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A Process for Assessment and Quality Improvement of the Clerkship Curriculum Reliance on the apprenticeship model of education in the clerkship years of medical education persists despite concerns with variability in educational delivery and outcomes. Although many institutions are addressing this variability, there needs to be a clear and objective method to assess what is working. Evaluating these educational experiences is an essential component to ensure that students graduate prepared to enter residency. In 2014, A.T. Still University’s School of Osteopathic Medicine in Arizona (ATSU-SOMA) introduced a curricular change to address clerkship variability by implementing an online curricular component for the core clerkship courses in the third and fourth years of medical student education. Subsequently, a new structured and objective process to evaluate these courses was designed to improve student learning outcomes in the clerkship years. A Curriculum Year Three and Four Work Group was created to develop the new process for curricular evaluation of the clerkship courses. In the pilot phase of its implementation, described herein, the process fostered stakeholder participation and buy-in, enhanced communication of expectations, increased accountability in clerkship course design, and effectively employed objective evaluation tools in determining what curricular changes were needed. The Curriculum Year Three and Four Work Group continues to revise the tools and methods to enhance the efficiency of the evaluation process and to analyze whether recommended course revisions have improved student outcomes. Background Reliance on the apprenticeship model of education in the clerkship years of medical education persists despite concerns with variability in educational delivery and outcomes.

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Although calls for change are occurring, they are slow. 1 The primary goal of the learning environment in these 2 years is working with students to practice situated cognition, in which students learn to apply what they previously learned in the classroom to actual patient care under the guidance of experienced physicians. 2 A downside of this apprenticeship model is that student learning during this time tends to be inconsistent. Some factors may be the variety of physicians with whom students spend time and the variability in training, practice, and teaching patterns. [2][3][4] Another factor is that students' didactic exposure in the third and fourth years is often dependent on the educational sessions and patient cases available at their rotation site. Furthermore, accurate assessment of student clinical competence requires a multifaceted approach that extends beyond the use of activity logs 5 and preceptor evaluations, 6 especially because these types of evaluations fail to differentiate between learner abilities. 7 The Flexner Report in 1910 and the subsequent Carnegie Foundation report in 2010 both emphasize the importance of standardization and integration of formal and clinical learning. 8 Context To address these concerns and to generally improve student outcomes on national board examinations, in 2014, A.T. Still University's School of Osteopathic Medicine in Arizona (ATSU-SOMA) decided to enhance its educational offerings by adding weekly, required coursework via an online platform in the third and fourth years. The 11 core clerkships for which online content was designed included Family Medicine, Internal Medicine, Pediatrics, Obstetrics and Gynecology, General Surgery, Psychiatry, Neurology,

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Cardiology, Critical Care, Emergency Medicine, and Osteopathic Principles and Practice. The Clerkship Course Directors (CCDs) for these clerkship courses created the learning content and delivered it through the school's learning management system (LMS), Blackboard, so all students could easily access it from locations across the country and internationally. This was the primary responsibility of the CCDs. Unlike other institutions, 9 at ATSU-SOMA, preceptor oversight and development does not fall under the purview of the CCDs and instead is the responsibility of the Regional Directors of Medical Education, who work directly with preceptors in furthering their development as clinical educators. To help close the loop of communication, the CCDs created short written guides for preceptors to better support the online course curricula and increase consistency across sites. Problem After the first year CCDs taught these new clerkship courses, ATSU-SOMA realized that there was a marked variation between courses and that the Curriculum Committee needed a systematic method to evaluate and document course 2 Journal of Medical Education and Curricular Development effectiveness. The Curriculum Committee typically interviewed the CCD and reviewed data points of interest for the course, such as examination scores. However, there was no way for the Curriculum Committee to systematically and objectively rate the data and discussion points to define minimum expectations and determine what improvements the CCDs might need to make. Thus, we saw the need to create a more robust and documented evaluation system to decrease ambiguity for both CCDs and the Curriculum Committee. This article describes the innovative, structured, and objective process

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that ATSU-SOMA created in 2016 to evaluate the core clerkship courses and details how this work led to enhanced communication, encouraged stakeholder engagement, increased accountability of course directors, and effectively employed objective evaluation tools to determine what curricular changes were needed. Creating the Evaluation Process Forming Curriculum Committee work groups Curriculum committees at most educational institutions generally maintain oversight of the curriculum and usually include administrators and faculty who have critical roles in the planning, creation, and delivery of curricular content. 10 After noting how other institutions effectively reorganized their curriculum committees, 11 ATSU-SOMA formed working groups of the Curriculum Committee, based on the experiences of these other institutions, to subdivide the work of curriculum review to allow for a deeper evaluation of different academic years and assessment of specific tasks by those with pertinent experience. The Curriculum Committee created a Curriculum Year One and Two Work Group and a Curriculum Year Three and Four Work Group to evaluate the curriculum in those respective years. Evaluation of the clerkship courses was a collaborative process between the CCD and the Curriculum Year Three and Four Work Group (hereafter referred to as the Work Group), and the Curriculum Committee reviewed and approved any final recommended changes. The Curriculum Committee purposefully selected Work Group members who had varied roles, perspectives, and levels of experience to ensure that the clerkship evaluation process addressed the diverse needs and concerns of CCDs, administrators, and students. The formation of these work groups allowed faculty and other key stakeholders who did not hold formal

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seats on the Curriculum Committee to have a say in the process of curriculum evaluation. The chair of the Work Group was a clinical faculty representative of the Curriculum Committee who also happened to be a CCD. Members of the Work Group included 3 clinical faculty who were also CCDs of clerkship courses, 1 basic science faculty who had experience as a course director for firstyear and second-year courses, 1 osteopathic medicine faculty who could evaluate the integration of osteopathic principles in each of the courses, the director of the Office of Evaluation and Effectiveness (assessment) who had analytic experience in developing methods that ensured usable outcomes, the staff member who facilitated delivery of course materials, and the assistant and associate deans of Clinical Education who had oversight of the third-and fourth-year curriculum. The Work Group also included the associate dean of Curricular Integration as an ex officio member who needed to stay informed of the Work Group's progress. Like other institutions that have created teams or focus groups of students to obtain input on the curriculum, 12 ATSU-SOMA recognizes the value of student input in curricular reform and has used student focus groups for feedback of courses from the first and second years. The chair of the Work Group considered adding a student to the team but found that students on the Curriculum Committee were rarely available to attend or contribute because of academic obligations, and focus groups are more challenging to form during the third and fourth years when education is so widely distributed

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at ATSU-SOMA sites across the country. Thus, the Work Group used components of the course feedback survey from students as the primary and most efficient source of student input (Table 1). At this early stage of development, the ultimate stakeholders, the patients, were not involved in the initial course evaluation process, but we hope to include patient feedback in the future. Creating a timeline and communication plan At the beginning, it took 6 months to develop the course evaluation process. Creating an annual project timeline for reviewing and revising the clerkship courses was also essential and ensured that the Work Group would meet the project's goals and deadlines each year ( Figure 1). The group used backward planning to ensure it would meet target dates. During the period for course reviews from July to December, the group met for 2 hours every other week to evaluate 11 clerkship courses. The role of the CCD for each course typically accounted for 0.1 to 0.2 full-time equivalent depending on the duration and complexity of the course they were developing. Because this was not their sole teaching responsibility, CCDs typically needed several weeks to months to make course changes in this early phase of curricular development. To minimize surprises and disruptions to usual workload and schedules and to increase the willingness to make changes, the Work Group determined that all persons involved or impacted by the evaluation process needed to know what to expect. Therefore, to ensure clear and accurate communication of the course evaluation process, the chair created

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a document that described the committee's process for curricular evaluation of the clerkship courses and shared it with all CCDs, curriculum committee members, and administrators. This document provided clarity by describing the step-by-step process to evaluate clerkship courses and indicated who the responsible parties were for each step in the process. Throughout the clerkship course review process, the Work Group documented and shared the notes from each weekly meeting with group members. A rapid editorial and dissemination procedure allowed for accurate recall of the discussions, and any questions or concerns could be addressed before the process was complete. With such a wide representation of stakeholders on the committee, web conferencing was essential for equal participation of those located on or off campus and broadened our pool of potential input. Identifying necessary data The Work Group and CCDs worked collaboratively to define what data were needed for the evaluation process. At ATSU-SOMA, because it takes a full academic year for an entire class of students to complete a given clerkship course, a full set of performance data and student feedback results for an individual course was not available until July of each academic year. To compensate for this delay of information, the Work Group provided mid-year course data packets to CCDs in January to allow them to assess student progress mid-year ( Figure 1). The course data packets included the following reports for each clerkship course: 1. Clerkship course final grade distribution histogram; 2. Histogram of clerkship national subject examination performance; 3. Histogram for weekly coursework scores

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delineated by week; 4. Summative student feedback with comments from the Student Evaluation of the Rotation form; 5. Histogram of Level 2 national board examination scores with discipline subscores; 6. Anonymous student feedback comments. Depending on the goals and interests of any given institution, different data sets may need to be included in the review packet to capture each school's unique programs. Developing the course evaluation process and evaluation tools Establishing and codifying the evaluation processes ensured clear communication of expectations for all involved. The clerkship course evaluation process included reviewing essential information about each course and CCD commentary about his or her course. We decided that a course rating evaluation system based on predetermined objective criteria was necessary. The course had clear objectives and goals stated in the syllabus or online in the lMS. The reading, written, and other assignments complemented the course learning objectives. i was able to complete my coursework and logs in a timely manner. The knowledge/concepts in the course were applicable to clinical situations i encountered during the rotation. My knowledge base has improved significantly as a result of this experience. Comments: (free text) lMS, learning management system. The overall results of student feedback determined the rating assigned for the first item in the Clerkship Course Evaluation rubric. July to June Students complete all clerkship courses in the academic year. Each course is 4-8 weeks. One year required for all students to cycle through each course. CCDs grade student assignments discovering where future changes are needed. July to December Work Group

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biweekly meetings to evaluate courses using data from the prior academic year and current course documents. Recommendations for change delivered to the CCDs in December. January to June Mid-year data packets shared with CCDs in January. CCDs make changes to courses to prepare for start of the next academic year. Work Group revises questionnaire and evaluation rubric for use in next academic cycle. Journal of Medical Education and Curricular Development When creating the evaluation tools, we determined that they needed to be collaborative to ensure that expectations were clear between the CCDs being evaluated and the evaluators. The purpose of the evaluation tools was to gain insight into the course, answer key questions about course design and outcomes, and provide objective means of evaluation. Using Google Forms, the Work Group created and distributed a questionnaire for CCDs to complete ( Table 2). The goal of the questionnaire was to gain insight into the course development process of the CCDs. The questionnaire included several items about course design, such as CCD goals for the course, methods of communication and feedback to students, methods of student assessment, interpretation of the course outcomes, and planned changes for the future. While creating the questionnaire, group members were especially interested in the CCDs' impressions of the questions and worked to revise those that created ambiguity, such as the differentiation of course goals from programmatic competencies. The questionnaire represents a portion of what ATSU-SOMA wanted to evaluate in this given year; however, the questionnaire could easily be modified to meet the unique

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goals and interests of a given institution. Next, the Work Group created a course evaluation rubric to evaluate courses consistently and objectively (Table 3). Like the questionnaire, the rubric includes a portion of what ATSU-SOMA wanted to evaluate for this particular year and could be modified as needed. Identifying benchmarks in rubric format had not been previously performed in curricular reviews, resulting in inconsistent evaluations between courses. During the first year of the evaluation process, therefore, the intention of the rubric was to ensure a basic level of consistency between courses rather than to evaluate the academic content of the courses. The rating level of "exceeds expectations" was included in the rubric to encourage clerkship directors to set goals above the minimum expectations and stimulate innovation. Although evaluation categories might vary from year to year depending on the specific goals and needs of the course reviews, we felt it was important to gather baseline information about course goals and objectives, structural design, and student feedback during this pilot phase of using this new evaluation process. Complete evaluation of courses required the Work Group to review questionnaire responses of the CCDs, course performance data, and course documents provided to the students through the LMS. After reviewing this information, the Work Group conducted live meetings with each CCD to discuss the information and ask questions about the clerkship course curriculum. Scheduled meetings adhered to the following process: 1. CCD gave a general overview of the intention for the curriculum and the process used in planning the course; 2.

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CCD provided suggestions for recommended changes for the next year; 3. Work Group members asked questions of the CCD as needed; 4. Work Group members shared and discussed their rubric ratings; 5. Work Group members made suggestions for changes to the clerkship course curriculum, which may have included or excluded recommended changes from the CCD. Course evaluations took place from July to December (Figure 1). During the first year of evaluations using this new process, meetings lasted 1 hour per course. In the next year, the time was reduced as the Work Group and CCDs were familiar with the process and asked fewer questions. In some cases, no meeting was needed with specific CCDs because there were no apparent deficiencies and the CCD provided sufficient information in the questionnaire, course data packet, and other course materials. At the end of each course review meeting, the Work Group and CCD discussed the proposed recommended course changes. After all courses were reviewed, Work Group members voted on all the proposed course changes. Waiting to decide on final recommended changes to each course allowed for analysis of patterns between courses and for recommendations to focus on the best practices. These recommendations were subsequently shared with the Curriculum Committee, which reviewed and modified them as necessary. The Curriculum Committee's final recommendations were then shared with each of the CCDs in January to allow time to make course changes before the start of the next academic year in July (Figure 1). Documenting the process and findings Google Forms and Google Sheets

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were essential for keeping track of the Work Group responses and for thoroughly documenting procedures and recommendations for the Curriculum Committee, especially when questions arose. Google Forms were used to collect the ratings of each of the rubric items for individual Work Group members. This method ensured that decision-making contributions were made by all members of the group rather than using group discussion of each item in the rubric where input from those who were less vocal or less opinionated may not have been accounted for. During the first evaluation year, all Work Group members rated all of the rubric categories, and an average score was calculated for each category. In the second year, to reduce the time commitment, each group member worked on an average of 2 items in the rubric that were evaluated across courses. The chair selected qualified evaluators for each item and allowed Work Group members to provide additional commentary on other rubric items. Google Sheets were used to track progress and improvement from one year to the next and to share information with Glaser 5 multiple individuals using a live document. This method of information sharing was especially beneficial in the case of group member turnover and for keeping the Curriculum Committee and administrators informed of progress. Each course had its own tab containing the evaluation rubric in the Google Sheet and included a column for CCD comments and a column for the Work Group members' comments. The Work Group created a separate Google Sheet to keep a summary of discussions

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from each meeting and record attendance. The group's decision-making process was democratic and decisions made were kept in a separate tab in the Google Sheet with dates and times. what were the 3 most common positive themes expressed by students regarding the clerkship curriculum? (information may be gleaned from logs, student evaluations, or course director interactions with students and rDMEs) what were the 3 most common negative themes expressed by students regarding the clerkship curriculum? (information may be gleaned from logs, student evaluations, or course director interactions with students and rDMEs) Assessment Other than the national subject examination at the end of the rotation, how else is student learning of the curricular content in the course assessed (describe your weekly assignments or any other mode of assessment)? what were some of the strengths and weaknesses of the course assessments? Based on the results of the course data packet (end of rotation examinations, CrE, coursework assessments, and SEr results), which of the items you wanted students to learn from the course (ie, your personal course goals from above) were achieved and what evidence supports this conclusion? Based on the results of the course data packet (end of rotation examinations, CrE, coursework assessments, and SEr results), which of the SOMA curricular goals listed in the syllabus were achieved and what evidence supports this conclusion? Conclusions what was the best thing about the course that you plan to continue and why? what needs to be improved and why? what changes are you planning on making for the next year?

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list Curriculum Committee recommendations from the last year and how they were or were not addressed. Any final remarks? CrE, clinical rotation evaluation; Gi, gastrointestinal; rDME, regional Director of Medical Education; SEr, student evaluation of the rotation; SOMA, School of Osteopathic Medicine in Arizona. Impact of the Clerkship Course Evaluation Process and Limitations During the pilot phase of this new process, the holistic view the Work Group gained from this innovative clerkship course evaluation process ( Figure 2) indicated that much work remains to be done to improve the clerkship courses. In particular, the responses to the questionnaires for CCDs revealed that several CCDs needed additional guidance in course design and pedagogy. Thus, the Work Group created a document that detailed clerkship course design and content expectations for CCDs as a basic guide to structuring clerkship courses. A key feature of this document was to guide CCDs in creating assignments with SMART (specific, measurable, achievable, relevant, and time bound) design structure. Feedback during group CCD meetings indicated that CCDs appreciated the clarity of expectations and guidelines to follow for their courses. As a result of this process, the Work Group noted that improvements were made in course design and student assessment methods, and CCDs reported receiving fewer student complaints the next year. The evaluation process also revealed that additional work on curriculum mapping for the clerkship courses needs to occur at ATSU-SOMA. Currently, our mapping involves using the 7 osteopathic core competency domains and includes 33 competencies we have defined as SOMA curricular goals. When we

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looked at which SOMA curricular goals were addressed by all parts of the clerkship curricula, we identified 1 goal of the 33 goals that was not addressed; the missing goal was in the area of systems-based practice. Furthermore, future steps will likely involve investigating how our curricula address the Entrustable Professional Activities of undergraduate medical education. In addition, we are assessing how clerkship courses address ATSU's Core Professional Attributes, which are the unifying competencies expected of all graduates of our university regardless of degree program. 13 Another positive outcome of using this evaluation process was that CCDs considered more deeply what they were trying to accomplish in their clerkship curriculum. The process made them more accountable for their courses and the potential impact on student educational outcomes. This accountability also enhanced faculty collaboration because CCDs became more engaged in discussions with their peers about course design and online student assessment methods. One of the most important benefits of using a working group for our clerkship course evaluation process included the buy-in from an array of stakeholders, which ensured the success of the process. Furthermore, the collaboration between the Work Group and CCDs led to other improvements in the overall structure of the clerkship curriculum, including necessary adjustments to the global clerkship grading scales, revision of preceptor evaluation forms, and enhancement of the student rotation feedback surveys. We did encounter challenges during the process. For example, a few Curriculum Committee members, who were not in the Work Group, may not have read the Google Sheets and meeting

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notes and had questions about the clerkships when we presented our findings to the Curriculum Committee. These discussions resulted in their offering solutions without having the Work Group's earlier input. In addition, the Work Group was not as empowered in evaluation and decision-making as it had initially presumed, and the Curriculum Committee's additional recommendations for change came as a surprise to the CCDs, resulting in CCD dissatisfaction. In the future, the Work Group will need the explicit support of the Curriculum Committee to effectively conduct the tasks asked of it. To ensure that repeated cycles of curricular evaluation adequately determine the effectiveness of the process, commitment from the group members and the CCDs is essential. At ATSU-SOMA, we were pleased to have 100% participation of the CCDs; each CCD had a vested interest in seeing the curriculum and, ultimately, student outcomes improve. However, the nature of academia, with its frequently shifting roles, responsibilities, and changes in leadership, places newly implemented changes at risk of dissolution. Overall, ATSU-SOMA's new curriculum evaluation process enhanced communication, stakeholder engagement, and accountability of course directors; and it effectively employed objective evaluation tools to determine what curricular changes were needed. Ideally, the Curriculum Year One and Two Work Group will successfully adopt and implement a similar evaluation process for their classroom-based curriculum in the next academic year. What is clear is that for sustained, robust 8 Journal of Medical Education and Curricular Development curricular change to occur, the CCDs, department heads, and deans need to support and direct a rigorous course review

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process, such as that described in this article. Subsequent evaluation and research at ATSU-SOMA is ongoing and will involve assessing the impact this process has had on student learning outcomes by monitoring score improvements on national board examinations, subject examinations, and preceptor evaluations of student performance. Other enhancements being considered are the use of student focus groups to gain deeper insight from students about how the clerkship course curricula can be improved. Ultimately, solicitation of patient feedback and input would also be included in this process. Furthermore, the Work Group will revise the course evaluation rubric each year to address evolving areas of needed evaluation. We hope that having described our process will enable other institutions to develop and revise methods of evaluating their course curriculum.

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Trading Capsule for Increased Cytotoxin Production: Contribution to Virulence of a Newly Emerged Clade of emm89 Streptococcus pyogenes ABSTRACT Strains of emm89 Streptococcus pyogenes have become one of the major causes of invasive infections worldwide in the last 10 years. We recently sequenced the genome of 1,125 emm89 strains and identified three major phylogenetic groups, designated clade 1, clade 2, and the epidemic clade 3. Epidemic clade 3 strains, which now cause the great majority of infections, have two distinct genetic features compared to clade 1 and clade 2 strains. First, all clade 3 organisms have a variant 3 nga promoter region pattern, which is associated with increased production of secreted cytolytic toxins SPN (S. pyogenes NADase) and SLO (streptolysin O). Second, all clade 3 strains lack the hasABC locus mediating hyaluronic acid capsule synthesis, whereas this locus is intact in clade 1 and clade 2 strains. We constructed isogenic mutant strains that produce different levels of SPN and SLO toxins and capsule (none, low, or high). Here we report that emm89 strains with elevated toxin production are significantly more virulent than low-toxin producers. Importantly, we also show that capsule production is dispensable for virulence in strains that already produce high levels of SPN and SLO. Our results provide new understanding about the molecular mechanisms contributing to the rapid emergence and molecular pathogenesis of epidemic clade 3 emm89 S. pyogenes. U nderstanding the evolutionary genetic changes underpinning emergence of new pathogenic strains is of practical importance because it provides useful information for development of effective strategies

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to control infectious diseases in humans, domesticated animals, and crops. The combination of population genomics, epidemiology, evolutionary biology, molecular genetics, and microbial pathogenesis makes it possible to precisely delineate these genetic changes at the nucleotide level. Streptococcus pyogenes (group A streptococcus [GAS]) is a human pathogen that causes many diseases ranging in severity from minor skin and throat infections to fatal invasive episodes (1). Based on variation in the emm gene encoding the antiphagocytic M protein, S. pyogenes can be classified into~200 emm types (2). For reasons that are not well understood, in the last 10 years emm89 strains have rapidly become one of the major emm types causing severe invasive S. pyogenes infections in several geographic regions (3)(4)(5)(6)(7)(8)(9)(10)(11)(12)(13)(14)(15)(16)(17)(18). We recently sequenced the genomes of 1,125 emm89 strains isolated on two continents from 1995 to 2013 and discovered the existence of three major phylogenetic groups of emm89 strains, designated clade 1, clade 2, and epidemic clade 3 (19). We discovered that clade 3 strains emerged, disseminated extensively, and rapidly displaced clade 1 and clade 2 strains in an epidemic wave of emm89 disease. Population genomic analysis revealed two major features of epidemic clade 3 emm89 strains. First, all clade 3 emm89 strains have the variant 3 nga promoter region sequence, which is identical to the nga promoter region present in pandemic emm1 strains. The variant 3 promoter region is associated with elevated production of SPN (S. pyogenes NADase) and SLO (streptolysin O), two potent secreted cytolytic toxins that contribute to virulence (19). Second, all clade 3

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emm89 strains studied lack the hasABC gene region containing hyaluronic acid (HA) capsule synthesis genes. We hypothesized that the genetic changes in the nga promoter region and capsule synthesis genes have altered the virulence phenotype of emm89 strains. To test this hypothesis, we constructed a panel of isogenic mutant strains that vary in level of production of secreted SPN and SLO cytolytic toxins and HA capsule. These mutant strains recapitulate the level of SPN and SLO toxin and capsule production made by genomically representative members of each of the three emm89 clades. The isogenic mutant strains were tested for virulence in a mouse model of necrotizing fasciitis. RESULTS Variant 3 nga promoter sequence is associated with increased production of secreted SPN and SLO toxins. Based on our analysis of 1,125 genome sequences, emm89 strains cluster into three major phylogenetic groups referred to as clades. Strains in each clade have a distinct nga promoter sequence (2). That is, clade 1 strains all have the variant 1 pattern, clade 2 strains have the variant 2 pattern, and epidemic clade 3 strains have the variant 3 pattern. Three reference strains, MGAS11027 (clade 1), MGAS23530 (clade 2), and MGAS26844 (clade 3), whose genomes were sequenced to closure at high fold coverage, were cho-sen for subsequent analyses. The nga, slo, and intervening ifs genes are organized as an operon and expressed as a single transcript (20-22) (see Fig. 2A). nga and slo encode SPN and SLO, respectively (22). In principle, variation in the promoter region sequence could alter the level of

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nga and slo transcripts and thereby SPN and SLO production. To test this idea, we first compared the secreted SPN (NADase) activities of 27 emm89 isolates belonging to clade 1, clade 2, and clade 3 (All 27 strains are wild type in transcriptional regulators known to control SPN, SLO production, and capsule production, i.e., covR/S and rocA [23][24][25][26]). Compared to clade 1 and clade 2 isolates, clade 3 isolates produced increased NADase activity (Fig. 1). We next examined the transcript levels of nga and slo and SPN and SLO production in the three reference strains representing the three clades. Consistent with the hypothesis, strain MGAS26844 (variant 3 pattern) had a significantly higher nga and slo transcript levels and production of SPN and SLO compared to strain MGAS23530 (variant 2 pattern) and strain MGAS11027 (variant 1 pattern) (Fig. 2B). To further test the hypothesis that variation in the promoter region alters gene transcript levels, we generated isogenic mutant strains by replacing the nga promoter of the variant-3 parental (Fig. 3). NADase assays and capsule assays were performed in triplicate on three separate occasions. Replicate data are expressed as the mean Ϯ SD. Zhu et al. strain 26844. The results (Fig. 2C) show that converting the nga promoter region of strain MGAS26844 to either variant 1 sequence or variant 2 sequence significantly reduced both nga and slo transcript levels and production of SPN and SLO cytotoxins. We next generated isogenic mutant strains by converting the nga promoter region of parental strain MGAS23530 (variant 2) to variant 1 sequence

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and variant 3 sequence. Consistent with the hypothesis, the isogenic mutant strain with the variant 3 sequence expressed significantly more nga and slo transcript and SPN and SLO proteins (Fig. 2D). In contrast, conversion to the variant 1 nga promoter sequence did not significantly alter the level of transcript or SLO and SPN production. Collectively, these results show that sequence variation in the nga promoter region alters transcription of nga and slo and production of SPN and SLO. Epidemic clade 3 emm89 strains with the variant 3 nga promoter pattern have the highest nga-slo transcript level and produce the largest amount of secreted SPN and SLO. A 38-bp deletion in the upstream region of hasA is associated with increased production of HA capsule. Epidemic clade 3 strains lack the hasABC genes for capsule synthesis, whereas clade 1 and clade 2 strains have these genes. However, the abundance of capsule production varies from strain to strain among clade 1 and clade 2 strains (Fig. 1B). For example, representative strain MGAS23530 (clade 2) produces a moderate amount of capsule (~0.3 g/ml), and HA capsule production by representative strain MGAS11027 (clade 1) is~40 times higher (~12 g/ml) (Fig. 3C). Analysis of the genome sequence data found two distinct hasA promoter region patterns present in clade 1 and clade 2 strains. We designate these two variants as pattern A and pattern B. Patterns A and B are present in both clade 1 and clade 2 strains. Compared to pattern A, pattern B has a 38-bp deletion that may remove a

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transcriptional terminator (Fig. 3A). Consistent with this idea, we found that regardless of clade assignment, strains with the 38-bp deletion produce a higher level of capsule ( Fig. 1B and 3A). Falaleeva et al. showed that deletion of this transcriptional terminator region resulted in increased capsule production (27). To directly study if the 38-bp deletion results in increased production of capsule, we generated an isogenic mutant derivative of parental reference strain MGAS23530 (23530-CapH) by replacing the hasA promoter with the promoter sequence present in pattern B strains. Deletion of the 38-bp region resulted in an~15fold increase in hasA expression and~40-fold increase in HA capsule production, levels similar to those obtained for the naturally occurring pattern B strain MGAS11027 ( Fig. 3B and C). These results demonstrate that removal of the 38-bp region upstream of the hasA promoter region results in increased hasA transcript and HA capsule synthesis. An isogenic acapsular emm89 strain with the variant 3 nga promoter region is highly virulent in a mouse model of necrotizing fasciitis. Two key genetic features of the epidemic emm89 clone are absence of HA capsule synthesis genes and presence of the variant 3 nga promoter region that results in increased production of the secreted SPN and SLO toxin virulence factors. We hypothesized that the absence of capsule together with increased SPN/SLO production (phenotypes that occur naturally in clade 3 strains) significantly increases strain virulence of emm89 S. pyogenes. We have previously shown that reduction of SPN/SLO production in the genetically representative clade 3 strain MGAS26844 significantly reduced its

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virulence in a mouse infection model (19). These data confirmed our hypothesis that increased toxin production is associated with increased virulence. However, strain MGAS26844 is acapsular, as are the isogenic mutant derivatives, which means that the effect of capsule loss on virulence in these strains was not determined. We next generated a panel of isogenic mutant strains of clade 2 strain MGAS23530 (weakly encapsulated, low SPN/SLO production) by altering the nga promoter region sequence and deleting the hasABC locus ( Fig. 4; Table 1). The virulence of these strains was compared using a mouse model of necrotizing fasciitis (Fig. 5). Consistent with our hypothesis, the acapsular variant 3 strain (23530-V3-CapN) caused significantly greater near mortality than the acapsular or weakly encapsulated strains that express comparatively smaller amounts of SPN and SLO toxin (Fig. 5A, 23530-V1-CapN, 23530-V1, 23530-V2-CapN, and 23530-V2). Similarly, gross and microscopic examination of infected limbs found that the acapsular variant 3 isogenic mutant strain (23530-V3-CapN) caused significantly larger lesions with more tissue destruction ( Fig. 5E to J). Interestingly, loss of HA capsule did not result in decreased virulence (Fig. 5). To the contrary, loss of capsule caused a substantial increase of virulence in an emm89 strain with the variant 3 nga promoter ( Fig. 5D and 6). This result suggests production of a low level of capsule is not sufficient to protect emm89 organisms from host innate immunity. Taken together, these data strongly support our hypothesis that the epidemic clade 3 genotype (absence of capsule combined with strong nga promoter expression) confers an

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increased virulence phenotype to the epidemic emm89 clade 3 organisms. HA capsule production is dispensable for full virulence in emm89 strains that produce high levels of SPN and SLO toxins. The data show that transition from low HA capsule production to complete absence of capsule did not result in decreased virulence (Fig. 5). To further study if HA capsule production plays a role in the virulence in emm89 strains, we compared the virulence levels of heavily encapsulated emm89 strains (generated by deleting a 38-bp region upstream of hasA [ Fig. 4; Table 1]) and acapsular organisms. Our virulence study results show that for emm89 strains that produce low levels of toxins (strains with a variant 1 or variant 2 nga promoter region), high capsule production rendered the organisms more virulent (Fig. 6A and B). However, for emm89 strains that produce high levels of SLO and SPN cytolytic toxins (strains with a variant 3 nga promoter region), the acapsular isogenic mutant strain is equally virulent to the heavily encapsulated strain ( Fig. 6C and D). In other words, production of a high level of HA capsule did not increase virulence in strains producing large amounts of SLO and SPN. Again, intriguingly, the poorly encapsulated strain was less virulent (23530-V3) ( Fig. 6C and D). Taken together, capsule production (regardless of whether high or low) is dispensable for virulence in emm89 strains that produce high levels of the secreted cytolytic toxins SLO and SPN. DISCUSSION One distinctive feature of the epidemic emm89 population is the lack of the

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hasABC locus responsible for production of HA capsule. Capsule has been studied for more than 100 years and has long been considered an important virulence factor for S. pyogenes strains, in part because of its role in resisting phagocytosis and killing by human polymorphonuclear leukocytes (PMNs) (28)(29)(30). Many lines of evidence, including virulence studies using isogenic mutant strains, have strongly supported the concept that HA capsule production is a crucial factor in the complex interaction between pathogen and host (28)(29)(30). These extensive studies led to the generally accepted idea that HA capsule is required for invasive infections and colonization. However, Flores et al. (31) recently reported that human disease isolates of emm4 and emm22 S. pyogenes lack the hasABC genes, a discovery effectively ruling out the idea that production of HA capsule is obligatory for virulence. In addition, the investigators showed that emm4 and emm22 strains proliferated ex vivo in human blood. Our work here adds emm89 strains to the list of S. pyogenes organisms that do not require HA capsule to cause human infections or, unexpectedly, epidemic disease. A second distinctive feature of epidemic clade 3 emm89 strains is that they all have the variant 3 nga promoter region pattern. Compared to the variant 1 and variant 2 nga promoter region sequences present in clade 1 and clade 2 strains, respectively, the variant 3 promoter region sequence is associated with increased expression of nga and slo and increased production of SPN and SLO cytotoxins. SPN and SLO have been reported to protect S. pyogenes from

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phagocytic killing and increase intracellular survival (32,33). In the absence of HA capsule, a high level of SPN and SLO production could provide a critical defense against host immunity. In this regard, we note that for emm89 strains that are high producers of SPN and SLO, HA capsule production is dispensable for virulence in a mouse infection model. The acapsular strain 23530-V3-CapN is as virulent as heavily encapsulated strain 23530-V3-CapH, as assessed by ability to cause near mortality and tissue destruction. In contrast, for strains that produce relatively smaller amounts of SPN and SLO, a high level of HA capsule production is important for virulence. Population genomic analysis showed that epidemic clade 3 emm89 evolved from a clade 2 strain (19). The precise nature and order of the molecular events that generated epidemic clade 3 is not known. In particular, we do not know if loss of the hasABC capsule synthesis genes preceded acquisition of the variant 3 nga-slo region or vice versa. Our inability to differentiate between these two possibilities is due in part to a lack of strains that have one of these two genotypic characteristics in the absence of the other. That is, our analysis did not identify strains with the hasABC capsule genes together with the variant 3 ngaslo region or which have the variant 2 nga-slo region but lack the HA capsule synthesis genes. The mouse infection data suggest that increased production of SPN and SLO is a more im-portant contributor to virulence than HA capsule production. This leads us to

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speculate that the key event immediately preceding the successful emergence of epidemic clade 3 organisms was gain of the variant 3 nga-slo region by horizontal gene transfer and homologous recombination in an emm89 strain that lacked the hasABC genes. It is possible that analysis of additional emm89 strains may identify strains that will permit us to differentiate between the possibilities. We note that it is a formal possibility that the evolutionary genetic events occurred simultaneously, although this seems unlikely. One intriguing finding of our study is that low HA capsule production is not sufficient to provide adequate protection against host defenses for emm89 S. pyogenes. For strains that produce low levels of SPN and SLO, poorly encapsulated strains are as attenuated as acapsular strains (Fig. 5 and 6). Interestingly, for high-toxin producers, poorly encapsulated strain 23530-V3 is significantly less virulent than acapsular strain 23530-V3-CapN (Fig. 5 and 6). One potential explanation for this unusual phenomenon is that acapsular emm89 organisms that produce high levels of SPN and SLO are more cytotoxic for host cells. Under this idea, compared to capsule-positive strains (even poorly encapsulated strains), acapsular organisms attach to the host cell better and are more likely to be internalized and intoxicate the host cell. For high-toxin producers, more internalization may result in more abundant cytotoxicity, tissue destruction, and host pathology. In summary, by analyzing isogenic mutant strains that recapitulate key genetic aspects of emm89 strains from different phylogenetic clades, we discovered that an acapsular emm89 strain pro-ducing high level of SPN and SLO (the two

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main features of epidemic emm89 strains) is highly virulent in a mouse model of necrotizing fasciitis. In addition, our data show that HA capsule production is dispensable for virulence in emm89 strains that produce high levels of SPN and SLO cytotoxins. The sum of the data provide additional evidence to support the idea we recently put Construction of isogenic mutant strains. Strain MGAS23530 was used as the parental organism for construction of isogenic mutant derivatives. Isogenic mutant strain 23530-V1 was generated by converting the nga promoter to the variant 1 pattern. Briefly, using MGAS23530 genomic DNA as the template, overlap extension PCR was performed with primer sets ngapro-1/V1A and ngapro-V1B/4 to generate a 1,097-bp fragment flanking the nga promoter region. SNPs G-27A and T-22G were introduced into the amplicon with primers ngapro-V1A and ngapro-V1B. The double-SNP-containing amplicon was digested with BamHI and ligated into the BamHI site of plasmid pBBL740 (34). The ligation product was column purified and transformed into strain MGAS23530. Allelic exchange was performed as previously described (34). The resulting derivative strain, 23530-V1, was sequenced (sequencing primer, ngapro-seq) ( Table 2) to ensure that the native MGAS23530 sequence GACTAT was replaced with the derived two-single-nucleotide polymorphism (SNP) sequence AACTAG located in the Ϫ10 and Ϫ35 spacer region of the nga promoter. (The changed residues are underlined.) Analogous to the procedure used to generate 23530-V1, strain 23530-V3 was generated by converting the nga promoter to the variant 3 pattern. SNPs G-27A and T-18C were introduced into the nga promoter region with primers ngapro-V3A and ngapro-V3B.

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Capsule-negative derivative strain 23530-V3-CapN was generated by deleting the has operon (hasA, hasB, and hasC) genes. Briefly, primer sets hasABCdel-1/2 and hasABCdel-3/4 ( Table 2) were used for overlap extension PCR to amplify and join the two DNA fragments upstream and downstream of the hasABC locus. PCR product was cloned into the BamHI site of suicide plasmid pBBL740 and then transformed into the designated S. pyogenes strains. Allelic exchange was performed as previously described. PCR (primer set hasABCdel-1/4) (Table 2) was used to screen for hasABC-negative strains. Capsule-negative derivative strains 23530-CapN and 23530-V1-CapN were made acapsular by disrupting the hasA gene with suicide plasmid pBBL740. Briefly, primers hasA-1 and hasA-2 ( Table 2) were used to amplify an~350-bp internal region of hasA. The PCR product was cloned into the BamHI site of pBBL740. Recombinant plasmid was used to transform S. pyogenes strains and disrupt hasA. Isogenic mutant strain 23530-V3-CapH was constructed by converting the hasA promoter region of strain 23530-V3 to the sequence present in strain MGAS11027. Briefly, using genomic DNA of strain MGAS11027 as the template, primers 11027has-1 and 11027has-2 were used to amplify a 1.4-kb fragment that spans the hasA promoter region. The amplicon was ligated into the BamHI site of plasmid pBBL740 and transformed into strain MGAS23530. Allelic exchange was performed as previously described (34). Isogenic mutant strains 23530-V1-CapH and 23530-CapH were generated by the same method. The genome of all mutant strains was sequenced, and no spurious mutations were identified. qRT-PCR analysis of nga, slo, and hasA transcripts in emm89 S. pyogenes

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strains grown in THY broth. RNA prepared from S. pyogenes cultures grown to an optical density at 600 nm (OD 600 ) of 0.5 was extracted with an RNeasy minikit (Qiagen) and converted into cDNA using a highcapacity cDNA reverse transcription (RT) kit (Applied Biosystems). Quantitative RT-PCR (qRT-PCR) was performed with TaqMan Fast Universal PCR master mix (Applied Biosystems) and an ABI 7500 Fast System (Life Technologies) instrument. The sequences of the TaqMan primers and probes used are listed in Table 3. Each experiment was performed in triplicate with the mean values (Ϯ standard deviation [SD]) shown. Statistical differences between strains were determined using Student's t test. Western immunoblot analysis of SLO and SPN in the culture supernatant. Western immunoblot analysis was done as described previously (19). Briefly, cell-free supernatants of S. pyogenes cultures grown to OD 600 of 0.5 were collected by centrifugation at 4,000 rpm for 10 min and filtered through a 0.2-m filter. Proteins in the supernatant were concentrated with 2-ml centrifugal filters (Amicon) and assayed for presence of SLO and SPN with anti-SLO antibody (American Research Product) and anti-NADase antibody (Abcam), respectively. Measurement of SLO and NADase activities in the culture supernatant. SLO activity and NADase activity in the culture supernatant was assayed as described previously (35). Capsule assay. Hyaluronic acid capsule production was assayed as described previously (31). Mouse virulence experiments. Six-week-old female CD1 mice (Harlan Laboratories) were used for the necrotizing fasciitis studies as described previously (36). This study was approved by the Institutional Animal Care and Use Committee of

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the Houston Methodist Research Institute (AUP-0615-0041). Mice were randomly assigned to treatment groups and inoculated in the right hindlimb to a uniform depth with 2.5 ϫ 10 8 CFU of emm89 strains in 100 l phosphate-buffered saline (PBS). Stocks of each strain were prepared at known CFU and stored at Ϫ80°C. Inocula were prepared immediately before infection by diluting frozen stocks in PBS to the desired number of CFU. For survival experiments (n ϭ 15 mice/strain for variant 1 and 2 strains and n ϭ 40 mice/ strain for variant 3 strains), near mortality was determined by observation using predefined internationally recognized criteria for sacrificing mice, including loss of Ͼ10% of body weight, failure to eat or drink for 24 h, reduction of body condition score by Ͻ2, becoming unable to ambulate, rupture of the abscess at the inoculation site, or rupture of an abscess at a (37). Data are shown as a Kaplan-Meier survival curve, with statistical differences between strain groups determined with the log-rank test. The larger number of mice used for the variant 3 strains was determined using a power calculation after conducting a pilot experiment. For histology evaluation (n ϭ 3 mice/strain), lesions were excised, visually inspected, and photographed. The tissue was fixed in 10% phosphate-buffered formalin, decalcified, and embedded in paraffin using standard automated instruments. Histology was scored by a pathologist blinded to the strain treatment groups as described previously (38). Data are shown as means Ϯ standard errors of the means (SEM), with statistical differences between strain groups determined using

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the Wilcoxon rank sum test.

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Predictors of Response to Oral Medications and Low-Histamine Diet in Patients with Chronic Urticaria Background Chronic urticaria (CU) is comprised of diverse phenotypes, and thus, a shift towards a precision medical approach is warranted in its management. Methods This study enrolled 78 patients with CU. Serum erythrocyte sedimentation rate, hemoglobin, hematocrit, eosinophil count, IgE, antinuclear antibody (ANA), and serum diamine oxidase (DAO) levels of the patients were measured and were compared according to the patient's response to second-generation antihistamines (sgAH), corticosteroids, leukotriene receptor antagonist (LTRA), H2 blockers, and low-histamine diet. Results Age- and sex-adjusted logistic regression analysis showed that patients with duration of CU > 3 years (adjusted odd ratio [aOR] = 4.39) and a DAO level < 10 U/mL (aOR = 3.90) were significantly associated with a good sgAH response. Age > 50 years (aOR = 0.02), duration of chronic urticaria > 3 years (aOR =0.06), and an ANA titer ≥ 1 : 80 (aOR = 0.03) were significantly and inversely associated with corticosteroid response. A low-histamine diet response was significantly associated with LTRA response (aOR = 67.29). In addition, a DAO level < 5.4 U/mL (aOR = 71.95) was significantly associated with H2 blocker response. Furthermore, concomitant angioedema (aOR = 10.56), multiple food triggers (aOR = 11.69), and a DAO level < 5.4 U/mL (aOR = 3.78) were significantly associated with a low-histamine diet response. Conversely, dermatographic urticaria and a hematocrit level < 36% were significantly and inversely associated with low-histamine diet response. Conclusions Several promising biomarkers were identified in this study to predict

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the efficacy of chronic urticaria treatment. DAO could be a novel biomarker for predicting the efficacy not only of dietary intervention but also for antagonists of H1 and H2 receptors. Introduction Chronic urticaria (CU) has traditionally been defined as episodes of urticaria rash that occur almost daily for more than six weeks. CU is a common disease and is prevalent in approximately 1% of the general population. However, treatment options are limited. The recommended treatment out-lined in the CU guidelines includes second-generation H1 antihistamines (sgAH), omalizumab, and cyclosporine [1,2]. Due to the cost of omalizumab and the adverse effects of cyclosporin, most patients with CU choose sgAH as their only treatment. However, many of them are dissatisfied with sgAH monotherapy because it cannot eliminate all symptoms. Furthermore, a number of patients are unable to tolerate sgAH because of its sedating effects, even at low doses. Additionally, CU is comprised of different phenotypes. For example, 40% of patients with CU have concomitant angioedema, 20% have CU that is physically triggered, and some have long-lasting wheals. Standard care may not always fit all phenotypes. Thus, there is a need to shift toward a precision medicine approach in management of CU. Diamine oxidase (DAO) is the main enzyme involved in histamine catabolism, and DAO deficiency has been suspected as a probable cause of histamine intolerance. Beyond a critical level, histamine can induce a variety of symptoms in individuals with histamine intolerance, including urticaria, migraine, and gastrointestinal upset. A significant inverse correlation (r = −0:335, p = 0:001) between DAO

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and serum histamine level was reported by Cho et al. [3]. However, the application of serum DAO levels in CU management has yet to be determined. Therefore, the aim of this study was to investigate the predictors of treatment efficacy for oral medications and dietary interventions for patients with CU. The role of serum DAO levels in CU management was also explored. Characteristics of the Patients and Treatment. Adult patients 18 years of age or older with CU were recruited from two regional hospitals in Taiwan. The diagnosis of CU was based on the definition given in current CU guideline [1,2]. All participants were asked about the duration of their CU and whether they had concomitant angioedema, the duration of wheals, and exacerbation of CU with certain foods. All patients were prescribed up to a twofold dose of sgAH and were asked to follow the recommendations of a low-histamine diet. Montelukast and H 2 blocker was also prescribed if necessary. Low-dose corticosteroids were used only to relieve symptom. Patients who can achieve symptom free by regular antihistamine treatment were defined as good response to sgAH; those who reported improvement but not symptom free were defined as partial response to sgAH; and those who reported no improvement were defined as no response. The response to montelukast, H 2 blocker, or a low-histamine diet was evaluated by a decrease in sgAH for individuals with good response to sgAH. All participants signed informed consent according to a study protocol approved by the Institutional Review Board of Dalin Tzu Chi

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Hospital, Buddhist Tzu Chi Medical Foundation (No. B10902014-2) or Taipei Tzu Chi Hospital, Buddhist Tzu Chi Medical Foundation (No. 09-C-095). Measurement of Serum DAO and Laboratory Parameters. The following laboratory tests were performed as follows: serum erythrocyte sedimentation rate (ESR), hemoglobin (Hb) and hematocrit (Hct) levels, eosinophil count, IgE, and antinuclear antibody (ANA) and DAO levels. The serum was separated and kept at −20°C until needed. Laboratory blood parameters and serum DAO levels were measured in the following way. Briefly, total and specific IgE were analyzed by fluorimetric enzyme-linked assay (FEIA) (Phadia™, Thermo Fisher, Uppsala, Sweden). DAO levels were measured using the DAO ELISA kit (Immundiagnostik AG, Bensheim, Germany), according to the manufacturer's protocol. A dose-response curve of optical density versus a standard concentration was generated, using the values obtained from the standard. The reference values from the manufacturer's specification for DAO are as follows: histamine intolerance is highly likely in patients with DAO activity < 3 U/mL, likely in patients with DAO activity < 10 U/mL, and improbable in patients with DAO activity > 10 U/mL. Other index data were obtained from the routine laboratory examinations. Statistical Analysis. Comparative analyses were conducted using chi-square tests for categorical variables and independent t-test for continuous variables. Pearson's correlations were performed to analyze the correlation between continuous variables. To identify significant predictors of treatment efficacy in patients with CU, five separate ageand sex-adjusted logistic regression analyses were performed with the following dependent variables: good response to sgAH, corticosteroid response, leukotriene receptor antagonist response, H 2 blockers, and low-histamine

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diet response. When the logistic regression model failed to converge for binary outcomes, firth logistic regression was used [4]. The procedure was implemented through the IBM SPSS extension command STATS FIRTHLOG. A p value < 0.05 was considered statistically significant. All statistical analyses were conducted using IBM SPSS version 28.0 (IBM Corp., Armonk, NY, USA). Results The basic characteristics of the patients with CU are shown in Table 1. A total of 78 patients with CU completed the study. Of them, 54 were females (69.2%) and the overall mean age was 45.8 years (range: 19 to 74 years). The mean level of DAO was 8.91 U/mL, and the range was 1.14 U/mL to 63.15 U/mL. In addition, Table 2 shows that DAO levels were significantly lower in male patients than in their female counterparts (p = 0:004). They were significantly lower in patients with CU who were >50 years of age compared with those ≤ 50 years of age (p = 0:033). Patients with dermatographic urticaria had a significantly lower DAO compared to those with chronic spontaneous urticaria (p = 0:006). Moreover, the mean ESR level was significantly lower in patients with dermatographic urticaria compared to those with chronic spontaneous urticaria (mean 5.36 and SD 3.73 mm/hr vs mean 11.17 and SD 9.58 mm/hr, p = 0:001, respectively). DAO was also significantly correlated to ESR (p = 0:004) and inversely correlated with Hb (p = 0:022) and Hct (p = 0:016). Of 11 patients with available montelukast response, six reported having a good response (54.5%), and

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5 reported having a poor response (45.5%). The results of the multiple logistic regression analysis showed that a significantly higher risk of montelukast response was observed in patients with CU who benefited from a low-histamine diet (aOR = 67:29, 95% CI 2.07-64667, p = 0:012). Of eight patients with available H 2 blocker response, three (37.5%) reported having a good response, and 5 (62.5%) reported a poor response. The results of the multiple logistic regression analysis showed that patients with CU who had a DAO level < 5:4 U/mL (the median of DAO levels) were significantly more likely to respond to H 2 blockers (aOR = 71:95, 95% CI 1.55-89411, p = 0:027). Among the 78 participants, the most common food trigger reported was shrimp (n = 12), followed by alcohol (n = 6 ), peanuts (n = 5), and processed food with food additives (n = 5). These foods were included in this study due to intermittent eruptions despite the elimination of culprit foods. Four individuals with a shrimp trigger had specific IgE (sIgE) to shrimp detected. No individuals with the peanut trigger had sIgE to peanut detected. Patients with exacerbations after consuming shrimp were more likely to benefit from a low-histamine diet (aOR = 11:28, 95% CI 1.29-1489.21, p = 0:024). Patients with exacerbation after pumpkin consumption were less likely to respond to a lowhistamine diet (aOR = 0:06, 95% CI 0.00-0.79, p = 0:032). In the subgroup with DAO levels below 5.4 U/mL, patients who had reactions to processed foods with food additives

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had significantly lower DAO levels (mean 1.80 and SD 0.88 U/mL vs mean 3.58 and SD 1.11 U/mL, p = 0:004). The prevalence of angioedema was significantly higher in patients who had exacerbation of symptoms after consuming peanuts (3/5; 60% vs 10/73; 13.70%; p = 0:030). Patients with exacerbations after consuming plant-derived foods were more likely to have contact dermatitis with culprit foods or herbal medicines (4/24; 16.67% vs 1/54; 1.85%; p = 0:029). The prevalence of eczema was higher among patients who had exacerbations with alcohol consumption (2/6; 33.33% vs 5/72; 6.94%; p = 0:030). Patients with eruptions triggered predominantly by heat were more likely to have exacerbations of symptoms when eating spicy foods (2/5; 40% vs 4/72; 5.56%; p = 0:046). Discussion Currently, most studies of predictive markers of treatment efficacy in CU focus on antihistamines, omalizumab, and cyclosporine. This study is aimed at identifying clinical and blood biomarkers that are promising predictors of response to use of antihistamines, corticosteroids, LTRA, H 2 blockers, and a low-histamine diet. We also analyzed serum DAO levels in patients with CU to identify its associations with response to a low-histamine diet. We noted that the response to oral agents and a low-histamine diet among those with CU was associated with a number of biomarkers and factors, including age, sex, duration of the disease, concomitant angioedema, a phenotype of dermatographism, the presence of long-lasting wheals, DAO, Hct levels, ESR levels, eosinophil count, IgE, ANA, and a history of exacerbation when a particular food was consumed. Some of these

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factors may be useful in predicting the efficacy of treatment. Serum DAO levels may be useful when selecting patients with CU who are good candidates for a low-histamine diet. In clinical practice, the response to sgAH in patients with CU varies, even though it is the first-line treatment recommended in all phenotypes of CU. Consequently, up to 50% of patients with CU need other therapeutic options [5]. Several studies have been published addressing biomarkers of sgAH efficacy in patients with CU. A high urticarial activity score (UAS-7), longer duration of wheals, eosinopenia, baso-penia, increased C5a fraction, lower IgE levels, and autologous serum skin test (ASST) positivity were reported to be biomarkers of sgAH-resistant patients compared to sgAHsensitive patients [6][7][8]. Increased levels of D-dimer, fibrinogen, ESR, and C-reactive protein were reported to be predictors of nonresponders to a standard dose of sgAH [9]. Duration of disease was also suggested as a predictive marker of severity due to the greater improvement reported in patients who had CU less than one year [10]. In the present study, having symptoms of CU for more than three years was found to be a clinical marker of poor response to corticosteroid. However, a favorable response to sgAH was likely to be seen in this group. In contrast to the result of a previous study [6], an association of low IgE with an insufficient sgAH response was observed in patients with IgE levels below <300 U/mL alone (OR = 0:98, 95% CI 0.97, 1.00; p = 0:006). Meanwhile, an association of high IgE

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levels with an insufficient sgAH response was observed in patients with CU who had an IgE level > 100 U/mL (OR = 1:00, 95% CI 1.00, 1.01; p = 0:029), suggesting that there may be a Ushaped relationship between IgE and insufficient sgAH response. In the present study, no association of insufficient sgAH response with elevated ESR levels or eosinopenia was observed. Corticosteroids, potent immunosuppressants, can be the only rescue for some severe eruptions. However, resistance to corticosteroids can be seen in some patients with CU. In our study, older age seems to be a predictor of less favorable response to corticosteroid in patients with CU. In this respect, corticosteroid should be cautiously used in patients with CU, particularly older adults, due to not only the susceptibility of potential adverse effects but also because of less favorable efficacy. In clinical practice, when a patient with CU has wheals for a long time, elevated ESR, or positive ANA levels, underlying autoimmune diseases usually come to mind, which can be an indication for a corticosteroid. In fact, we found that patients with CU who had an ANA titer ≥ 1 : 80 were more likely to have a long duration of wheals (4/12; 33.33% vs 5/66; 7.58%; p = 0:010). However, the association of resistance to corticosteroid with ANA titers ≥ 1 : 80 was also observed. In the meantime, we have not observed the association of responders to corticosteroid with a long duration of wheals or elevated ESR. A possible explanation could be that ANA may be

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another marker of CU severity that results in resistance to corticosteroids at a lower dose prescribed. Unlike allergic diseases, an increased eosinophil count may not be helpful in selecting patients with CU who would be good candidates for corticosteroids. Even though LTRA has been recommended as a secondline treatment of CU in the BSACI guideline [1], its efficacy was found to be less reliable in a survey among clinicians in UK. According to our previous survey, 63.2% of allergists/ immunologists and 71.7% dermatologists stated that less than 25% of patients with CU were able to benefit from LTRA [11]. However, in real-world experience, there is a subgroup of patients with CU who can benefit from montelukast. Akoglu et al. have reported a significant decreased urinary LTE4 after a low-pseudoallergen diet in responders [12]. In our study, we observed an association of LTRA Journal of Immunology Research Dietary intervention has been reported to be beneficial in patients with CU. The response rate of an elimination diet among patients with CU varied widely in different studies, ranging from 31% to 100% [13]. A recent study focusing on a low-histamine diet for CU reported a 75% response rate [14]. Adherence is often a major issue in dietary intervention. A biomarker for the prediction of efficacy would be required to prompt patients with CU to adhere to the dietary recommendations. In comparison with healthy controls, significantly lower DAO levels in individuals with histamine intolerance benefiting from a histamine-limited diet have been reported (mean 7.04 and SD 6.90 U/mL vs mean

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39.50 and SD 18.16 U/mL; p = 0:003). However, patients with CU were excluded from the study [15]. In our study, no differences in response to dietary intervention could be observed between groups when the manufacturer's recommended cutoff was used. However, when we used the median of DAO levels as the cutoff, the difference in response to dietary intervention reached statistical significance between groups. Therefore, DAO appears to be a potential biomarker for the efficacy of a low-histamine diet in patients with CU. However, the cutoff level in clinical practice has yet to be determined. Interestingly, an association of DAO insufficiency with favorable response to sgAH and H 2 blockers was also observed in patients with CU, suggesting antagonists of H 1 and H 2 receptors might be helpful in dealing with excessive histamine when histamine metabolism is impaired. However, their efficacy may be insufficient when the capacity of histamine catabolism is excessively impaired, because the association of insufficient sgAH response with low DAO was observed in patients with DAO levels < 10 U/mL (OR = 0:74, 95% CI 0.56, 0.97; p = 0:031). Hemoglobin and Hct level analysis were included in this study because polycythemia could result in increased histamine formation. In the present study, an association of low Hct level with histamine tolerance was observed. Based on the inverse correlation between DAO and Hb, DAO consumption by excessive histamine in the process of erythrocyte metabolism might be considered. Additionally, a positive correlation was also observed between ESR, an acute phase reactant affected by erythrocyte

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number, and the level of DAO. Daschner et al. reported a significant correlation between IL-6, a proinflammatory cytokine, and DAO levels in patients with CU who were Anisakis sensitization (Rho = 0:57; p = 0:003) [15], which could be an alternative explanation for the significant correlation between DAO and ESR. While IL-6 and C-reactive protein have been reported to be biomarkers of CU severity, [7] DAO was reported as not associated with UAS [16]. Theoretically, pseudoallergic reactions are not evident immediately after consuming histamine-rich foods because the symptoms principally result from a buildup of histamine. However, exacerbation of CU followed by specific food intake has been reported by a number of patients. In the study of Magerl et al., fewer than 10% of patients with chronic spontaneous urticaria had reported food as a trigger [17]. In our study, 55.1% of patients with CU reported exacerbations of symptoms when they consumed specific foods. In our real-world experience, it could be difficult to identify the association between food consumption and CU exacerbation in the absence of adequate treatment, including sgAH and dietary intervention. In theory, any food rich in histamine may trigger the eruption in patients with histamine intolerance. As a result, a history of eruption with multiple food triggers might be another predictor of low-histamine diet efficacy. Food that can be a trigger for a CU attack might be a source of pseudoallergens, including salicylate, histamine, and other ingredients not clearly defined. Of course, IgE-mediated food allergy combined with CU should be ruled out. The food trigger

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reported the most frequently in our study was shrimp. Shrimp is a commonly seen as food allergen, while shellfish has been reported as a histamine releaser, as well as being a source of histamine [18]. Pseudoallergic reaction was preferred to food allergy, based on the association of low-histamine diet efficacy with CU exacerbated by shrimp consumption. Interestingly, reported foods that trigger exacerbation of CU were not on the top of the histamine-rich food list. Notably, most of these foods had been classified as histamine releasers [19]. Compared to histamine-rich foods, histaminereleasing foods may be more likely to immediately trigger pseudoallergic reactions. Instead of chronic spontaneous urticaria (CSU), dermatographic urticaria was classified as chronic inducible urticaria (CindU). While there is no identified trigger in patients of CSU, CindU can be triggered by physical stimuli. The differences observed in the efficacy of diet intervention between CSU and dermatographic urticaria suggested that a low-histamine diet may be less likely to be beneficial in CindU compared to CSU. In addition, we observed a trend for a lower prevalence of angioedema in patients with dermatographic urticaria (0/13; 0% vs 14/65; 21.54%; p = 0:065) and vice versa. While the association of concomitant angioedema with benefit from a low-histamine diet was observed, patients with dermatographism were less likely to benefit from a low-histamine diet. Conclusion A number of biomarkers identified in our study can be helpful to achieve better CU control. DAO appears to be a novel biomarker in CU, predicting the efficacy of sgAH, H 2 blocker, and a low-histamine diet.

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This is not surprising because DAO is the key enzyme in histamine metabolism. Furthermore, with appropriate patient selection, the efficacy of LTRA, H 2 blockers, and a low-histamine diet in patients with CU should be evaluated with additional studies. Data Availability The data used to support the findings of this study are included within the article.

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Two weight commutators for Beurling--Ahlfors operator We establish the equivalent characterisation of the weighted BMO space on the complex plane $\mathbb{C}$ via the two weight commutator of the Beurling--Ahlfors operator with a BMO function. Our method of proofs relies on the explicit kernel of the Beurling--Ahlfors operator and the properties of Muckenhoupt weight class. Introduction and statement of main result The theory of singular integrals in harmonic analysis has had its origin closely related to other fields of mathematics such as complex analysis and partial differential equations. A typical example is the Hilbert transform which has arisen from the complex conjugates of harmonic functions in the real and complex parts of analytic functions. The higher dimensional version of the Hilbert transform is the Riesz transform on the Euclidean space R n . The Hardy space H 1 and its dual space, the BMO space (BMO is abbreviation for bounded mean oscillation) have played an important role for the end-point estimates of singular integrals as they are known as the substitutes of the spaces L 1 and L ∞ . For singular integrals, weighted estimates are important and the A p class of Muckenhoupt weights has provided the appropriate class of weights for the study of Calderón-Zygmund operators. One can use two weight estimates on commutators of BMO functions with certain singular integral operators to characterise BMO spaces. The recent paper [7] gives a notable result which characterised weighted BMO spaces through two weight estimates on commutators of BMO functions and the Riesz transform. More specifically, consider

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the j-th Riesz transform on R n given by R j = ∂ ∂x j ∆ −1/2 , the weights λ 1 , λ 2 in the Muckenhoupt class A p , 1 < p < ∞, and the weight ν = λ the commutator of the Riesz transform R i and the function b ∈ BMO ν (R n ), i.e., the Muckenhoupt-Wheeden weighted BMO space (introduced in [8]). The main result in [7], Theorem 1.2, says that there exist constants 0 < c < C < ∞ such that where the constants c and C depend only on n, p, λ 1 , λ 2 . This result extended previous results of Bloom [1], Coifman, Rochberg and Weiss [2] and Nehari [11]. While the upper bound of the two weight commutator can be obtained for a large class of singular integral operators, the lower bound is delicate and its proof for each specific operator can be quite different and depends on the nature of the operator. For example, the proof for the lower bound of the commutator with the Riesz transform used the spherical harmonic expansions for the Riesz kernels, which relies on the property of the Fourier transform of the Riesz kernels. In this paper, we consider the Beurling-Ahlfors operator B (see for example [9,10]) which plays a notable role in complex analysis and is given by convolution with the distributional kernel p.v. 1 z 2 , i.e., for x ∈ C, Here, for simplicity, we just use dy to denote Lebesgue measure

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on C. For other works on the Beurling-Ahlfors operator, see for example [9] where they established a sharp weighted estimate of B, which is sufficient to prove that any weakly quasiregular map is quasiregular. We now recall the Muckenhoupt-Wheeden type weighted BMO space on C. For ν ∈ A 2 (C), BMO ν (C) is defined (see [8]) as the set of all f ∈ L 1 loc (C), such that where the supremum is taken over all cubes Q ⊂ C and A natural question is as follows. Q: Can we establish the characterisation of two weight commutator and the related weighted BMO space for the Beurling-Ahlfors operator, i.e. obtain (1.1) with the Beurling-Ahlfors operator in place of the Riesz transform? Our following main result gives a positive answer to this question. We provide the proof of the above theorem in Section 2. Then in Section 3, we provide the application of Theorem 1.1: the weak factorisation of the weighted Hardy space via the bilinear form in terms of the Beurling-Ahlfors operator, which extends the classical result of Coifman, Rochberg and Weiss [2]. Throughout the paper, we denote by C and C positive constants which are independent of the main parameters, but they may vary from line to line. For every p ∈ (1, ∞), we denote by p ′ the conjugate of p, i.e., 1 p ′ + 1 p = 1. If f ≤ Cg, we then write f g or g f ; and if f g f , we write f

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≈ g. Proof of the main theorem We first recall the definition and some basic properties of the Muckenhoupt A p (C) weights. where the supremum is taken over all cubes Q in C. We denote by [w] Ap the smallest constant C such that (2.1) holds. The class A 1 (C) consists of the weights w satisfying for some C > 0 that for any cubes Q ⊂ C. We denote by [w] A 1 the smallest constant C such that the above inequality holds. If w ∈ A p (C) with p > 1, then the "conjugate" weight Note that ν ∈ A 2 (C). Since B is a Calderón-Zygmund operator, following the result in [7] we obtain that there exists a positive constant C such that for b ∈ BMO ν (C), When λ 1 = λ 2 , then the upper bound with precise information about the constant C as a function of the A p characteristic was obtained in [4]. We now prove the lower bound. Suppose that b ∈ L 1 loc (C) and that [b, B] is bounded from L p λ 1 (C) to L p λ 2 (C). It suffices to show that for every cube Q ⊂ C, there exists a positive constant C such that To see this, without lost of generality, we now consider an arbitrary cube Q ⊂ C centered at the origin. Then we have where We now denote Γ Q (x) := sgn(b(x) − b Q ). Then for the term

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I 1 , we obtain that where in the first inequality we use Holder's inequality with the index 1 p + 1 p ′ = 1, in the second inequality we use the boundedness of [b, B] from L p λ 1 (C) to L p λ 2 (C) and the fact that |Γ Q (x)| ≤ 1 for any x ∈ C, and in the last inequality we use the fundamental fact in (2.4). As for the term I 2 , similarly, we have Again, for the term I 3 , using similar argument, we get that As a consequence, combining the estimates of I 1 , I 2 and I 3 , we get that Applications: Weak factorization of the weighted Hardy space We recall the weighted Hardy space, then prove that it is the predual of BMO ν (C). Note that based on the results in [8] and the recent result in [5], there are also other equivalent characterisations of the weighted Hardy space, for example, via Littlewood-Paley area functions, maximal functions, etcetera. For simplicity, we just define the weighted Hardy space via atoms as follows. where B is a ball in C; (2) C a(x)dx = 0; We say that f belongs to the weighted Hardy space H 1 ν (C) if f can be written as |α j | : f has the representation as in (3.1) . We also recall the John-Nirenberg inequality for the BMO ν (C). According to [5], we know that for v ∈ A 2 (C)

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and for 1 ≤ r ≤ 2, where b BMOν,r(C) := sup Proof. This duality result follows from a standard argument, see for example [3]. By completeness, we provide the proof as follows. We first show that In fact, for any g ∈ BMO ν (C), define where a is an ν-weighted (1, 2)-atom. Assume that a is supported in a cube Q. Then from Hölder's inequality and ν ∈ A 2 (C), we see that Thus L g extends to a bounded linear functional on H 1 ν (C). Conversely, assume that L ∈ H 1 ν (C) * . For any cube Q, let Then we see that for any f ∈ L 2 0, ν (Q), the function . From the Riesz representation theorem, there exists [ϕ] ∈ [L 2 0, ν (Q)] * = L 2 0, ν −1 (Q)/C, and ϕ ∈ [ϕ], such that for any f ∈ L 2 0, ν (Q), Let Q fixed and Q j = 2 j Q, j ∈ N. Then we have that for all f ∈ L 2 0, ν (Q) and j ∈ N, It follows that for almost every x ∈ Q, ϕ Q j (x) − ϕ Q 0 (x) = C j for some constant C j . From this we further deduce that for all j, l ∈ N, j ≤ l and almost every x ∈ Q j , Thus, ϕ is well defined. Moreover, since C = ∪ j Q j , by Hölder's inequality

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and ν ∈ A 2 (C), we see that for any c and any cube Q, Taking the infimum over c, we have that ϕ ∈ BMO ν (C) and ϕ BMO ν (C) ≤ C L . The main result of this section is as follows. Theorem 3.3. Suppose 1 < p < ∞, λ 1 , λ 2 ∈ A p (C) and ν = λ . For every f ∈ H 1 ν (C), there exist sequences {α k j } j ∈ ℓ 1 and functions h k j ∈ L p λ 1 (C), g k j ∈ L p ′ λ ′ 2 (C) with p ′ = p p−1 and in the sense of H 1 ν (C), where Π(g k j , h k j )(x) is the bilinear form defined as Moreover, we have that where the infimum is taken over all possible representations of f from (3.4). It is well known that this theorem follows from the duality between H 1 ν (C) and BMO ν (C) and the equivalence between BMO ν (C) and the boundedness of the commutator, provided in Theorem 1.1. We omit the details of this proof.

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Prenatal diagnosis of ultrasound soft markers in a single medical center of mainland China Background There are a few studies on the chromosomal aberration of Ultrasound soft markers (USMs). The aim of this study was to determine the detection rate of clinically significant chromosomal abnormalities (CSCA) in fetuses with different USMs. Methods This study included fetuses with USMs who underwent invasive prenatal diagnosis for karyotype and/or chromosomal microarray (CMA) by categorizing into two groups: a single USM (SUSM) and multiple USMs (MUSMs). Results Of the 358 cases with USMs, CSCA occurred in 3.09% (8/259) and 8.08% (8/99) of the SUSM and MUSM groups, respectively (P < 0.05). Of 16 cases identified with CSCA, theoretically 68.75% (11/16) could be detected by karyotype, while 31.25% (5/16) could be recognized only by CMA. Among CSCA cases, the most frequent USM was an absent or hypoplastic nasal bone (62.5%, 10/16). In cases with negative karyotypes and/or CMA, follow-up results were available in 307 cases, including 292 term deliveries, 6 preterm deliveries, 8 terminations of pregnancy due to USMs, and 1 still birth. Conclusion MUSMs increased the risk of chromosomal abnormalities. An absent or hypoplastic nasal bone was the most clinically significant marker either alone or in combination with other USMs. Most of SUSM had a good prognosis. Introduction The structural anomalies associated with genetic factors can be detected by prenatal ultrasound throughout pregnancy. With the advance in ultrasound devices and the improvement of sonographers' skills, the detection rate of ultrasound anomalies, even minor structural abnormalities, has increased. Unlike structural anomalies,

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