Rare variant associations with plasma protein levels in the UK Biobank.

Integrating human genomics and proteomics can help elucidate disease mechanisms, identify clinical biomarkers and discover drug targets1-4. Because previous proteogenomic studies have focused on common variation via genome-wide association studies, the contribution of rare variants to the plasma proteome remains largely unknown. Here we identify associations between rare protein-coding variants and 2,923 plasma protein abundances measured in 49,736 UK Biobank individuals. Our variant-level exome-wide association study identified 5,433 rare genotype-protein associations, of which 81% were undetected in a previous genome-wide association study of the same cohort5. We then looked at aggregate signals using gene-level collapsing analysis, which revealed 1,962 gene-protein associations. Of the 691 gene-level signals from protein-truncating variants, 99.4% were associated with decreased protein levels. STAB1 and STAB2, encoding scavenger receptors involved in plasma protein clearance, emerged as pleiotropic loci, with 77 and 41 protein associations, respectively. We demonstrate the utility of our publicly accessible resource through several applications. These include detailing an allelic series in NLRC4, identifying potential biomarkers for a fatty liver disease-associated variant in HSD17B13 and bolstering phenome-wide association studies by integrating protein quantitative trait loci with protein-truncating variants in collapsing analyses. Finally, we uncover distinct proteomic consequences of clonal haematopoiesis (CH), including an association between TET2-CH and increased FLT3 levels. Our results highlight a considerable role for rare variation in plasma protein abundance and the value of proteogenomics in therapeutic discovery.

Genotyping, sequencing and analysis of 140,000 adults from Mexico City.

The Mexico City Prospective Study is a prospective cohort of more than 150,000 adults recruited two decades ago from the urban districts of Coyoacán and Iztapalapa in Mexico City1. Here we generated genotype and exome-sequencing data for all individuals and whole-genome sequencing data for 9,950 selected individuals. We describe high levels of relatedness and substantial heterogeneity in ancestry composition across individuals. Most sequenced individuals had admixed Indigenous American, European and African ancestry, with extensive admixture from Indigenous populations in central, southern and southeastern Mexico. Indigenous Mexican segments of the genome had lower levels of coding variation but an excess of homozygous loss-of-function variants compared with segments of African and European origin. We estimated ancestry-specific allele frequencies at 142 million genomic variants, with an effective sample size of 91,856 for Indigenous Mexican ancestry at exome variants, all available through a public browser. Using whole-genome sequencing, we developed an imputation reference panel that outperforms existing panels at common variants in individuals with high proportions of central, southern and southeastern Indigenous Mexican ancestry. Our work illustrates the value of genetic studies in diverse populations and provides foundational imputation and allele frequency resources for future genetic studies in Mexico and in the United States, where the Hispanic/Latino population is predominantly of Mexican descent.

Rare variant contribution to human disease in 281,104 UK Biobank exomes.

Genome-wide association studies have uncovered thousands of common variants associated with human disease, but the contribution of rare variants to common disease remains relatively unexplored. The UK Biobank contains detailed phenotypic data linked to medical records for approximately 500,000 participants, offering an unprecedented opportunity to evaluate the effect of rare variation on a broad collection of traits1,2. Here we study the relationships between rare protein-coding variants and 17,361 binary and 1,419 quantitative phenotypes using exome sequencing data from 269,171 UK Biobank participants of European ancestry. Gene-based collapsing analyses revealed 1,703 statistically significant gene-phenotype associations for binary traits, with a median odds ratio of 12.4. Furthermore, 83% of these associations were undetectable via single-variant association tests, emphasizing the power of gene-based collapsing analysis in the setting of high allelic heterogeneity. Gene-phenotype associations were also significantly enriched for loss-of-function-mediated traits and approved drug targets. Finally, we performed ancestry-specific and pan-ancestry collapsing analyses using exome sequencing data from 11,933 UK Biobank participants of African, East Asian or South Asian ancestry. Our results highlight a significant contribution of rare variants to common disease. Summary statistics are publicly available through an interactive portal ( http://azphewas.com/ ).

Effects of protein-coding variants on blood metabolite measurements and clinical biomarkers in the UK Biobank.

Genome-wide association studies (GWASs) have established the contribution of common and low-frequency variants to metabolic blood measurements in the UK Biobank (UKB). To complement existing GWAS findings, we assessed the contribution of rare protein-coding variants in relation to 355 metabolic blood measurements-including 325 predominantly lipid-related nuclear magnetic resonance (NMR)-derived blood metabolite measurements (Nightingale Health Plc) and 30 clinical blood biomarkers-using 412,393 exome sequences from four genetically diverse ancestries in the UKB. Gene-level collapsing analyses were conducted to evaluate a diverse range of rare-variant architectures for the metabolic blood measurements. Altogether, we identified significant associations (p < 1 × 10-8) for 205 distinct genes that involved 1,968 significant relationships for the Nightingale blood metabolite measurements and 331 for the clinical blood biomarkers. These include associations for rare non-synonymous variants in PLIN1 and CREB3L3 with lipid metabolite measurements and SYT7 with creatinine, among others, which may not only provide insights into novel biology but also deepen our understanding of established disease mechanisms. Of the study-wide significant clinical biomarker associations, 40% were not previously detected on analyzing coding variants in a GWAS in the same cohort, reinforcing the importance of studying rare variation to fully understand the genetic architecture of metabolic blood measurements.

Efficacy and Safety of NVX-CoV2373 in Adults in the United States and Mexico.

BACKGROUND: NVX-CoV2373 is an adjuvanted, recombinant spike protein nanoparticle vaccine that was shown to have clinical efficacy for the prevention of coronavirus disease 2019 (Covid-19) in phase 2b-3 trials in the United Kingdom and South Africa, but its efficacy had not yet been tested in North America. METHODS: We conducted a phase 3, randomized, observer-blinded, placebo-controlled trial in the United States and Mexico during the first half of 2021 to evaluate the efficacy and safety of NVX-CoV2373 in adults (≥18 years of age) who had not had severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection. Participants were randomly assigned in a 2:1 ratio to receive two doses of NVX-CoV2373 or placebo 21 days apart. The primary objective was to determine vaccine efficacy against reverse-transcriptase-polymerase-chain-reaction-confirmed Covid-19 occurring at least 7 days after the second dose. Vaccine efficacy against moderate-to-severe disease and against different variants was also assessed. RESULTS: Of the 29,949 participants who underwent randomization between December 27, 2020, and February 18, 2021, a total of 29,582 (median age, 47 years; 12.6% ≥65 years of age) received at least one dose: 19,714 received vaccine and 9868 placebo. Over a period of 3 months, 77 cases of Covid-19 were noted - 14 among vaccine recipients and 63 among placebo recipients (vaccine efficacy, 90.4%; 95% confidence interval [CI], 82.9 to 94.6; P<0.001). Ten moderate and 4 severe cases occurred, all in placebo recipients, yielding vaccine efficacy against moderate-to-severe disease of 100% (95% CI, 87.0 to 100). Most sequenced viral genomes (48 of 61, 79%) were variants of concern or interest - largely B.1.1.7 (alpha) (31 of the 35 genomes for variants of concern, 89%). Vaccine efficacy against any variant of concern or interest was 92.6% (95% CI, 83.6 to 96.7). Reactogenicity was mostly mild to moderate and transient but was more frequent among NVX-CoV2373 recipients than among placebo recipients and was more frequent after the second dose than after the first dose. CONCLUSIONS: NVX-CoV2373 was safe and effective for the prevention of Covid-19. Most breakthrough cases were caused by contemporary variant strains. (Funded by Novavax and others; PREVENT-19 ClinicalTrials.gov number, NCT04611802.).

Key subphenotypes of bipolar disorder are differentially associated with polygenic liabilities for bipolar disorder, schizophrenia, and major depressive disorder.

Bipolar disorder (BD) features heterogenous clinical presentation and course of illness. It remains unclear how subphenotypes associate with genetic loadings of BD and related psychiatric disorders. We investigated associations between the subphenotypes and polygenic risk scores (PRS) for BD, schizophrenia, and major depressive disorder (MDD) in two BD cohorts from Sweden (N = 5180) and the UK (N = 2577). Participants were assessed through interviews and medical records for inter-episode remission, psychotic features during mood episodes, global assessment of functioning (GAF, function and symptom burden dimensions), and comorbid anxiety disorders. Meta-analyses based on both cohorts showed that inter-episode remission and GAF-function were positively correlated with BD-PRS but negatively correlated with schizophrenia-PRS (SCZ-PRS) and MDD-PRS. Moreover, BD-PRS was negatively, and MDD-PRS positively, associated with the risk of comorbid anxiety disorders. Finally, SCZ-PRS was positively associated with psychotic symptoms during mood episodes. Assuming a higher PRS of certain psychiatric disorders in cases with a positive family history, we further tested the associations between subphenotypes in index BD people and occurrence of BD, schizophrenia, or MDD in their relatives using Swedish national registries. BD patients with a relative diagnosed with BD had: (1) higher GAF and lower risk of comorbid anxiety than those with a relative diagnosed with schizophrenia or MDD, (2) lower risk of psychotic symptoms than those with a relative diagnosed with schizophrenia. Our findings shed light on the genetic underpinnings of the heterogeneity in clinical manifestations and course of illness in BD, which ultimately provide insights for developing personalized approaches to the diagnosis and treatment.

Alveolar macrophage-derived extracellular vesicles inhibit endosomal fusion of influenza virus.

Alveolar macrophages (AMs) and epithelial cells (ECs) are the lone resident lung cells positioned to respond to pathogens at early stages of infection. Extracellular vesicles (EVs) are important vectors of paracrine signaling implicated in a range of (patho)physiologic contexts. Here we demonstrate that AMs, but not ECs, constitutively secrete paracrine activity localized to EVs which inhibits influenza infection of ECs in vitro and in vivo. AMs exposed to cigarette smoke extract lost the inhibitory activity of their secreted EVs. Influenza strains varied in their susceptibility to inhibition by AM-EVs. Only those exhibiting early endosomal escape and high pH of fusion were inhibited via a reduction in endosomal pH. By contrast, strains exhibiting later endosomal escape and lower fusion pH proved resistant to inhibition. These results extend our understanding of how resident AMs participate in host defense and have broader implications in the defense and treatment of pathogens internalized within endosomes.

100,000 Genomes Pilot on Rare-Disease Diagnosis in Health Care - Preliminary Report.

BACKGROUND: The U.K. 100,000 Genomes Project is in the process of investigating the role of genome sequencing in patients with undiagnosed rare diseases after usual care and the alignment of this research with health care implementation in the U.K. National Health Service. Other parts of this project focus on patients with cancer and infection. METHODS: We conducted a pilot study involving 4660 participants from 2183 families, among whom 161 disorders covering a broad spectrum of rare diseases were present. We collected data on clinical features with the use of Human Phenotype Ontology terms, undertook genome sequencing, applied automated variant prioritization on the basis of applied virtual gene panels and phenotypes, and identified novel pathogenic variants through research analysis. RESULTS: Diagnostic yields varied among family structures and were highest in family trios (both parents and a proband) and families with larger pedigrees. Diagnostic yields were much higher for disorders likely to have a monogenic cause (35%) than for disorders likely to have a complex cause (11%). Diagnostic yields for intellectual disability, hearing disorders, and vision disorders ranged from 40 to 55%. We made genetic diagnoses in 25% of the probands. A total of 14% of the diagnoses were made by means of the combination of research and automated approaches, which was critical for cases in which we found etiologic noncoding, structural, and mitochondrial genome variants and coding variants poorly covered by exome sequencing. Cohortwide burden testing across 57,000 genomes enabled the discovery of three new disease genes and 19 new associations. Of the genetic diagnoses that we made, 25% had immediate ramifications for clinical decision making for the patients or their relatives. CONCLUSIONS: Our pilot study of genome sequencing in a national health care system showed an increase in diagnostic yield across a range of rare diseases. (Funded by the National Institute for Health Research and others.).

Lessons Learned Implementing Syringe Services Programs at Rural Health Departments in Kentucky.

Until recently, most syringe services programs (SSPs) in the United States operated in metropolitan areas. This study explores how SSP implementers at rural health departments in Kentucky secured support for SSP operations. In late 2020, we conducted in-depth, semi-structured interviews with 18 people involved with rural SSP implementation in Kentucky. Participants were asked to reflect on their experiences building support for SSP operations among rural health department staff and community members. Participants reported that attitudes and beliefs about SSP implementation among rural health department staff shifted quickly following engagement in educational activities and interaction with SSP clients. Participants explained that successful SSP implementation at rural health departments required sustained educational activities among community members and authorizing authorities. Future work should explore how rural communities may advocate for low-threshold and evidence-based policies that support the provision of harm reduction services.

Use of an intelligent tutoring system for a curriculum on augmentative and alternative communication: Feasibility for implementation.

BACKGROUND: Over 8 million children with disabilities live in Africa and are candidates for augmentative and alternative communication (AAC), yet formal training for team members, such as speech-language therapists and special education teachers, is extremely limited. Only one university on the continent provides postgraduate degrees in AAC, and other institutions provide only short modules at an undergraduate level. The need for an introductory training course on AAC that is accessible by university students continent-wide was identified. An online programme, namely an intelligent tutoring system (ITS), was identified as a possible option to facilitate interactive learning without the need for synchronous teaching. The use of an ITS is shown to be effective in developing knowledge and clinical reasoning in the health and rehabilitation fields. However, it has not yet been applied to student teaching in the field of AAC. AIM: To determine both the feasibility of an ITS to implement an AAC curriculum for students in four African countries, and the usability and effectiveness of such a system as a mechanism for learning about AAC. METHOD & PROCEDURES: The study included two components: the development of a valid AAC curriculum; and using the ITS to test the effectiveness of implementation in a pre- and post-test design with 98 speech-language therapy and special education students from five universities. OUTCOMES & RESULTS: Statistically significant differences were obtained between pre- and post-test assessments. Students perceived the learning experience as practical, with rich content. CONCLUSIONS & IMPLICATIONS: The findings suggest that the ITS-based AAC curriculum was positively perceived by the students and potentially offers an effective means of providing supplementary AAC training to students, although modifications to the system are still required. WHAT THIS PAPER ADDS: What is already known on the subject Professionals typically lack formal training in AAC. In Africa, this presents a serious challenge as there are over 8 million children who are candidates for AAC. A need for an introductory training course on AAC, which can be accessed by university students continent-wide, was identified. What this paper adds to existing knowledge An AAC curriculum was developed and integrated into an ITS, an online programme allowing interactive learning through asynchronous teaching. Students from four African countries completed the AAC ITS curriculum. The curriculum was positively received by the students and statistically significant changes in knowledge were identified. What are the practical and clinical implications of this work? This feasibility study shows that the use of an ITS is an effective means of providing AAC training to university students in these African countries. The results provide a valuable contribution toward ensuring the equitable distribution of AAC training opportunities in the African context. This will have a significant positive impact on those who are candidates for AAC.

Collaboration for Health Equity: A Qualitative Assessment of Local Leaders in Community-Based Organizations, Health Care, and Public Health in Michigan.

A qualitative study was conducted using semi-structured interviews leaders in community-based organizations, health care, and local public health to understand organizational perspectives of collaboration for health equity and identify opportunities to improve collaboration. Twelve leaders were interviewed from March through May 2023 in Kent County, Michigan. All leaders saw collaboration as valuable for advancing health equity. Key themes that affected collaboration and could be a facilitator or barrier to advancing health equity in the community were inclusion, power, relationships, resources, and organizational traits. Leaders articulated the following factors as those that support collaboration for health equity: authentic inclusion, shared decision-making, taking time to foster trusting relationships, adequate resources to support the infrastructure needed for collaborations, organizational flexibility, and individual commitment. Building partnerships with these facilitators in mind may result in more robust, sustainable, and resilient collaboratives.

Retrospective Cohort Analysis of the Impact of Puberty on Plexiform Neurofibroma Growth in Patients with Neurofibromatosis Type 1.

OBJECTIVE: To assess the hypothesis that plexiform neurofibroma (PN) growth rates increase during puberty. STUDY DESIGN: PN growth rates before and during puberty were compared in a retrospective cohort of children with neurofibromatosis type 1 with puberty defined by Tanner staging. Of 33 potentially eligible patients, 25 had adequate quality magnetic resonance imaging for volumetric analysis and were included in ≥1 anchor cohort. Volumetric analysis was performed for all available imaging studies within the 4 years before and after puberty, and before and after 9- and 11-year-old anchor scans. Linear regression was performed to estimate the slope of change (PN growth rate); growth rates were compared with paired t test or Wilcoxon matched-pairs signed rank test. RESULTS: There were no significant difference in rates of PN growth in milliliters per month or milliliters per kilogram per month in the prepubertal vs pubertal periods (mean, 1.33 ± 1.67 vs 1.15 ± 1.38 [P = .139] and -0.003 ± 0.015 vs -0.002 ± 0.02 [P = .568]). Percent increases of PN volumes from baseline per month were significantly higher prepubertally (1.8% vs 0.84%; P = .041) and seemed to be related inversely to advancing age. CONCLUSIONS: Puberty and its associated hormonal changes do not seem to influence PN growth rate. These findings support those previously reported, but from a typical population of children with neurofibromatosis type 1 with puberty confirmed by Tanner staging.

Engaging Payors and Primary Care Physicians Together in Improving Diabetes Prevention.

BACKGROUND: Type 2 diabetes can be prevented through lifestyle programs like the Diabetes Prevention Programs (DPP), but few people with prediabetes participate in them, in part because their insurance does not reliably cover DPPs. Prior studies have not focused on payor-level barriers. OBJECTIVE: To understand barriers to DPP uptake that exist and intersect at different levels (patients, PCPs, and payors) to inform strategies to improve diabetes prevention in primary care settings through interviews with PCPs and payors. DESIGN: From May 2020 to October 2021, we conducted remote, semi-structured interviews with PCPs and payors. PARTICIPANTS: PCPs were from primary care practices affiliated with one mid-Atlantic academic system. Payor leaders were from regional commercial, Medicare, and Medicaid plans. APPROACH: Using a standardized interview guide focused on barriers, facilitators, and potential intervention components, interviews were audio-recorded using Zoom and professionally transcribed. Two reviewers double-coded transcripts using the framework analytic approach. KEY RESULTS: We interviewed 16 PCPs from 13 primary care clinics and 7 payor leaders representing 6 insurance plans. Two themes emerged from PCP reports of patient-level barriers: (1) lack of programs and insurance coverage of resources to address nutrition and exercise and (2) inadequate resources to address social determinants of health that impact diabetes prevention. Among barriers PCPs faced, we identified two themes: (1) low PCP knowledge about DPPs and misperceptions of insurance coverage of DPPs and (2) inadequate clinical staff to address diabetes prevention. Barriers common to PCPs and payors included (1) absence of prediabetes quality measures and (2) inadequate engagement of PCPs and patients with payors. CONCLUSIONS: Discussions with PCPs and payors revealed systemic barriers that suggest important priorities to improve prediabetes clinical care, including universal coverage of DPPs, clarity about coverage benefits, data reporting and outreach by payors to PCPs, and adoption of appropriate prediabetes quality measures.

Hydrogen Peroxide Disproportionation Activity Is Sensitive to Pyridine Substitutions on Manganese Catalysts Derived from 12-Membered Tetra-Aza Macrocyclic Ligands.

The abundance of manganese in nature and versatility to access different oxidation states have made manganese complexes attractive as catalysts for oxidation reactions in both biology and industry. Macrocyclic ligands offer the advantage of substantially controlling the reactivity of the manganese center through electronic tuning and steric constraint. Inspired by the manganese catalase enzyme, a biological catalyst for the disproportionation of H2O2 into water and O2, the work herein employs 12-membered tetra-aza macrocyclic ligands to study how the inclusion of and substitution to the pyridine ring on the macrocyclic ligand scaffold impacts the reactivity of the manganese complex as a H2O2 disproportionation catalyst. Synthesis and isolation of the manganese complexes was validated by characterization using UV-vis spectroscopy, SC-XRD, and cyclic voltammetry. Potentiometric titrations were used to study the ligand basicity as well as the thermodynamic equilibrium with Mn(II). Manganese complexes were also produced in situ and characterized using electrochemistry for comparison to the isolated species. Results from these studies and H2O2 reactivity showed a remarkable difference among the ligands studied, revealing instead a distinction in the reactivity regarding the number of pyridine rings within the scaffold. Moreover, electron-donating groups on the 4-position of the pyridine ring enhanced the reactivity of the manganese center for H2O2 disproportionation, demonstrating a handle for control of oxidation reactions using the pyridinophane macrocycle.

Increased Efficiency of a Functional SOD Mimic Achieved with Pyridine Modification on a Pyclen-Based Copper(II) Complex.

A series of Cu(II) complexes with the formula [CuRPyN3]2+ varying in substitution on the pyridine ring were investigated as superoxide dismutase (SOD) mimics to identify the most efficient reaction rates produced by a synthetic, water-soluble copper-based SOD mimic reported to date. The resulting Cu(II) complexes were characterized by X-ray diffraction analysis, UV-visible spectroscopy, cyclic voltammetry, and metal-binding (log ß) affinities. Unique to this approach, the modifications to the pyridine ring of the PyN3 parent system tune the redox potential while exhibiting high binding stabilities without changing the coordination environment of the metal complex within the PyN3 family of ligands. We were able to adjust in parallel the binding stability and the SOD activity without compromising on either through simple modification of the pyridine ring on the ligand system. This goldilocks effect of high metal stabilities and high SOD activity reveals the potential of this system to be explored in therapeutics. These results serve as a guide for factors that can be modified in metal complexes using pyridine substitutions for PyN3, which can be incorporated into a range of applications moving forward.

Normal and Abnormal Appearances of the Ovaries during Assisted Reproduction: Multimodality Imaging Review.

Infertility is a common diagnosis that prompts many couples and individuals to seek assisted reproductive technology (ART) for assistance with conception. These technologies have become increasingly used in the United States in the past several decades, with 326 468 ART cycles performed in 2020, resulting in 75 023 live births. This ubiquity of ART also increases the likelihood that radiologists will encounter both normal and abnormal imaging findings associated with these treatments. Thus, radiologists of all subspecialties should be familiar with the multimodality appearance of the ovaries and pelvis in patients undergoing ART treatments. Furthermore, it is imperative that radiologists understand the appearance expected during different stages of the ART process. During stimulated ovulatory cycles, it is normal and expected for the ovaries to appear enlarged and to contain numerous cystic follicles, often with a small to moderate volume of pelvic free fluid. After oocyte retrieval, hemorrhagic ovarian follicles and a small to moderate volume of blood products in the cul-de-sac can be expected to be seen. Multiple nonemergency and emergency complications are related to ART, many of which can be seen at imaging. The most encountered emergency complications of ART include ovarian hyperstimulation syndrome, ectopic pregnancy, heterotopic pregnancy, multiple gestations, ovarian torsion, and procedural complications related to oocyte retrieval. These complications have important clinical implications, thus necessitating accurate and timely detection by the radiologist and the clinical team. ©RSNA, 2023 Supplemental material is available for this article. Quiz questions for this article are available through the Online Learning Center.

Growing pains: strategies for improving ergonomics in minimally invasive gynecologic surgery.

PURPOSE OF REVIEW: To evaluate factors contributing to the development of work-related musculoskeletal disorders (WMSDs) and review strategies for mitigating ergonomic strain in minimally invasive gynecologic surgery. RECENT FINDINGS: Factors associated with increased ergonomic strain and the development of WMSDs include increasing patient body mass index (BMI), smaller surgeon hand size, noninclusive design of instruments and energy devices and improper positioning of surgical equipment. Each type of minimally invasive surgery (laparoscopic, robotic, vaginal) confers its own ergonomic risk to the surgeon. Recommendations have been published regarding optimal ergonomic surgeon and equipment positioning. Intraoperative breaks and stretching are effective in reducing surgeon discomfort. Formal training in ergonomics has not yet been widely implemented, but educational interventions have been effective in reducing surgeon discomfort and can improve surgeon recognition of suboptimal ergonomics. SUMMARY: Considering the serious downstream effects of WMSDs on surgeons, it is imperative to implement strategies for WMSD prevention. Optimal positioning of the surgeons and operative equipment should be routine. Intraoperative breaks and stretching should be incorporated during procedures and between every case. Formal education in ergonomics should be provided to surgeons and trainees. Additionally, more inclusive instrument design by industry partners should be prioritized.

Straw cigarette branding: misleading descriptors and a new Marlboro man.

BACKGROUND: Straw cigarettes are hand-rolled tobacco in a corn straw, common in Brazil and believed to be less harmful by consumers. Traditionally consumed in rural areas and by men, a recent increase in use among young people in urban areas was observed. This study assessed marketing appeals of straw cigarette packaging in Brazil. METHODS: Following a systematic protocol, unique straw cigarette packs were purchased in 2013, 2016 and 2019 from three cities: Manaus, Salvador and São Paulo. Packs were double-coded for the absence/presence of marketing appeals (ie, luxury/quality, masculinity, classic/timeless, flavours and less harm). RESULTS: The number of unique straw cigarette packs purchased as part of the overall sample increased in the most recent collection: 2.3% (n=3), 1.3% (n=2) and 15.5% (n=27) in 2013, 2016 and 2019, respectively. Across the years, all packs presented luxury/quality elements (eg, images of crests); 93.8% (n=30) included the word 'artisanal'. Masculine appeal (eg, images of men) was present on 56.3% (n=18) of packs. Almost half had classic/timeless elements, including the words 'tradition'/'original'. In 2019, 59.3% (n=16) of the packs were flavoured compared with none in previous years. Over a quarter of all packs presented a less harm appeal (eg, 'smooth'). CONCLUSION: We identified an increase in availability of unique straw cigarette packs, strong branding and use of misleading descriptors on the packs. The adoption of plain packaging and the implementation of Brazil's additive ban could help to reduce the appeal of straw cigarettes and curb the current increase in use among youth.

Tobacco companies' creation of additional communication space: a content analysis of cigarette pack inserts and onserts.

OBJECTIVES: Pack inserts and onserts-removable items placed inside or on the outside of packs-are a communicative strategy used by tobacco companies that provide them with additional marketing space. A content analysis of these items was conducted across several years, countries and brands to assess how these items are used to communicate with consumers. METHODS: Between 2013 and 2020, cigarette packs were systematically collected using the Tobacco Pack Surveillance System protocol. Packs with inserts or onserts (n=178) were identified from 11 low and middle-income countries. Packs were coded for tobacco company strategies, physical pack characteristics and imagery and lexical marketing appeals. RESULTS: Of the 5903 packs, 3% (n=178) had an insert or onsert. 171 of these (96%) were inserts. While most (78%) pack exteriors were entirely in English, over half (51%) of the inserts/onserts were entirely in the local (non-English) language from where the pack was collected. The most common appeals on the inserts/onserts were product dependability (64%), luxury/aspirational (55%) and machinery/technology (37%). Product images were prevalent as well as images or words mentioning filters (22%). The most used appeals involved featuring aspects of a product (66%), addressing customers directly (52%) and informing customers about new aspects of a product (31%). CONCLUSIONS: Cigarette pack inserts/onserts are unregulated in many countries and provide additional space for tobacco companies to extend and innovate their advertising. Tobacco advertising and packaging policies such as plain and standardised packaging should expand to address inserts/onserts to protect consumers more fully from industry promotion of deadly products.