liver diseases other than HBV infection. Histologic grading of necroinflammation (G0–G4) and staging of liver fibrosis (S0–S4) were performed according to the Scheuer scoring system by two senior liver pathologists blinded to clinical data/biological data. Patients with he AASLD , and APA different-tests when the data were normally distributed; otherwise, the Mann–Whitney test was used. Categorical variables were compared using the χ2 test or Fisher's exact test as appropriate. Univariate logistic regression was performed to identify the risk factors of significant histological abnormalities followed by multivariate logistic regression to adjust potential confounding effects. The optimum cutoff values for identification of significant histological changes were selected by maximizing the sum of sensitivity and specificity. All statistical analyses were performed using GraphPad Prism 7.0  or SPSS 24.0 . A two-tailed P value of <0.05 was considered statistically significant.Categorical variables were described as count (percentages). Continuous variables with normal distribution were presented as mean ± standard deviation (SD), otherwise as median . Means for continuous variables were compared using independent group N = 236, 60.2%) and low HBsAg groups . Baseline characteristics are shown in A total of 827 patients with HBeAg-negative chronic HBV infection were screened and liver biopsy results were reviewed and 392 of them were eligible for analysis . PatientP=0.021) with higher ALT (P=0.005), AST (P=0.027), GGT (P=0.001), and HBV DNA (P=0.004) levels compared to those in the low HBsAg group. Besides, the proportion of significant liver histological abnormality in the high HBsAg group was significantly higher than that in the low HBsAg group .Patients in the high HBsAg group were significantly younger (P=0.002) or fibrosis  was more frequently observed in patients with high HBsAg than in those with low HBsAg, respectively .Significant necroinflammation , AST , and HBV family history  were identified as independent predictors for significant liver histopathology under multiple logistic regression analysis with stepwise selection (P=0.0001), AST , or age  was identified as an independent predictor for significant necroinflammation and HBsAg level , AST , or HBV family history  for significant fibrosis , AST level (higher or lower than 25 IU/L), and age (higher or lower than 40 years) as shown in G2, fibrosis ≥ S2, and necroinflammation ≥ G2 or fibrosis ≥ S2 were 83%, 79%, and 92%, respectively. 30 (7.7%) patients had HBsAg ≤1000 IU/mL, age <40 years, and AST <25 IU/L. The proportion of patients with necroinflammation ≥ G2, fibrosis ≥ S2, and necroinflammation ≥ G2 or fibrosis ≥ S2 were 57%, 47%, or 63%, respectively.Among patients with ALT ≤2ULN, there were 76 (19.4%) patients with HBsAg ≥1000 IU/mL, age >40 years, and AST >25 IU/L. The proportion of patients with necroinflammation ≥ G2, fibrosis ≥ S2, and necroinflammation ≥ G2 or fibrosis ≥ S2 were 81%, 77%, and 91%, respectively. 30 (7.7%) patients had HBsAg ≤1000 IU/mL, age <40 years, and AST <25 IU/L. The proportion of patients with necroinflammation ≥ G2, fibrosis ≥ S2, and necroinflammation ≥ G2 or fibrosis ≥ S2 were 57%, 47%, and 63%, respectively.Among patients with ALT ≤ ULN, there were 43 (15.8%) patients with HBsAg ≥1000 IU/mL, age >40 years, and AST >25 IU/L. The proportion of patients with necroinflammation ≥ Predictive values for serum levels of HBsAg, AST, and age for identifying significant histological changes are shown in qHBsAg has been proposed as a new diagnostic tool for the characterization of the HBV disease state. Recent reports suggest that HBsAg quantification might be a useful complement to HBV DNA quantification for clinical assessment and treatment monitoring in chronic HBV infection patients –9. This Our study found that 77.8% treatment-naive HBeAg-negative chronic HBV infection patients with ALT ≤2ULN had a significant liver histological change. It is worth noting that 74.3% of patients with ALT ≤ ULN had a significant liver histological change, which is the criterion for starting an antiviral treatment. Therefore, it is critical to distinguish these patients using virological or biochemical markers without liver biopsy, so as to improve their clinical outcomes. Also, the proportion of patients with significant liver fibrosis and inflammation in the high HBsAg group was significantly higher than the low HBsAg group. Moreover, the high HBsAg group had higher ALT, AST, and GGT levels than the low HBsAg group, indicating that the HBsAg level can reflect the liver necroinflammatory activity. This is consistent with previous studies showing that the HBsAg level reflects a clinical stage and liver disease severity , 9. HoweAs recommended by the AASLD guidelines, HBeAg-negative patients with ALT ≤2ULN are recommended to monitor the levels of ALT and HBV DNA or go through liver biopsy, instead of starting the antiviral treatment immediately . HoweverThe chances of histological abnormalities in our cohort indicated that the overall diagnostic accuracy for identifying significant liver histological abnormalities by the HBsAg level was better than the combination with HBV DNA or HBV DNA alone. Using HBsAg, AST, and age as parameters to identify patients with significant histological abnormalities showed a PPV of 92.1% and specificity of 93.1%, indicating it as an effective way for screening patients in need of antiviral treatment. According to our results, whether ALT ≤2ULN or ALT ≤ ULN, patients with HBsAg ≥1000 IU/mL, age >40 years, and AST >25 IU/L can start antiviral treatment without having biopsy, as about 90% of them had significant liver histological abnormalities. Patients who meet any two of the above criteria should assess disease severity with noninvasive tests or liver biopsy. Patients with HBsAg <1000 IU/mL, age <40 years, and AST <25 IU/L have a lower chance of histological abnormalities but still need close monitor of serum markers and perform a liver biopsy when necessary.The current study has some limitations. As a retrospective cross-sectional study, the analysis was based on data from a single time test. The relatively higher percentage of patients with significant liver histological changes compared to other studies could be related to this reason. Also, data such as HBV genotype were missing because this test was not performed routinely in clinical practice. Although using HBsAg and AST levels and age as parameters to identify patients with significant histological changes showed high specificity and PPV, the sensibility and NPV are relatively low. Prospective studies in the future are still needed.In conclusion, qHBsAg level helps to identify histological significant abnormalities in treatment-naive HBeAg-negative chronic HBV infection patients with normal or mildly elevated levels of ALT. An algorithm based on the qHBsAg, AST, age, and HBV family history further improves discriminative accuracy and could be a valuable tool to guide antiviral treatment.QG, YH, and CZ share co-first authorship.


The risk of transmission is relatively high in African countries, mainly due to unreliable screening methods of blood donations. In Burkina Faso, predonation screening using rapid diagnostic tests (RDTs) is widespread, raising the major question of the transfusion safety in the country. The objective of this study was to assess the risk of transmission of HBV, HCV, and HIV through blood transfusion in the context of the use of RDTs for screening of the blood donations.In this cross‐sectional study, a total of 417 serum samples obtained from blood donors tested negative for HBsAg, anti‐HCV, and anti‐HIV using RDTs were retested for the same markers using chemiluminescent immunologic assays. Total antibodies to HBV core (anti‐HBc) were tested on randomly selected samples. HBV‐DNA and HCV‐RNA viral loads (VLs) were quantified on HBsAg and anti‐HCV positive samples, respectively. To assess possible occult hepatitis B infection (OBI), HBV‐DNA‐VL was quantified on 313 randomly selected HBsAg‐negative samples.HBsAg and anti‐HCV were found respectively in 6  and 11  samples. No samples were reactive for anti‐HIV. Total anti‐HBc were detected in 217 out of the 319 randomly selected samples . HBV‐DNA was detected in four  samples, including two  of the six HBsAg positive samples and two  of the HBsAg‐negative samples, suggesting two cases of occult HBV infection. All anti‐HCV antibody‐positive samples were HCV‐RNA negative.This study shows that RDTs are not sufficiently sensitive for the screening of blood donations. Our results highlight the urgent need to think about the extension of sensitive immunological tests in all blood transfusion centers and also the implementation of nucleic acid amplification techniques. Each year, about 1.34 million people die from hepatitis infections and 96% of these deaths are attributable to hepatitis B virus (HBV) and hepatitis C virus (HCV).To address this situation, the WHO advocates the implementation of various strategies including mandatory screening of all blood donors for major transfusion–transmissible infections by high‐quality laboratory testing.In Burkina Faso, with respective prevalence rates of 9.1%, 3.6%, and 0.8% in the general population, HBV, HCV, and HIV infections remain major public health problems.22.1This cross‐sectional descriptive study was conducted between November 2019 and March 2020 in four health centers of Burkina Faso, the Regional Hospital (RH) of Banfora in the “Cascades” region, the RH of Dédougou in the “Boucle du Mouhoun” region, the Medical Center (MC) of Pô in the Centre‐South region and the MC of Gourcy in the North region , HCV antibodies (anti‐HCV), and HIV‐1/2 antigen/antibodies (anti‐HIV) using RDTs, and who signed an informed consent form to participate in the study. Venous whole blood sample was collected from each participant into a dry tube. Sera were obtained after centrifugation of the samples at 4000 rpm for 5 min and were stored at −80°C until analysis. In addition, sociodemographic characteristics and knowledge about viral hepatitis were recorded using a structured questionnaire. These data included age, sex, education, occupation, marital status, viral hepatitis transmission routes, and immunization status. Detailed information about the RDTs used during the study period at each site are presented in Table 2.3All serum samples were tested for HBsAg, anti‐HCV, and anti‐HIV using the CLIA on the Cobas® 6000 analyzer . Total anti‐HBc were tested on samples randomly selected using CMIA . HBV‐DNA and HCV‐RNA viral loads (VLs) were quantified respectively on HBsAg and anti‐HCV‐reactive samples using the Roche Molecular System . To detect possible occult hepatitis B infection, HBV‐DNA‐VL was quantified in HBsAg‐negative samples randomly selected (COBAS AmpliPrep/COBAS Taqman HBV Test Version 2.0). The manufacturer's reported limits of detection for HBsAg, HIV (p24 antigen), HBV DNA, and HCV RNA were 0.1 (0.88ng/ml), ≤2, 20, and 15 IU/ml, respectively. As for Elecsys® Anti‐HCV II, the sensitivity and specificity were 100% and 99.9%, respectively, and 100% for both for Elecsys® Anti‐HBc II. With respect to the cost of testing, serological markers have been estimated to cost approximately $7.00 per test, and approximately $25.00 for molecular testing. All tests were performed according to the manufacturer's instructions.2.4Statistical analyses were performed using STATA SE version 14.0 software. We used proportions to describe categorical variables and means (standard deviation) to describe continuous variables. Risk of transmission was computed for each pathogen considering CLIA as a gold standard. Statistical inferences were based on 95% confidence intervals (CIs).2.5This study received the approval of the Ethics Committee of the “Institut de Recherche en Sciences de la Santé” before its implementation (17‐2019/CEIRS‐20062019). All participants and legally authorized representatives of illiterate participants provided written informed consent and donors who were tested positive for one or several markers, were referred to a Health Center. The research was performed in accordance with the Declaration of Helsinki.33.1n = 122/417; 29.2%), Dédougou , Pô , and Gourcy  were enrolled. The mean age of the donors was 33.45 years (age range: 18–57 years), 297  were male and 211  were students. The age group from 18 to 29 years was the most represented with 303  participants. Among the donors, 214  had heard of viral hepatitis and 102 had knowledge of transmission routes. As for their hepatitis B immunization status, only 14  of the participants reported being fully vaccinated. The detailed characteristics of the study population are summarized in Table During the study period, a total of 2994 blood donors were tested, and 57 (1.9%) were found positive for HIV, 252 (8.4%) for HBV, 90 for anti‐HCV (3.0%), and 46 (1.5%) for syphilis. Of the blood donors screened negative for these markers (2549), 417 from Banfora  and 11 . No samples were reactive for HIV‐1/2 infection. Of the 319 randomly selected samples, including the six HBsAg‐reactive samples, total anti‐HBc was detected in 217 . HBV‐DNA was detected in four  samples including two  in the sikx reactive HBsAg samples and two  in the nonreactive HBsAg samples, this latter result arguing for two cases of occult hepatitis B. In HBsAg‐reactive samples, HBV‐DNA‐VL were 16.400 and 932 IU/ml, and for occult hepatitis B infection (OBI) cases, they were 21 and 38 IU/ml. All HBV‐DNA detectable samples were reactive for anti‐HBc. HCV‐RNA was not detected in any of the anti‐HCV‐reactive samples. The serological and molecular results are summarized in Table A multivariable logistic regression analysis reported a statistically significant correlation between knowledge of viral hepatitis (transmission routes) and HBsAg seronegativity  in Burkina Faso and also has WHO prequalification. In addition, a national screening algorithm for HIV using this test is available and is evaluated regularly contributing to reduce the risk of false results. Another explanation could be the low HIV prevalence estimated to 0.8% in the general population.No case of HIV infection was detected, suggesting the good quality of the Except for SD Bioline® tests, all tests used for HBV and HCV predonation screening have not been evaluated and approved in Burkina Faso, and do not have WHO prequalification. Consequently, information on their diagnostic performance on local samples are lacking and there is a risk of missing positive cases with these tests. The routine use of not registered tests in the country could be explained by the fact that the ordering of tests is mostly managed by nonspecialists in the field. Another reason may be the level of corruption in the health sector.An overall prevalence of 68.02% of total anti‐HBc was found, indicating an important circulation of HBV in Burkina Faso.Our results showed that HBV DNA was not detected on four HBsAg‐reactive samples. This could be due to the very low level of viral DNA that could not be detected by the COBAS Version 2.0 used in this study  for molecular analysis. In addition, since a confirmatory test for HBsAg was not used, false positive results cannot be excluded.Based on these overall results, the implementation of sensitive methods in blood centers in Burkina Faso should be strongly considered. CLIA tests, because of their operating cost and capacity, are not suitable for small blood banks. However, they could be used in regional centers, and made accessible to peripheral blood banks through a centralized testing system. High‐performance RDTs could therefore significantly improve patient blood safety. To date, NAAT tests are available in almost all health centers in Burkina because of the response to the COVID‐19 pandemic. Thus, the detection of OBI cases by applying NAAT to pooled samples could be an interesting option since studies have reported transfusion transmission of HBV with blood components from donors with OBI.In our study, assessment of participants' knowledge about viral hepatitis revealed that half of the donors 48.68%) had never heard of viral hepatitis and 75.54% had no information on transmission routes, confirming data we reported earlier.8.68% hadTaking into account the objectives, the major limitation of our study was the relatively small sample size, which did not allow having a comprehensive picture of the magnitude of the transmission risk. Large‐scale studies are therefore needed. Another limitation is the absence of confirmation step HBsAg, anti‐HCV, and anti‐HIV assays.In conclusion, our study showed a potential risk of transmission of HBV (1.44%) when using RDTs for qualification of blood donations. In addition, although HCV RNA was not detected in any samples confirming HCV infection, 11 anti‐HCV‐reactive samples were missed by the rapid tests. These results highlight the need to implement sensitives techniques in blood transfusion in Burkina Faso. OBI cases were also diagnosed in at least 0.6% of the donors. As Burkina Faso is a country of high endemicity for HBV, further confirmed here by high anti‐HBc positivity in our study cohort (about 68%), the implementation of NAAT in blood transfusion centers for the detection of OBI cases, while cost‐effective, should be definitely considered. This will constitute the main point of a high quality of our blood transfusion system.Armel M. Sanou: Conceptualization; data curation; formal analysis; funding acquisition; investigation; methodology; writing – original draft; writing – review and editing. Achille S. Nikiéma: Conceptualization; data curation; investigation; methodology; writing – review and editing. Seimbou Zalla: Conceptualization; data curation; investigation; methodology; writing – review and editing. Mamadou Ouattara: Methodology; formal analysis; software; writing – review and editing. Mariam Seynou: Methodology; formal analysis; software; writing – review and editing. Alice Kiba‐Koumare: Supervision; writing – review and editing. Delphine Napon‐Zongo: Supervision; writing – review and editing. Roger Sombié: Supervision; writing – review and editing.The authors declare no conflict of interest.Armel M. Sanou affirms that this manuscript is an honest, accurate, and transparent account of the study being reported; that no important aspects of the study have been omitted; and that any discrepancies from the study as planned  have been explained. All authors have read and approved the final version of the manuscript. Armel M. Sanou had full access to all of the data in this study and takes complete responsibility for the integrity of the data and the accuracy of the data analysis.


Non‐motor fluctuations (NMF) in people with Parkinson's disease (PwP) are clinically important yet understudied.To study NMF in PwP using both the Movement Disorder Society Non‐Motor Rating Scale (MDS‐NMS) NMF subscale and wearable sensors.We evaluated differences in overall burden of NMF and of specific NMF across disease durations: <2 years (n = 33), 2–5 years (n = 35), 5–10 years (n = 33), and > 10 years (n = 31). In addition, wearable triaxial sensor output was used as an exploratory outcome for early morning “off” periods.P < 0.001), and specifically for depression, anxiety, fatigue and cognition, with both NMF prevalence and burden increasing in those with longer disease duration. Whereas only 9.1% with a short disease duration had NMF (none of whom had dyskinesia), in PwP with a disease duration of >10 years this was 71.0% (P < 0.001). From a motor perspective, dyskinesia severity increased evenly with increasing disease duration, while NMF scores in affected individuals showed an initial increase with largest differences between 2–5 years disease duration (P < 0.001), with plateauing afterwards. Finally, we observed that the most common NMF symptoms in patients with sensor‐confirmed early morning “off” periods were fluctuations in cognitive capabilities, restlessness, and excessive sweating.Significant between‐group differences were observed for MDS‐NMS NMF total scores (Non‐motor fluctuations prevalence in PwP increases with disease duration, but in a pattern different from motor fluctuations. Moreover, NMF can occur in PwP without dyskinesia, and in those with NMF the severity of NMF increases most during years 2–5 after diagnosis. Parkinson's disease (PD) is a neurodegenerative disorder characterized by progressive extrapyramidal symptoms and a range of non‐motor symptoms (NMS), such as neuropsychiatric and autonomic symptoms, sleep disturbances and pain.The most common clinical scenario in people with Parkinson's (PwP) is NMF accompanying motor fluctuations, although the pattern of NMF may vary.Data used in the current analyses were from the dataset used for the validation of the MDS‐NMS and have been described before.For a subset of PwP who were included in the MDS‐NMS validation study, wearable sensor data was available through the Parkinson's Kinetigraph™ Registry (PKG Registry) study which is currently active at the Parkinson's Foundation Centre of Excellence at King's College Hospital London, UK. This registry obtained ethical approval from the London – Riverside Research Ethics committee (REC reference: 17/LO/1010). All patients gave written informed consent, and the study was conducted in line with the Declaration of Helsinki. For a total of 81 participants in the study a PKG reading within 4 months of the MDS‐NMS completion was available. The cut‐off of 4 months has been deployed in previous studies,The primary aim of the current study was to evaluate differences in overall burden of NMF, and of specific NMF, as measured by the NMF subscale of the MDS‐NMS, across different disease durations of PD. For this, we divided the patient cohort (n = 132) into four different groups, depending on disease duration based on previously published cut‐offsFor the wearable sensor outcomes, we compared: (a) dyskinesia severity scores across different disease durations in PD, in line with the MDS‐NMS analysis outlined above; (b) differences in recording dyskinesia severity between the wearable sensor and classical motor scales (MDS‐UPDRS); and (c) NMF severity in participants with and without early morning “off” periods (EMO) as a marker of motor fluctuations. The first epoch (06:00–09:00) was not used for dyskinesia analysis as some participants may still have been asleep during this time potentially providing unreliable readings.U test for total NMF scores, and the degree of change for each of the eight separate NMF were compared using Chi‐Square test. For all correlations, we used Spearman's test.To assess the primary aim, group comparisons were made using Kruskal‐Wallis test. For comparison of the distribution of each of the five severity levels of NMF across the four different disease durations we used Chi‐Square test. With the wearable sensor data, group comparisons were made using Kruskal‐Wallis test. Group differences in NMF total scores between participants with and without EMO were compared through Mann–Witney P‐value ≤0.05 was considered statistically significant, with a correction for multiple testing using Benjamini‐Hochberg procedure, where relevant. Data are presented as mean ± standard deviation, median (interquartile range) or number (percentage), unless otherwise specified. All data were analyzed using SPSS Version 27 For all analyses, a The data that support the findings of this study are available from the corresponding author upon reasonable request. Restrictions apply to the availability of these data, which were used for this study. Data are available only with the permission of the Movement Disorders Society.P = 0.655), disease duration (P = 0.120), LEDD (P = 0.104), MDS‐NMS scores (P = 0.379), MDS‐NMS NMF scores (P = 0.645), or MDS‐UPDRS part III and IV scores (P = 0.751 and P = 0.488).A total of 132 participants with PD were recruited to the MDS‐NMS validation study at King's College Hospital and were included in the current analyses. For 81 of the participants, wearable sensor data was available; the majority of participants had the wearable sensor recording within 30 days of the clinical MDS‐NMS assessment (74.1%). Demographics are provided in Table P < 0.001; Table P < 0.001). In addition, higher dyskinesia and MDS‐UPDRS part IV total scores were observed in those with a longer disease duration (P < 0.001)  Table ; Fig. 1.1) Table ; Fig. 1.P ≤ 0.040), with the >10 years duration group having the highest number of PD participants experiencing these NMF. Moreover, the daily time spent in non‐motor “off” state was higher in the groups with longer disease duration, with only 29.0% of PD participants with a disease duration of over 10 years having <10% non‐motor “off” time .We observed a moderate correlation between MDS‐NMS NMF scores and MDS‐UPDRS part IV scores , despite the lack of between‐group differences in age, disease duration, and LEDD which are usually associated with EMO . We observed a trend towards a statistically significant increase in average dyskinesia scores measured through a wearable sensor over time, with higher scores in PwP with a longer disease duration (P < 0.001), and MDS‐UPDRS part IV scores .We observed weak associations between wearable sensor dyskinesia scores and MDS‐NMS NMF scores  for both motor and NMF.The plateauing of NMF prevalence and severity in participants with longer disease duration in our analysis could perhaps be explained by an increase in the severity of non‐motor symptoms during ON periods in the more advanced stages, with a consequent reduction in the magnitude of symptom changes among On and Off states, coupled with a more severe motor phenotype as has been suggested by previous authors.Our study is among the first to examine the association of specific NMF with the occurrence of EMO in PwP, which we measured using a novel approach by using wearable sensor data to define EMO. We found that those with EMO had higher occurrence of NMF, which reached statistical significance for fluctuations in cognition, restlessness and fatigue. Whilst we did not measure NMF during the occurrence of EMO, our findings are in line with Rizos et al. who showed that 88.0% of PwP with EMO had worsening of NMS during the early morning period, mostly with an increase in urinary urgency and anxiety, but also dribbling of saliva, pain, low mood, limb paraesthesia and dizziness.Some limitations to our study need to be acknowledged, foremost the fact that our analyses were performed on a cross‐sectional cohort of participants and further studies with a long‐term longitudinal design to examine the evaluation of NMF should be performed. In addition, we used wearable sensor outcomes that did not necessarily coincide with the assessment of NMF, and some data was obtained up to 4 months from NMF assessment, reflecting clinical practice of use of wearables and in keeping with other studies.In summary, by using the novel NMF subscale of the MDS‐NMS we showed that NMF prevalence and severity increases with disease duration in a non‐linear pattern with the largest increase in severity occurring after the first 2 years after PD diagnosis, mirroring NMS burden progression. Moreover, NMF can occur in PwP without dyskinesia showing that the two are not necessarily linked. This analysis also underlines the feasibility of the MDS‐NMS scale with its dedicated NMF subscale in detecting NMF. In addition, we showed that this increase in NMF was largely driven by fluctuations in bladder symptoms, pain, and hyperhidrosis NMF increasing over time, with stable prevalence of other NMF. Further research is needed into the factors determining the progression of NMF and how impact on quality of life might chance over time.(1) Research Project: A. Conception, B. Organization, C. Execution; (2) Statistical Analysis: A. Design, B. Execution, C. Review and Critique; (3) Manuscript: A. Writing of the first draft, B. Review and Critique.DvW: 1A, 1B, 1C, 2A, 2B, 2C, 3A, 3B.SR: 1B, 1C, 2C, 3A, 3B.AS: 2C, 3A, 3B.AR: 2C, 3A, 3B.PMM: 1A, 1C, 2C, 3A, 3B.DW: 2C, 3A, 3B.KRC: 2C, 3A, 3B.Data used in the current analyses were obtained from the validation of the MDS‐NMS and the PKG Registry studies. For the MDS‐NMS study, each site contributing to the study received approval of their respective ethics committee/IRB for participation. In the UK, the study was approved for adoption to the UK Clinical Research Network (UKCRN No 18003). The PKG Registry obtained ethical approval from the London – Riverside Research Ethics committee (REC reference: 17/LO/1010). All patients gave written informed consent, and the study was conducted in line with the Declaration of Helsinki. We confirm that we have read the Journal's position on issues involved in ethical publication and affirm that this work is consistent with those guidelines.The current data analyses were not supported by funding. There is no conflict of interest.In the past year D.v.W. has received speaker fees from Bial and Britannia pharmaceuticals, and research grants from NIHR BRC and CHDI; S.R. has nothing to disclose; A.S. has nothing to disclose; A.R. has received support from the National institute of Health Research (NIHR) Clinical Research Network (CRN) South London, Guy's Hospital, Great Maze Pond, London UK and International Parkinson and Movement Disorder Society (MDS); P.M.M. has nothing to disclose; In the past year D.W. has received research funding or support from Michael J. Fox Foundation for Parkinson's Research, Alzheimer's Therapeutic Research Initiative (ATRI), Alzheimer's Disease Cooperative Study (ADCS), International Parkinson and Movement Disorder Society (IPMDS), National Institute on Health (NIH), Parkinson's Foundation; U.S. Department of Veterans Affairs and Acadia Pharmaceuticals; honoraria for consultancy from Acadia Pharmaceuticals, Alkahest, Aptinyx, Cerevel Therapeutics, CHDI Foundation, Clexio, Clintrex LLC (Otsuka), EcoR1 Capital, Eisai, Ferring, Gray Matter Technologies, Great Lake Neurotechnologies, Intra‐Cellular Therapies, Janssen, Merck, Sage, Scion, Signant Health and Vanda; and license fee payments from the University of Pennsylvania for the QUIP and QUIP‐RS. K.R.C. reports advisory board for AbbVie, UCB, GKC, Bial, Cynapsus, Lobsor, Stada, Zambon, Profile Pharma, Synovion, Roche, and Therevance, Scion, as well as honoraria for lectures for AbbVie, Britannia, UCB, Zambon, Novartis, Boeringer Ingelheim, Bial, Kyowa Kirin, SK Pharma, and frants (Investigator Initiated) from Bial, EU Horizon 2020, Parkinson's UK, NIHR, Parkinson's Foundation, Wellcome Trust, and royalties or licenses (ongoing) from Oxford (book), Cambridge publishers (book), MAPI institute , and payment for expert testimony for the General Medical Council (UK).


ACTG A5288 was a strategy trial conducted in diverse populations from multiple continents of people living with HIV (PLWH) failing second‐line protease inhibitor (PI)‐based antiretroviral therapy (ART) from 10 low‐ and middle‐income countries (LMICs). Participants resistant to lopinavir (LPV) and/or multiple nucleotide reverse transcriptase inhibitors started on third‐line regimens that included raltegravir (RAL), darunavir/ritonavir (DRV/r) and/or etravirine (ETR) according to their resistance profiles. At 48 weeks, 87% of these participants achieved HIV‐1 RNA ≤200 copies/ml. We report here long‐term outcomes over 144 weeks.Study participants were enrolled from 2013 to 2015, prior to the availability of dolutegravir in LMICs. “Extended Follow‐up” of the study started after the last participant enrolled had reached 48 weeks and included participants still on antiretroviral (ARV) regimens containing RAL, DRV/r and/or ETR at that time. RAL, DRV/r and ETR were provided for an additional 96 weeks , with HIV‐1 RNA measured at 48 and 96 weeks and CD4 count at 96 weeks after entry into Extended Follow‐up. Proportion of participants with HIV‐1 RNA ≤200 copies/ml was estimated every 24 weeks, using imputation if necessary to handle the different measurement schedule in Extended Follow‐up; mean CD4 count changes were estimated using loess regression.3, and HIV‐1 RNA was 4.6 log10 copies/ml. Median follow‐up was 168 weeks (IQR: 156–204); 15 (6%) participants were lost to follow‐up and 9 (4%) died. 27/246 (11%), 26/246 (11%) and 13/92 (14%) of participants who started RAL, DRV/r and ETR, respectively, discontinued these drugs; only three due to adverse events. 87%, 86%, 83% and 80% of the participants had HIV‐1 RNA ≤200 copies/ml at weeks 48, 96, 144 and 168 (95% CI at week 168: 74–85%), respectively. Mean increase from study entry in CD4 count at week 168 was 265 cells/mm3 (95% CI 247–283).Of 257 participants , at study entry, median CD4 count was 179 cells/mmThird‐line regimens comprising of RAL, DRV/r and/or ETR were very well tolerated and had high rates of durable virologic suppression among PLWH in LMICs who were failing on second‐line PI‐based ART prior to the availability of dolutegravir. StudiesTo address this gap, the ACTG A5288 study enrolled participants at 19 urban sites in LMICs in Africa , Latin America  and Asia (India and Thailand) during 2013–2015, prior to the availability of dolutegravir (DTG). A5288 included evaluation of third‐line regimens containing DRV/r plus raltegravir (RAL) with either ETR or two NRTIs in PLWH experiencing virologic failure (VF) on their PI‐based second‐line regimen with PI resistance and/or resistance to multiple NRTIs. In primary results, 87% of participants on these regimens achieved HIV‐1 RNA ≤200 copies/ml at week 48 . Here, w22.1The design of ACTG A5288 and primary results at week 48 have been published . In brieFor participants experiencing VF, another resistance test was performed with the possibility of changing treatment based on resistance results, similar to the process performed at study entry Figure . All parThis study was approved by site‐specific ethics committees. All participants gave their written informed consent.2.2This report describes long‐term outcomes of 257 study participants who initially received one or more of RAL, DRV/r and ETR in cohorts B, C and D . Changes in CD4 count were estimated using loess regression.33, HIV‐1 RNA was 4.6 log10 copies/ml and 38% were females. Median time on ART prior to study entry was 8.0 years. All participants in cohorts B and C and 48% of participants in cohort D showed DRV susceptibility.Characteristics at study entry are presented in Table Overall, 233 (91%) completed study follow‐up, 9 (4%) died and 15 (6%) were lost to follow‐up. Median follow‐up (including Extended Follow‐up) was 168 weeks. Thirty‐five participants (14%) permanently discontinued one or more drugs in the regimen started in the study. Reasons for discontinuation were death (4%), adverse events (3%), loss to follow‐up (2%), non‐compliance (2%), VF with new resistance mutations (1%) and other reasons (1%). Of participants who started RAL, DRV/r and ETR, 27/246 (11%), 26/246 (11%) and 13/92 (14%), respectively, discontinued these drugs; only three due to adverse events . Only three participants (one in cohort B2 and two in cohort D) discontinued any of these drugs due to resistance. The one in cohort B2 developed INSTI resistance in Extended Follow‐up identified on a resistance test obtained outside of the study, discontinued RAL, DRV/r and ETR, and was subsequently discontinued from the study. Of the two in cohort D, one developed the Y181C mutation and changed from ETR to RAL and one developed the E92Q and N155H mutations and changed from RAL to DRV/r.Any grade serious adverse event, Grade≥3 signs and symptoms, Grade≥3 laboratory abnormalities and Grade≥3 diagnoses occurred in 23%, 15%, 29% and 23% of participants, respectively. Clinical events included AIDS‐defining events (7%), non‐AIDS‐defining events (14%), hospitalizations (16%) and pregnancies (4%).An estimated 87%, 86%, 83% and 80% of participants had HIV‐1 RNA ≤200 copies/ml at weeks 48, 96, 144 and 168 (95% CI at week 168: 74–85%), respectively Figure  and b. ACohort D, which had the most extensive resistance, generally had the lowest proportion of participants with HIV‐1 RNA suppressed throughout follow‐up. The two randomized cohorts  had similar suppression rates: 88% in both cohorts at week 48, and 80% versus 78% at week 168.3 at weeks 48, 96, 144 and 168, respectively.There was a gradual increase in CD4 count over time in all cohorts Figure  and d: m4In LMICs where frequent HIV RNA testing is not accessible, access to antiretroviral (ARV) regimens with robust efficacy and durable HIV RNA suppression is critical. Although DTG is currently recommended globally for both first‐line and second‐line therapies after failure of non‐DTG‐containing first‐line regimens, our results remain relevant for PLWH who have experienced treatment failure on both NNRTI‐ and PI‐based regimens. A5288 is an important trial in LMICs of treatment options for PLWH of various ethnicities, cultures and socio‐economic backgrounds failing second‐line PI‐based regimens with PI resistance and/or resistance to multiple NRTIs, which used real‐time genotyping testing to select third‐line regimens using an algorithmic approach . We founThese results are of critical importance in showing that, if highly tolerable suppressive ART is available, virologic suppression can be achieved even after sequential treatment failures, with beneficial results not only for delaying HIV progression but also in preventing onward HIV transmission. The high proportion of participants achieving long‐term virologic suppression in this study is in line with that seen in observational studies in LMICs. In South Africa, 82.9% of PLWH on salvage ARV regimens achieved HIV‐1 RNA <400 copies/ml with median follow‐up of 2.5 years . ZimbabwOur findings are particularly important because data regarding long‐term outcomes after more than 3 years of third‐line ART in LMICs are scarce. In Extended Follow‐up, HIV‐1 RNA was only monitored annually, reflecting clinical practice in many LMICs. Therefore, the robust durability and tolerability of our third‐line regimens are likely generalizable to these settings. However, some limitations should be acknowledged. First, RAL, which was used in this study because of its availability when the study was initiated, is more expensive than generic DTG and requires twice daily rather than once daily dosing. Second, we did not have data on drug concentrations and genotypic drug‐resistance testing at VF in Extended Follow‐up, so we cannot evaluate whether the VF was due to poor adherence, or emergence of new mutations. However, the majority (63%) of participants with HIV‐1 RNA >200 copies/ml at week 48 were re‐suppressed to <200 copies at week 144 suggesting adherence issues.In conclusion, among PLWH from LMICs who failed NNRTI‐ and PI‐based regimens, third‐line regimens containing RAL, DRV/r and/or ETR were well tolerated and provided a high rate of durable virological suppression over 3 years.ACC has received a research grant from Bristol‐Myers Squibb and honoraria from Merck & Co. for Data Monitoring Committee membership. JWM is a consultant to and grant recipient from Gilead Sciences and owns shares in Abound Bio (unrelated to the current work) and has received share options in Infectious Disease Connect (unrelated to the current work). All other authors declare no competing interests.AA contributed to the literature search, participant recruitment, data collection and interpretation, and manuscript drafting and revision. MDH contributed to the study design, data analysis and interpretation, and manuscript drafting and revision. CM contributed to the figures, data analysis and interpretation, and manuscript drafting and revision. RS, EH, CG and RTS contributed to the study design, data interpretation and manuscript revision. PM contributed to the study design, participant recruitment and data collection, and manuscript revision. SWC, AB, MM, SBF, VM, BWN, SNF, WS, RS, MvS, RM, LM, JV, PS, EM, CM, MC, BRS, NK and CK contributed to participant recruitment, data collection and interpretation, and manuscript revision. JWM contributed to the study design, virological resistance studies, data interpretation and manuscript revision. CLW contributed to the literature search, study design, virological resistance studies, data interpretation, and manuscript drafting and revision. ACC contributed to the literature search, study design, data interpretation and manuscript revision. BG contributed to the literature search, study design, participant recruitment, data collection and interpretation, and manuscript revision.Research reported in this publication was supported by the National Institutes of Allergy and Infectious Diseases of the National Institutes of Health under Award numbers .The United States’ National Institutes of Health were the study funders and had an oversight role in the development and monitoring of the study. One author (CG) was an employee of this sponsor and a member of the study team involved in the conduct, analyses and interpretation/conclusions of the study. The corresponding author had full access to all the study data and had final responsibility for the decision to submit for publication.The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Allergy and Infectious Diseases. AbbVie, Gilead Sciences, Janssen Pharmaceuticals and Merck & Company provided the study drugs.