Pyroptosis is a gasdermin-mediated, pro-inflammatory form of cell death distinct from apoptosis. In recent years, increasing attention has shifted toward pyroptosis as more studies demonstrate its involvement in diverse inflammatory disease states, including retinal diseases. This review discusses how currently known pyroptotic cell death pathways have been implicated in models of age-related macular degeneration, diabetic retinopathy, and glaucoma. We also identify potential future therapeutic strategies for these retinopathies that target drivers of pyroptotic cell death. Presently, the drivers of pyroptosis that have been studied the most in retinal cells are the nucleotide-binding and oligomerization domain-like receptor family pyrin domain-containing 3 (NLRP3) inflammasome, caspase-1, and gasdermin D (GSDMD). Targeting these proteins may help us develop new drug therapies, or supplement existing therapies, in the treatment of retinal diseases. As novel mechanisms of pyroptosis come to light, including those involving other inflammatory caspases and members of the gasdermin protein family, more targets for pyroptosis-mediated therapies in retinal disease can be explored. Traditionally, apoptosis was equated to PCD because it was the most well-studied and well-characterized form of PCD. Research from the 1990's suggested that apoptosis was the main mechanism of regulated cell loss in retinal degeneration and this remained a popular view for most of the 21 century . The Nom century . Necropt century . Ferropt century . Finally century –9. As th century ; howeverSalmonella-infected macrophages were found to undergo a caspase-1-dependent form of cell death that was associated with pore formation and was distinguishable from apoptosis  and DFNB59  in humans. Gasdermin proteins share 45% sequence homology, and all members (except for Pejvakin) contain C-terminal and N-terminal domains , 15. TheMultiple different mechanisms can lead to gasdermin cleavage in pyroptosis. The two most well-studied mechanisms are the canonical and non-canonical inflammasome pathways. The canonical inflammasome pathway, leading to canonical pyroptosis, is mediated by caspase-1. Inflammasomes are multimeric protein complexes, composed of a central sensor protein, an adaptor protein ASC (apoptosis-associated speck-like protein containing a caspase activation and recruitment domain), and pro-caspase-1. The most well-studied sensor proteins known to assemble canonical inflammasomes are NLRP1, NLRP3, NLRC4, AIM2, and pyrin , 24. Othin vitro and in vivo studies showed that pyroptosis could be induced by GSDME expression and cleavage of GSDME into GSDME-N by caspase-3  is the most common cause of irreversible vision loss among the elderly in the developed world, and is projected to affect 288 million people globally by 2040 . AMD is Alu RNA, non-coding RNA transcripts expressed by the Alu retrotransposon, in RPE from human donor eyes with GA. Alu RNA accumulation, in turn, resulted in RPE degeneration in both humans and mice  restored susceptibility to Alu RNA-induced RPE degeneration. Full-length GSDMD cannot induce pyroptosis; thus, it must exert its effects on Alu RNA-induced RPE toxicity through another mechanism. Administration of mature IL-18 to Gsdmd−/− mice restored Alu RNA-induced RPE degeneration and led to the appearance of annexin V positive, propidium iodide (PI) negative staining RPE cells. This suggested that GSDMD plays a role in Alu RNA-induced cytotoxicity via IL-18-dependent apoptosis, rather than pyroptosis, in RPE , a component of drusen. After the NLRP3 inflammasome was activated by repeated intravitreal injections of Aβ into the eyes of Long-Evans rats, RPE-choroid protein lysates from Aβ-injected animals showed significantly increased levels of GSDMD-N and decreased levels of full-length GSDMD . This suThere is also evidence that GSDME-mediated, rather than GSDMD-mediated, pyroptosis occurs in RPE in the all-trans retinal (atRAL) model of AMD. atRAL is generated during the visual (retinoid) cycle and can accumulate in visual cycle anomalies, causing RPE atrophy in AMD. Cleavage of GSDME was detected at 6 and 12 h in lysates of atRAL-treated ARPE-19 cells but GSDMD remained intact, suggesting that atRAL triggers pyroptosis in ARPE-19 cells by activating the caspase-3/GSDME pathway of pyroptosis . ResearcDiabetic retinopathy (DR) is a leading cause of preventable vision loss in working-age adults and can be broadly classified into two clinical stages: non-proliferative diabetic retinopathy (NPDR) and proliferative diabetic retinopathy (PDR) . Early iin vivo and in vitro models of DR , the neurons that communicate visual information from the retina to the brain . It is aTLR4−/− mice reduced inflammasome production and RGC death after ischemic injury  using c injury . TLR4 dec injury  also shoc injury . In suppc injury . Thus, bc injury . Aside fxon loss . These fCasp1 inhibitor (Z-YVAD-fmk) markedly reduced cleavage of IL-1β and GSDMD, and restored RGC numbers during RIR injury. Furthermore, genetic deletion of GSDMD significantly increased retinal thickness and decreased RGC death after RIR injury  development and subretinal hemorrhage compared to wild-type (WT) mice in a laser-induced model of wet AMD. Furthermore, intravitreal injections of IL-18-neutralizing antibodies after laser-induced CNV resulted in significantly increased CNV development in WT mice, suggesting that IL-18 may protect against CNV through the downregulation of VEGF. Indeed, treatment with IL-18 significantly decreased the amount of VEGF secreted by human ARPE-19 cells as well as mouse brain microvascular endothelial cells (hyper mice (We know that inflammasome activation and release of inflammatory cytokines are associated with pyroptotic cell death; thus, we may expect that inhibiting these factors would have a protective effect in retinal disease. On the contrary, al cells . Therefoper mice . In 2014per mice  further per mice  also shoper mice . This shWhile the above research is promising, it has been met with some controversy. Tarallo et al.  found thIn this review, we have outlined the role of pyroptosis as a gasdermin-mediated inflammatory form of PCD in three common retinal diseases–age-related macular degeneration, diabetic retinopathy, and glaucoma. In AMD, GSDMD-mediated pyroptosis appears to occur in RPE when triggered by lysosomal destabilization or Aβ while GSDME-mediated pyroptosis occurs in the atRAL model of AMD. The research on pyroptosis in DR is in more preliminary stages, with most of its evidence for pyroptosis being limited to inflammasome activation rather than gasdermin activation in endothelial cells, pericytes, Müller cells, and RPE. Finally, there is support for gasdermin involvement in RGC loss in acute glaucoma, but evidence in chronic glaucoma models remains in its infancy. All in all, as our understanding of pyroptosis has grown and evolved, there is more support for its involvement in retinal disease. However, there are still many limitations in our understanding of pyroptosis in retinal disease that must be addressed.Firstly, the involvement of pyroptosis in retinal disease does not exclude the occurrence of other forms of PCD such as apoptosis, ferroptosis, and necroptosis. There is substantial evidence for the involvement of these other PCD pathways in retinal disease as well , 66, 92.We also need to be wary of the limitations in how we interpret the existing literature on pyroptosis in retinal disease. Because the essential role of gasdermin in pyroptosis was only recently established in 2015, earlier research on pyroptosis in retinal disease could only aim to identify inflammasome and caspase-1 activation in these diseases. We now know that activated inflammasomes can cause caspase-1 to cleave and release inflammatory cytokines without resulting in cell death , 28. ThuMZ sourced and analyzed the referenced literature, conceived the structure of the manuscript, and wrote the manuscript. SL was a significant contributor in reviewing the drafts of the manuscript, adding to section “Pyroptosis and Glaucoma” of the manuscript, and creating supplementary figures. JM was a significant contributor in refining the topic of the review, conceiving the structure of the manuscript, reviewing the drafts of the manuscript, creating supplementary figures, and providing funding for the study. All authors have read and approved the final manuscript.Authors acknowledge funding from Canadian Institutes of Health Research, Natural Science and Engineering Research Council, and National Institutes of Health Research.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.


Baseline post-exercise energy intake was associated with weight change over two years , but not with change in fat mass (p = 0.11). Appetitive traits were not associated with weight or body composition change (p > 0.22). State appetite cues were the strongest predictors of post-exercise energy intake, independent of body size. Future studies should examine whether long-term exercise programs enhance responsiveness to homeostatic appetite signals in youth with overweight and obesity, with a goal to reduce excess energy intake and risk for weight gain over time.Exercise may sensitize individuals with overweight and obesity to appetitive signals , overriding trait eating behaviors that contribute to overeating and obesity, such as uncontrolled eating. The objective of the current study was to measure predictors of objective ad libitum energy intake at a laboratory-based, post-exercise test-meal in adolescents ranging in weight status from overweight to severe obesity. We hypothesized that appetitive states, rather than appetitive traits, would be the strongest predictors of energy intake at a post-exercise test-meal, after controlling for body size. At Baseline, 30 adolescents , 43% non-Hispanic white (NHW), 83% with obesity (OB)) completed state and trait appetite measures and an ad libitum dinner meal following intensive exercise. Nineteen of those participants  completed identical assessments two years later (Year 2). Energy intake  at each time point was adjusted for fat-free mass index . Adjusted energy intake was reliable from Baseline to Year 2 (ICC = 0.84). Multiple pre-meal appetite ratings were associated with test-meal energy intake. In stepwise linear regression models, pre-meal prospective food consumption was the strongest and only significant predictor of test-meal energy intake at both Baseline (R Studies in youth with obesity suggest that there are trait-level differences in eating behavior  compared to healthy weight youth, leading to a vicious cycle of overeating and obesity ,2,3. ComBeyond trait-level eating behaviors, individual differences in appetitive states  and sensitivity to fluctuations in appetite can influence food-seeking behavior and energy intake ,16. AppeEnergy needs vary based on body size, growth, and physical activity levels, and are common drivers of energy intake. Fat-free mass is an indicator of body size and a primary determinant of resting metabolic rate and total energy expenditure, therefore contributing significantly to daily energy needs. Several studies have documented fat-free mass as a predictor of meal size, single meal food intake, and energy intake over the course of a day in both adults and children ,28,29,30The objective of the current study was to measure predictors of objective ad libitum energy intake at a post-exercise, laboratory-based test-meal in adolescents ages 10–16 years ranging in weight status from overweight to severe obesity. We investigated the contributions of both appetitive traits and states to post-exercise intake, independent of the effects of body size. We collected identical measures at a two-year follow-up visit to assess reliability and replicability of appetitive profiles and associations with post-exercise energy intake. We also tested whether baseline energy intake, appetitive states, and appetitive traits were associated with the change in adolescents’ weight and body fat over the two-year period. We hypothesized that appetitive states, rather than appetitive traits, would be the strongest predictor of energy intake at a post-exercise test-meal, after controlling for body size. We also hypothesized that baseline energy intake and appetitive traits would be positively associated with the change in weight and body fat over time.Study information was provided to potential participants at an orientation visit for a larger, prospective cohort study (NCT02784509) ,33, duriIf interested, parents and children signed an additional informed consent and written assent, respectively, for the ancillary study procedures at their baseline clinic visit. During this initial clinic visit, participants underwent a fasting blood draw and assessment of vital signs, anthropometrics to verify BMI percentile, dual energy x-ray absorptiometry (DXA) for body composition, and self-reported pubertal development. Participants also received a brief introduction to the cycle ergometer test with exercise testing staff. After completion of screening procedures at the initial clinic visit, the following additional eligibility criteria were assessed: ability to understand instructions and complete all study procedures; meeting physical (height and weight) restrictions for the cycle ergometer; no anemia; no pregnancy; not consuming a restrictive diet; no food allergies or intolerances; willing to consume test-meal foods; no contraindications to exercise testing ; and no Eligible participants returned 7–21 days later for an ancillary visit where cardiorespiratory fitness testing, questionnaires, behavioral tasks, and the ad libitum dinner test meal were completed. Recruitment continued until the target sample size of 30 participants was enrolled and completed study procedures. Families were contacted by phone once every 6 months for retention purposes. Year 2 follow-up visits for the parent longitudinal cohort study and this ancillary study were conducted within a ±6-month scheduling window depending on the family’s schedule and availability.Participants arrived at their clinic visits for the parent longitudinal study after an overnight fast (12 h), confirmed by parental report. The ancillary visits were conducted in the late afternoon  after a 3-h fast, confirmed by parental and self-report. The after-school timeframe was chosen to best accommodate family schedules, but also represents a time of day when youth this age would be most likely to engage in structured physical activity followed by an eating occasion. The timeline of study assessments is shown in 2) was calculated as FFM (kg) divided by height (m) squared and used as the indicator of body size [Height (cm), weight (kg), and waist circumference (cm) were measured at the clinic visit by trained staff according to standard clinical procedures. These values were used to calculate BMI and age- and sex-specific BMI percentiles . Adolescody size .Cardiorespiratory fitness (VO2peak) was determined with a graded cycle ergometer test with the use of standard open-circuit metabolic cart  until volitional fatigue. This protocol was designed to measure steady-state responses to exercise at multiple submaximal stages leading up to a maximum workload . Prior tTrait measures were assessed once at each time point, prior to the ad libitum test meal . The measures described herein were selected to capture ingestive behavior constructs of interest in obesity and weight regulation, including disinhibition, cognitive restraint, emotional eating, and food reward. Participants completed the Three-Factor Eating Questionnaire revised 18-item version 2 (TFEQ-R18v2) with subscales for Uncontrolled Eating, Cognitive Restraint, and Emotional Eating ,39. ThisTo assess disinhibition more generally, in addition to food-specific disinhibition , participants also completed a go/no-go inhibitory control task in E-Prime (version 2.0). The go/no-go tasks involve responding to a certain stimulus  and refraining from responding to another stimulus  . This taState measures were taken at multiple time points throughout each visit, including pre-exercise, post-exercise, pre-meal, and post-meal. The measures described herein were selected to capture subjective appetite and food wanting as potential drivers of food intake. For the purposes of this analysis, we only report the pre-meal appetitive state and explicit wanting outcomes as the most proximal to the initiation of the ad libitum test-meal.We assessed appetitive states with standard 100 mm visual analog scales (VAS): hunger, fullness, desire to eat, prospective food consumption, and satisfaction . These sIn addition to overall appetite ratings, explicit wanting for specific foods  was also assessed by VAS. Participants were instructed to look at each food image and then indicate whether or not they are familiar with that food. If yes, they were asked to rate how much they want to eat that food on a sliding scale with anchors marked “not at all” on the left and “want very much” on the right. Average explicit wanting was calculated for each time point. Data were collected and managed using REDCap.Objective energy intake  was measured at an ad libitum, multi-item dinner test-meal that occurred approximately one hour after the exercise test. Participants were offered standardized portions of common and familiar foods for this age group, including macaroni and cheese, chicken nuggets, broccoli with butter, and chocolate chip cookies with ketchup and barbeque sauce for dipping and water to drink see . Foods wThe following statistical approaches were applied to data collected at Baseline and Year 2, separately. Energy intake  was adjusted for FFMI by taking the unstandardized residual of the linear regression between the two variables. Pearson’s correlations were used to test associations between energy intake (adjusted for FFMI) with trait variables  and state variables . Significant correlates were entered into a stepwise linear regression model to determine the strongest profile that would predict energy intake (adjusted for FFMI) as the dependent variable, controlling for age and sex where appropriate. Retention of covariates was determined by stepwise linear regression, and age and sex were non-significant and therefore not included in any final models.p < 0.05.Intra-class correlation coefficients were computed using the two-way mixed procedure with absolute agreement. One-sample t-tests were used to examine changes in energy intake, body weight, and fat mass from Baseline to Year 2. In longitudinal analyses, linear regression models were used to test the effects of baseline energy intake at the post-exercise test-meal (adjusted for FFMI) on the change in weight and fat mass from Baseline to Year 2. Similarly, we tested the change in energy intake (adjusted for FFMI) from Baseline to Year 2 as the independent variable. Analyses were conducted in SPSS  and results were considered significant at p = 0.61), sex (p = 0.71), or weight status (p = 0.62) at baseline.Characteristics of the study sample are described in p = 0.002), they did not differ from the other participants in terms of change in weight, fat mass, or energy intake over time , nor was the time between visits associated with any of these outcome measures . Therefore, all analyses were conducted using the full sample.An average of 22.2 ± 3.7 months passed between visits. Four participants completed follow-up assessments in the summer of 2020, following the end of local stay-at-home orders and clinical research suspension at the start of the COVID-19 pandemic. While the time between visits for these four participants was significantly longer compared to the rest of the study sample . In this same time frame, they showed a significant increase in fat mass  and in FFMI . From Baseline to Year 2, there were no significant changes in test-meal energy intake .Participants experienced a significant increase in body weight over the two-year period  was 0.84, indicating good reliability over time. The correlation between Baseline and Year 2 is depicted in The intra-class correlation for energy intake (adj. for FFMI) at Baseline and Year 2 (p < 0.05). Pre-meal fullness  was negatively associated with test-meal energy intake (adj. for FFMI). There was a trend for pre-meal food wanting to be associated with test-meal energy intake . None of the go/no-go task outcomes  were associated with test-meal energy intake, regardless of whether it was a food-motivated (win a favorite or least favorite snack) or neutral condition . In stepwise linear regression, pre-meal prospective food consumption was the strongest and sole predictor of test-meal energy intake  included in the model , regardless of age (p = 0.69) and sex (p = 0.37).Correlations are presented in p < 0.05). Pre-meal food wanting was not associated with test-meal energy intake (p = 0.75). None of the TFEQ subscale scores, nor the go/no-go task outcomes, were associated with energy intake . In stepwise linear regression, pre-meal prospective food consumption was the strongest and sole predictor of test-meal energy intake (adj. for FFMI)  in the final model .Correlations are presented in p = 0.04) was positively associated with the change in body weight (kg) . None of the other baseline appetitive states or traits were associated with the change in body weight or fat mass .Baseline test-meal energy intake (adj. for FFMI) , nor with the change in fat mass (p = 0.22) over the two-year period.The change in test-meal energy intake (adj. for FFMI) from Baseline to Year 2 was not significantly associated with the change in body weight  was positively associated with actual consumption, regardless of body size. This pattern of association was replicated at the two-year follow-up. Baseline post-exercise energy intake (adj. for FFMI) was associated with the change in weight over two years. However, contrary to our hypothesis, baseline appetitive traits were not associated with change in weight or body fat.Test-meal energy intake adjusted for body size was consistent over the two-year period, with a strong correlation between residuals from Baseline and Year 2. This suggests that the homeostatic drive is retained despite the presence of obesity and the effects of growth and weight gain. At Baseline, approximately 25% of the variability in energy intake (controlling for body size) was explained by prospective food consumption. This increased to 41% of the variability at Year 2. Prospective food consumption is a subjective rating of the amount of food a person thinks they can eat at a particular moment. In this study, we used VAS ratings taken approximately 1 h after exercise, immediately before the test-meal. Participants with a higher rating of pre-meal prospective food consumption did consume more calories at the subsequent meal, suggesting an intentional awareness regarding the amount or portion size they planned to eat. This is counter to previous reports in people with obesity that intake of an objectively large amount of food is often driven by a loss of control over intake during an eating occasion ,14,44. TDespite weight gain over the course of the two-year period, these eating behavior profiles were preserved. In other words, weight gain over time did not result in a reduction in eating behaviors  that predispose individuals to additional weight gain. Adolescents who ate more food in relation to their body size continued to do so two years later. Studies in adults have posited that weight gain propensity should be diminished once weight gain has occurred, at least from a metabolic perspective . HoweverContrary to previous studies, we did not find an association between appetitive traits  and weight gain over time. This could be due to the fact that we used versions of these assessments that were adapted for children and adolescents and therefore may have captured slight differences in cognitive constructs of interest. We used a food motivation manipulation in the go/no-go task, but performance  across trials was not affected by the presence of food rewards (data not shown). For this reason, we treated the go/no-go task as a trait measure of disinhibition. Go/no-go task performance was not associated with energy intake, eating behaviors, or weight gain in this study sample. The revised 18-item version of the TFEQ that we used has only one overlapping subscale (cognitive restraint) with the original version developed by Stunkard and Messick . UncontrStrengths of the current study include the balanced racial and ethnic diversity of the study sample, as well as the representation of adolescents ranging in weight status from overweight to severe obesity. In addition, we assessed several state and trait eating behavior constructs in relation to an objective ad libitum test-meal after controlled exercise. Limitations of the current study include the small sample size and loss of participants at the Year 2 follow-up. However, the reliability of our eating behavior assessments and replication of correlations increases our confidence in the effects seen. Future larger studies could examine potential differences in post-exercise eating behaviors by race/ethnicity, age, or sex. We were unable to test the direct impact of reproductive hormones and physiological maturation on our study outcomes. However, self-reported Tanner stages collected for this study were strongly associated with both age and FFMI (data not shown), which we did account for in our analysis. A future study designed specifically to address the impact of the pubertal transition on eating behaviors and weight gain would be of interest. Finally, we also lacked a non-exercise control condition, and the exercise itself was not standardized in terms of duration nor energy expenditure. The current study also did not examine multiple exercise intensities. Future studies could examine differences in predictors of energy intake between various exercise and non-exercise conditions. This line of work could further support the hypothesis that exercise sensitizes individuals to appetitive state and determine the optimal exercise parameters  needed for such an effect . AdditioIn sum, we found that appetitive state was the strongest predictor of post-exercise energy intake and that energy intake at Baseline predicted two-year weight gain. Future studies could examine whether post-exercise prospective food consumption is modifiable through cognitive behavioral strategies, with a goal to reduce compensatory food intake and risk for weight gain over time.


Cancer studies reported mixed results on benefit finding (BF) and posttraumatic growth (PTG) prevalence and few were focused on long-term survivors.BF and PTG were assessed in a multi-regional population-based study in Germany with 6952 breast, colorectal and prostate cancer survivors, using the Benefit Finding Scale and Posttraumatic Growth Inventory. We calculated the age-adjusted prevalence, stratified by demographical and clinical characteristics.Overall, 66.0% of cancer survivors indicated moderate-to-high BF, and 20.5% moderate-to-high PTG. Age-adjusted prevalence of BF and PTG differed according to cancer type  and sex . BF and PTG prevalence were higher in younger than in older respondents; the age-adjusted prevalence was higher in respondents who survived more years after diagnosis. The strength and direction of associations of age-adjusted prevalence with cancer stage, disease recurrence, and time since diagnosis varied according to cancer type and sex.A substantial proportion of long-term cancer survivors reported moderate-to-high BF and PTG. However, the prevalence was lower in older and male cancer survivors, and during the earlier years after cancer diagnosis. Further longitudinal studies on PTG and BF in cancer survivors are warranted to address heterogeneity in survivors’ experience after cancer diagnosis. Cancer, as a life-threatening illness, has been recognised as a traumatic event . HoweverA challenging/traumatic event like cancer is crucial to trigger BF or PTG , 5. CancDue to the increasing prevalence of cancer, it is crucial to better understand the outcomes of cancer-related stress and coping, such as BF and PTG. Most of the pertinent studies on the prevalence of BF and PTG have focused on heterogeneous cancer types and had mixed results , 20. TheThe study population came from the CAESAR study  which was conducted by the German Cancer Research Center  in cooperation with population-based cancer registries in Germany. Participants in this study were registered in one of six population-based cancer registries in Germany , Rhineland-Palatinate, and Saarland) .Details of the recruitment of study participants including data collection have been described elsewhere . In brieThe questionnaire used in CAESAR study included questions regarding demographic and clinical factors. Clinical data such as cancer type, cancer stage, year of birth, and year of diagnosis were provided by the cancer registries. Disease recurrence was self-reported and is defined as any recurrence, metastases or new cancer after index cancer. Respondents’ age was calculated by deducting year of birth from year of survey; years since diagnosis equate to the year of survey minus the year of diagnosis.Benefit finding (BF) was measured by the German short form of the Benefit Finding Scale (BFS) . The oriPosttraumatic growth (PTG) was assessed by three subscales  of the German version of the Posttraumatic Growth Inventory (PTGI) . Two othThe intensity levels of BF and PTG were determined by the mean item score. The mean item score was calculated as the mean of non-missing items if at least half of the items had been completed, which was then dichotomised using a cut-off of 3 (indicated as ‘moderate’ according to a previous report ) into ‘nMultiple imputation with 25 repetitions was employed to handle missing values by using the Markov chain Monte Carlo method to reduce possible bias and increase precision of the replaced values . Before 2-tests were used to evaluate subgroup differences. A two-tailed P-value <0.05 was considered statistically significant.In the description of characteristics of the total study population, and by cancer type and sex, respondents’ were grouped according to age at survey: <60 years, 60–69 years, 70–79 years, or 80–89 years; and to their time since diagnosis: long-term cancer survivors  or very long-term cancer survivors . Age-adjusted prevalence of moderate-to-high BF and PTG by cancer type and sex were calculated according to the direct method using the age-specific proportions from the total sample described previously as standard population weights. Distributions of moderate-to-high BF and PTG prevalence according to age groups in cancer survivors overall and by cancer type and sex were also estimated. Detailed age-adjusted prevalence of moderate-to-high BF and PTG by characteristics of the study population on overall scale level were calculated accordingly, stratified by cancer type and sex. ChiThe imputed data was comparable to original data Table . From thP < 0.0001). Stratifying all survivors by sex, the age-adjusted prevalence was statistically significantly higher in female than in male cancer survivors, both for moderate-to-high BF  and moderate-to-high PTG .Overall, 66.0% of all survivors indicated moderate-to-high BF (95%CI: 65.8%–66.2%) and 20.5% moderate-to-high PTG (95%CI: 20.3%–20.7%) Fig. . SimilarPattern of age-specific prevalence: a significant trend by age was noted in which older survivors reported lower prevalence of moderate-to-high BF and PTG. When stratified by cancer type, the same pattern was observed in breast cancer survivors Figs.  and 3. CP < 0.0001) and PTG (P < 0.0001) as compared to stage I/II survivors . This pattern was found in all tumour groups but reversely in prostate cancer survivors  but positively associated with the prevalence of moderate-to-high BF in female colorectal cancer survivors. Time since diagnosis was positively associated with the prevalence of moderate-to-high PTG in breast and female colorectal cancer survivors but inversely associated in male colorectal cancer survivors (P = 0.0005).Regarding time since diagnosis, LTCS showed a significantly lower age-adjusted prevalence of moderate-to-high BF Fig.  and modeBased on data from a multi-regional population-based study, our study contributes detailed information on the prevalence of BF and PTG according to demographic and disease characteristics. When adjusted for respondents’ age, our results suggest that the prevalence of BF and PTG differed by cancer type  and sex . Generally, the prevalence of moderate-to-high BF and PTG is lower in older cancer survivors compared to younger age groups; the age-adjusted prevalence of moderate-to-high BF and PTG was higher in VLTCS than in LTCS. Cancer stage, disease recurrence and time since diagnosis were also associated with the prevalence of BF and PTG, although the strength and direction of the association varied according to cancer type and sex. To our knowledge, our study is the largest one to address the role of time since diagnosis on the prevalence of moderate-to-high BF and PTG in cancer survivors 5–16 years after diagnosis, with sufficient power to describe the prevalence by cancer type and sex in detail.Age, in general, had an inverse relationship with PTG and BF, which is comparable to previous studies , 30. TheIn our study, the prevalence of moderate-to-high PTG ranged between 16 and 24%. These rates are lower compared to previous studies in a recent review . It is pFollowing adjustment by respondents’ age, our study found prevalence differences in BF and in PTG according to sex and cancer type. Reflecting the sex distribution of the cancer types, the highest prevalence rates of BF and PTG were noted in breast cancer survivors, and the lowest prevalence in prostate cancer survivors in this study. The sex difference  also existed in colorectal cancer survivors. Life cycle events, physical changes, and developmental maturity associated with aging could also differ by sex. Females experience more stressful life events with physical changes, like pregnancy, giving birth or (peri)menopause , 35. TheCancer stage may also contribute to differences in the prevalence of PTG and BF. The lower chance of cancer-free survival associated with more advanced cancer stage could increase the sense of hopelessness and perception of illness in a negative way . As a reWe found that long-term cancer survivors, with or without recurrence, reported comparable prevalence of PTG, but those with cancer recurrence/metastasis generally had a higher chance to perceive BF. Previous studies also reported that long-term survivors with adolescent cancer who had recurrence/metastasis did not significantly differ in terms of their amount of PTG from those who did not experience such an episode , 39. HowWhile previous research only included short-term survivors (within 5 years) , 40, we Although our reports were from a population-based study, which allowed the detailed stratification according to several factors, several limitations still need to be noted. Firstly, we acknowledge that potential response bias could exist because of an over-selection of ‘fitter’ survivors who were able to complete the questionnaire or were more interested/motivated to give feedback. Secondly, there is a limited generalisation with regard to sex differences as we only looked at selected cancer types. Nevertheless, these three cancers are the most common cancers for males and females . Another factor related to cancer type is that the age distribution of samples differed between the cancers. To address this age difference, we reported age-adjusted prevalence when we stratified the groups by cancer type and sex. Thirdly, we did not use full instruments because we wanted to reduce participant burden by excluding overlapping subscales. However, the mean ratings of two excluded subscales of PTGI reported in other studies , 43 wereFuture studies on the prevalence of BF/PTG in cancer survivors need to address the abovementioned limitations. For example, they could use full instruments and include more types of cancers to verify the results. Future prospective studies should ideally assess baseline BF/PTG before the onset of cancer or at least at diagnosis and longitudinal studies with repeated BF/PTG assessments could verify the association between time since diagnosis and BF/PTG. Additional studies are also needed to test cognitive perception of illness severity in processing of BF and PTG.We found that moderate-to-high BF and PTG are common in cancer survivors 5–16 years after diagnosis and that the prevalence varies with survivors’ age, sex, cancer type, stage of disease, recurrence/metastasis, and time since diagnosis. Our results highlight the heterogeneity in survivors’ experience after cancer diagnosis. Prevalence of BF and PTG was lower in older and male cancer survivors, during the earlier years after diagnosis and in survivors with more advanced cancer diagnosis. Further longitudinal studies on PTG and BF in cancer survivors are warranted to address heterogeneity in survivors’ experience after cancer diagnosis. Better identification of vulnerable survivors, e.g. taking demographic and clinical factors into consideration, could assist in tailoring interventions appropriate to their needs.supplemental material


Participants reported enjoying feedback and video-based engagement during pedaling, with 83% of participants feeling that engagement motivated them to perform pedaling-based exercise. In conclusion, real-time cadence control feedback and video-based engagement during pedaling for healthy individuals may improve performance in targeted pedaling tasks. Through dissociation from the physical cues associated with exercise and fatigue, feedback and engagement may ultimately increase enjoyment and exercise compliance and adherence of pedaling-based exercise. The findings may be useful in prescription and maintenance of targeted pedaling exercises for stroke rehabilitation and exercise therapy.The use of video and music as an intrinsic, dissociative attentional stimulus during exercise is thought to distract from the physical discomfort of exercise, and contribute to improved exercise adherence; however, the effects of video-based feedback and engagement during pedaling on exercise performance and motivation are poorly understood. The aims of the present study were twofold. Firstly, to develop a novel video-based engagement regime for pedaling that links pedaling cadence with the play rate of a video, and secondly, to employ an instrumented pedaling device to assess the influence of the video engagement paradigm on cadence performance and exercise motivation. Eighteen healthy subjects participated in 15-min-duration pedaling sessions while targeting a specific low cadence (60 rotations per minute) and high cadence (100 rotations per minute), including pedaling with the provision of (i) target pedaling cadence information only, (ii) visual feedback on cadence control, including pedaling duration, pedaling cadence, and cadence deviation from target, and (iii) real-time engagement, which involved pedaling at the target speed to maintain the playback rate of a pre-recorded video. Cadence deviation from the target was evaluated, and self-reported exercise motivation examined with a post-exercise survey. Pedaling-cadence deviations significantly reduced with cadence feedback at both low and high cadence ( Pedaling is an activity used in lower-limb rehabilitation and training to improve aerobic capacity and cardiopulmonary function. It provides similar periodic muscle activation patterns to those during walking ,2,3, andObjective, real-time measurement and display of exercise performance during cycling, including cadence, cadence variability, power, distance and duration of the exercise, has been shown to increase exercise performance ,15,16,17Viewing videos during pedaling tasks has been shown to contribute to higher peak pedaling speeds for a given blood lactate level compared to pedaling without video interaction due, in part, to an altered perception of effort . In a stEighteen healthy individuals were recruited for testing  from the University of Melbourne. The participants had no history of lower limb pain, dysfunction or previous lower-limb surgery. All were non-smokers and had varying level of physical activity in their lives . This subject number was chosen as a sample of convenience, based on a previous pedaling study of similar sample size that detected significant differences in SpO2 with exercise . Ethical approval was obtained from the Western Health Ethics Advisory Group , and written, informed consent was obtained.Each subject was seated at a custom-designed pedal apparatus that provided real-time cadence performance feedback and video engagement during pedaling. The pedaling apparatus comprised a seat, pedals, a cadence data recorder, and a computer with a display positioned at head-level. The seat and pedal crank position were adjusted to each subject’s lower limb lengths for optimal knee flexion and comfort. A magnet and hall-effect sensor positioned on the crank was used to measure pedaling cadence and cadence using an open-source microcontroller board , and the output validated using an external pulse generator circuit. Pedaling cadence feedback data were provided by transmitting the following data in real-time to the PC using a custom exercise monitoring program : cycle count, pedaling cadence, duration, target cadence, and deviation from target cadence. The software program also facilitated real-time video engagement by displaying a video of the participant’s choice from a collection offered by the researchers . This reAll testing occurred at a standardised time in the afternoon (between 2 pm and 4 pm), and participants were requested not to consume any substances that may affect their performance in the 24 h prior, including alcohol or caffeine. Participants were given approximately one minute to familiarise themselves with the apparatus before testing. A series of three randomly allocated 15-min testing sessions of pedaling were then undertaken during which the subject was asked to maintain their pedaling at a target low cadence (60 RPM). Three testing conditions were established to evaluate the influence of real-time feedback of cadence and video-based engagement on cadence performance, physiology and motivation during pedaling, specifically, (i) base-line pedaling control case in which pedaling cadence feedback data were provided only in the first 20 s of each trial to allow participants to ‘establish’ their cadence, (ii) real-time visual feedback of cadence performance, including pedaling duration, pedaling cadence, and cadence deviation from target, and (iii) real-time engagement, which involved pedaling to control the playback speed of an audio-visual video (film) synchronised to the target cadence. The entire pedaling task, including the three random allocated testing conditions, was then repeated at a target high cadence (100 RPM). For all tests, the pedaling resistance was set at a moderate level, approximately half of the maximum. This was defined by a pedal load of 8.3N, producing a torque of 0.872 Nm, which was just sufficient to overcome static resistance and begin to move the crank, which was of length 10.5 cm. Rest periods of 30 s were provided every 5 min.Pedaling cadence was recorded throughout each trial, as well as the mean-cadence deviation from target, defined by the difference between the target cadence and the mean pedaling cadence of the session. Absolute cadence deviation was evaluated and defined as the absolute difference between individual cycle cadence and target cadence. Following testing, each participant was asked to complete a survey questionnaire  that evat-tests and Games–Howell tests for groups with unequal variances. CV and standard deviation were computed and used as a measure of the dispersion of data. Level of significance was defined at p ≤ 0.05. To demonstrate the influence of the feedback and engagement interventions during pedaling, a comparison between intervention group and baseline pedaling was drawn and effect size was computed for each variable  were used to assess the effect of intervention type  on cadence parameters while pedaling at slow and fast cadences. The cadence control dependent variables were mean-cadence deviation, coefficient of variation (CV) in mean-cadence, absolute cadence deviation and CV absolute cadence deviation. Analysis of variance was also used to assess interactions between the independent variables. Levene’s test for homogeneity of variances was performed, and a test for data normality was undertaken. Post hoc tests were undertaken using paired variable .p < 0.001), mean-pedaling-cadence deviation (p = 0.032) and CV (p < 0.001), while intervention type had a significant influence on mean-pedaling-cadence CV (p = 0.005), absolute pedaling-cadence deviation, and CV (p < 0.001) (p > 0.05). Reduction in the absolute pedaling-cadence deviation and its CV with cadence control feedback or video engagement intervention relative to baseline was dependent on pedaling cadence. For example, cadence control feedback significantly reduced the absolute pedaling-cadence deviation relative to baseline during low-cadence pedaling  (p = 0.002). Similarly, a significant reduction in absolute-cadence deviation was observed with video engagement relative to baseline, during low-cadence  as well as during high-cadence pedaling . One asterisk indicates p ≤ 0.05, two asterisks indicate p ≤ 0.01, while three asterisks indicate p ≤ 0.001.Pedaling cadence significantly affected the absolute-cadence deviation CV (< 0.001) . There w= 0.028) . A signiThe results showed that feedback and video-engagement helped the subject maintain the target 60 RPM cadence  Appendi with vidp = 0.029) and video engagement  relative to baseline pedaling. A similar trend was also observed during high-cadence pedaling. The CV of the absolute-deviation in pedaling cadence during low-cadence pedaling was significantly smaller with cadence control feedback  and video engagement  relative to that during baseline pedaling. Significant improvements in mean-cadence CV relative to baseline were observed with feedback  and engagement , but only during high-cadence pedaling.The CV of the absolute-deviation in pedaling cadence during low-cadence pedaling was significantly smaller with the provision of cadence control feedback , irrespective of the target cadence. Similar findings have been documented in virtual reality studies, with immersive artificial environments facilitating more constant pedaling cadence, as well as increased work output and time trial performance ,29,30. VThere are limitations of this study that ought to be considered. First, a limited set of videos were provided to participants during video feedback trials, and personal preferences as well as level of stimulation may have been a confounding factor in the engagement level. A specific selection of video clips were provided to assist in standardizing the testing protocol. Second, this study assessed within-subject differences due to performance feedback and video-based engagement during pedaling, with the reference (control) state defined by pedaling without feedback or engagement. Due to the nature of the experimental design, a cohort of control subjects was not required. Future studies ought to investigate the influence of video-engagement on rehabilitation in the clinical setting, including the response of patients with varying levels of cognitive function, and whether the findings observed translate to clinically meaningful outcomes. Finally, some of the mean differences in cadence between test conditions observed in this study were small, and their clinical relevance should be interpreted with caution.This study demonstrates that real-time visual feedback of cadence control as well as a novel video engagement protocol helps to improve targeted cadence pedaling performance by reducing pedaling cadence variability, which in the clinical setting, may ultimately help patients achieve targeted rehabilitation goals. These strategies may improve pedaling exercise effectiveness over a shorter time-period, though further research is warranted to quantify functional benefits, particularly over extended durations. Feedback and video-based engagement was also shown to increase exercise enjoyment and motivation. These findings suggest that feedback and video-based engagement may be useful in prescription and maintenance of targeted pedaling exercises for rehabilitation such as stroke therapy, and may ultimately help to improve exercise motivation and compliance.


Mycobacterium tuberculosis, which is difficult to rapidly diagnose and treat. There is an urgent need for new methods of diagnosis, sufficient in vitro models that capably mimic all physiological conditions of the infection, and high-throughput drug screening platforms. Microfluidic-based techniques provide single-cell analysis which reduces experimental time and the cost of reagents, and have been extremely useful for gaining insight into monitoring microorganisms. This review outlines the field of microfluidics and discusses the use of this novel technique so far in M. tuberculosis diagnostics, research methods, and drug discovery platforms. The practices of microfluidics have promising future applications for diagnosing and treating TB.Tuberculosis (TB) remains a global healthcare crisis, with an estimated 5.8 million new cases and 1.5 million deaths in 2020. TB is caused by infection with the major human pathogen  Mycobacterium tuberculosis is the causative agent of the human pulmonary infection tuberculosis (TB). TB is the second leading infectious killer since the global COVID-19 pandemic. The World Health Organisation (WHO) estimates that 1.5 million people died from TB in 2020 [ in 2020 . Additio in 2020  and WHO  in 2020 . There iM. tuberculosis is a rod-shaped acid-fast-staining bacterium of the Actinomycete family [M. tuberculosis contains a core composed of peptidoglycan and the highly branched polysaccharide arabinogalactan. This is covalently attached to the unique mycolic acids that cover the bacteria with a mycobacterial outer membrane which allows cellular integrity and virulence [M. tuberculosis evades the host and causes disease is complex, involving a dynamic range of immune cells. The organism infects the host after the inhalation of droplet nuclei spread by aerosolisation from an infected individual, which then resides in the respiratory tract [M. tuberculosis in individuals—one where the infection clears, one with an active infection treated with a course of antibiotics, and one which remains in a latent form [M. tuberculosis can avoid this host defence response by persisting in phagosomes and inhibiting lysosome fusion [e family . The uniirulence . This seirulence ,9. The mry tract . There aent form . Upon inent form . Throughent form . Howevere fusion . The sube fusion . The eare fusion . During e fusion ,15. M. tuberculosis can survive in a dormancy state known as non-replicating persistence (NRP). The external pressures such as hypoxia, nutrient deprivation, low pH, and high CO2 created by the hostile host environment induce this survival response of the bacteria [M. tuberculosis to effectively hide within the hostile environment of the granuloma is that not only does the immune system keep the bacteria trapped, it also physically restricts penetration by antimicrobials, thus protecting M. tuberculosis from antibiotic activity.Here, bacteria . The NRPbacteria . Houben bacteria . The issEarly diagnosis and accurate detection of TB infection is essential for effective treatment options, especially in low-income and high-burden countries. Conventional TB diagnostics include microscopy (Ziehl–Neelsen staining), which provides 22–43% low sensitivity for a single smear . Other mM. tuberculosis in the laboratory in the presence of different concentrations of antimicrobials, is traditionally used to detect drug-resistant strains. The turnover time for these results is extensive, by which point the patient’s health will have deteriorated [The phenotypic evaluation of clinical isolates, by culturing riorated . Improveriorated . Additioriorated . User-frExperimental modelling of TB has historically helped scientists to discover the pathogenicity, physiology, metabolism, and genetic make-up of the organism. Challenges arising for researchers studying mycobacteria are the characteristics of slow growth rate, hydrophobic aggregation of cells in the absence of non-ionic surfactant when grown in culture, and the need for the containment of aerosols which brings additional safety precautions, including a biosafety level 3 (BSL-3) facility . AdditioM. tuberculosis difficult. Efforts have been made to standardise testing; however, protocols are still time consuming [Research groups often use variable types of nutrient media, inoculum starting points, and reading endpoints, making the standardisation of antimicrobial testing for onsuming ,31. TB honsuming . There iM. tuberculosis research, such as new high-throughput methods of phenotypic assessment. Advancements in microtechnology, particularly at the micro and nanoscale, have had wide microbiological applications. Microfluidics is a rapidly growing field which comprises multidisciplinary expertise in biology, chemistry, physics, and engineering. A simple definition of microfluidics is the systematic manipulation of systems that have microscale channels where fluid volumes of nanolitres to attolitres can flow in geometric configurations [We require innovative and advanced technology to advance urations . Well su3) Different physical forces direct the behaviour of a fluid in a system. The essential behaviour of a hydrodynamic system and the dominant physical effects are typically analysed by characteristic, dimensionless numbers. These numbers compare the relative importance of competing forces or may be alternatively described as ratios of characteristic length, time, or energy scales. The most prominent number in microfluidics is the Reynolds number, describing the ratio of inertial forces and viscous forces : (1)Re=ρDue to the capacity of microfluidic systems and the corresponding slow flow velocities, the value of the number is typically hen Re<1 :(2)∇p+f=With the absence of turbulent flow, the mixing of parallel, laminar fluid flows in microfluidics only occurs by diffusion, which can be a slow process. The Péclet number (Pe) describes the ratio of the rates of convection and diffusion for suspended objects, and is given by :(3)Pe≡vIn Equation (4), Where mixing is desired, passive mixing can be introduced when designing channel geometries, such as ridges, network gradient generators, and vortex micromixers. Alternatively, active mixing can be introduced by external energy, for example, electrokinetic forces and thermal actuation . Active As the geometrical dimensions of a microchannel decrease, the fluidic resistance increases because of friction between the microchannel walls and the body of fluid. Generally, the surface area to volume ratio becomes larger as the channel geometry becomes more complex, and so does the fluidic resistance R=12μgiven by :(7)R=12μThe resistance in a microchannel with a circular cross-section can be calculated using:The study of multiphase flows, often termed droplet microfluidics, is a subset of the microfluidics field in which nano- to femtoliter volume droplets can be routinely generated by drop-making micronozzles in a carrier fluid at production rates exceeding 10 kHz . RecentlFor single-cell analysis applications using droplet microfluidics, liquid/liquid emulsions comprising a cell-friendly aqueous interior, and a surfactant-stabilised fluorous oil are often used. Inclusion of cells in the aqueous, dispersed phase, results in the encapsulation of individual cells within the emulsion. The droplet occupancy number can be controlled by altering the concentration of cells within the dispersed phase and calculated using Poisson statistics . To enabolutions :(9)Γ=−cRAs surfactant adsorbs to the interface, the interface rigidifies: the loss of mobility imposes a change in the boundary condition at the interface which slows it down. The origin of the rigidification is the so-called Marangoni effect: as a drop moves, the surfactant distribution is non-uniform, with an excess at the rear of the drop . The nonIn conjunction with the interfacial tension between the two phases, complex phenomena arise that are governed by various dimensionless numbers containing the surface tension. The balance of inertial, viscous, and interfacial tension forces govern droplet formation and subsequent droplet flow. The relationship between the inertial and interfacial tension forces of the aqueous phase is quantified by the Weber number :(10)We=ρy number : (11)Ca=e number :(12)Oh=WNumerous biomedical applications require materials such as solids or gels, and not liquids . Solid ppolymers . The WeiAnother relevant time scale As the flow velocity increases, elastic effects become more influential and y number  El=DeRe=The most common channel geometries used for microdroplet generation include the T-junction, flow-focusing, and co-flow nozzles, and step-emulsification devices , each wiDroplets can be produced hydrodynamically within a T-junction system in the squeezing, dripping, or jetting regimes, whereby ajetting . Using tA flow-focusing junction comprises two immiscible phases converging at a cross junction. The dispersed phase flows towards the junction in a single channel, and the continuous phase flows towards the junction in two diametrically opposed channels, each perpendicular to the dispersed phase . The disCo-flow droplet generators were first described by Cramer et al. , and utiStep emulsification generators  Figure  create dUsing an external input of energy can also dictate droplet generation, termed “active droplet generation” application of an external force can drive the creation of droplets. There are many techniques for active droplet formation including electrical ,68, magnDroplet sensing is important for the identification and/or manipulation of droplets, and for the automation of sequential droplet activities in microfluidic Lab-on-Chip devices and/or instruments. When performing time-dependent tasks such as the manipulation of specific droplets at a specific on-chip location, droplet sensing is crucial to ensure triggered actions have the correct timing. Further, as the number of manipulation events increases, the management and automation of droplet manipulation activities needs precise, reliable information about the location, size, frequency, velocity, and/or content of droplets at certain locations within the system . Two freThe efficient manipulation of droplets , i.e., tSome of the most frequently used materials in microfluidics include thermoplastics, polydimethylsiloxane (PDMS), inorganic materials such as glass or silicon, paper, and even devices made by 3D printing, a newer approach to fabrication . The mosSilicon micromachining was firstly developed for application within the field of microelectromechanical systems (MEMS) but was subsequently one of the first techniques to be used for the microfabrication of microfluidic systems . The welGlass has excellent analysis performance due to its biocompatibility, optical transparency, low fluorescence background, surface stability, and chemical resistance . HoweverSoft lithography is one of many techniques used to fabricate microfluidic chips, which has largely driven the use of PDMS as a commonly used microfluidic device material. By contact printing, replica modelling and embossing, soft lithography can be used to create micro-patterns . The proThermoplastics have been extensively researched, refined and used for the mass production of high-quality goods, since their initial industrial uses in the 1930s . VariousThe use and prevalence of paper-based microfluidics has increased significantly in recent years due to the compatibility of such devices in point-of-care or point of-use testing applications, plus their simplicity, fundamental low cost, biocompatibility, and hydrophilicity ,126. VarAn emerging microfabrication method which may overcome the limitations of prior microfluidics fabrication techniques is 3D printing, which enables the prototyping of devices at lower cost and fabrication time compared to techniques such as soft lithography or hot embossing . FurtherM. tuberculosis. Detecting the pathogen with robust and reproducible fluidic models offers capabilities for clinical procedures and scientific exploration. Interestingly, a bacteria enrichment microfluidic chip and a microfluidic immunoassay chip have detected airborne M. tuberculosis. Jing and colleagues (2014) validated a method whereby a micro-pump draws air containing bacteria into the enrichment fluidic chip and then a full immunoassay reaction is performed on a separate chip. The method offers the potential to accurately screen M. tuberculosis in the aerosol [M. tuberculosis currently requires long cultivation due to the low concentration in air samples. Capturing and directly detecting airborne M. tuberculosis will aid effective disease prevention and control as there is a requirement to detect samples directly from patients for quicker analysis. The small volume sizes in microfluidic chip cultivation provides rapid detection at lower sample concentrations. Diagnosing TB, especially in developing countries, requires low-cost point-of-care technologies. A paper-based microfluidics system detected sputum samples containing mycobacteria. The system used enabled the decontamination of non-mycobacteria and storage of the sputum sample [M. tuberculosis with high sensitivity within forty-five minutes. By creating an eight-chamber microfluidic electrochemical system with real-time loop-mediated isothermal amplification (LAMP), amplification of three respiratory related infections including M. tuberculosis could be monitored by measuring the electrochemical signal of methylene blue [Employing microfluidics is a promising approach for rapid and cost-effective diagnostics for  aerosol . Airbornm sample . A laserene blue . Here, aM. tuberculosis complex, were detected by combining a closed system of bead-beating, droplet fluidics, and surface-enhanced Raman spectroscopy. The spectral information obtained from the vibrational signals of the mycobacterial cell wall component, mycolic acid, effectively identified the different species. This is a promising step forward for ensuring the correct treatments are administered for the correct infections [M. tuberculosis in droplet microfluidics was developed by detecting cells that express the endogenous β-lactamase, BlaC—an enzyme marker naturally expressed by M. tuberculosis. By encapsulating a specific fluorescent probe of BlaC and samples of bacterial strains that express BlaC in droplets, the researchers could calculate the initial concentration of cells based on fluorescence [Six species of mycobacteria, including nontuberculous species and members of the fections . Small crescence .M. tuberculosis H37Ra with magnetic beads and differentiation of live/dead bacteria with propidium monoazide dye, followed by RT-PCR and optical detection within two hours. By measuring the threshold cycle number, a low detection limit of 14 colony-forming units per reaction was achieved [M. tuberculosis without the laborious need for PCR amplification has been achieved. For example, Domínguez et al. (2015) created a micro-cantilever platform, where hydration-induced stress could identify M. tuberculosis and RIF resistance within 1.5 h [Other researchers have combined PCR techniques with microfluidics. Ip et al. (2018) used a single chip comprising positive and negative reaction chambers, as well as small liquid handling chambers. They performed isolation of achieved . Besidesin 1.5 h . M. tuberculosis from clinical isolates was detected by an integrated system of microfluidics and electrochemical biosensing. The platform consisting of a monolithic chip and multiwall carbon nanotubes detects M. tuberculosis without the need for DNA amplification [M. tuberculosis. The protein is a biomarker for actively dividing mycobacteria, detected by electrochemical impedance spectroscopy and synthetic aptamers integrated with a microfluidic chamber [Previously, label-free DNA of fication . Another chamber . M. tuberculosis isolates and susceptible isolates by combining a microfluidic chip with post-PCR high-resolution melting analysis (HRMA). The authors’ “Light Forge” microfluidic DNA melting-based TB test showed better performance of melting temperature differences compared to conventional Sanger sequencing, as well as a HRMA device on its own and phenotypic drug susceptibility testing [M. tuberculosis. The lab-on-a-disc platform utilised separate fluidic chambers for ligation and amplification steps, which provided temperature control [2 colony-forming units per millilitre [Detecting drug-resistant strains early in the infection will aid clinical decision making and shorten the time for optimal drug treatment. Sophisticated detection of resistant strains will also transform drug discovery and innovation within the laboratory. Researchers detected single-nucleotide polymorphisms between RIF-resistant  testing . Additio control . Law et llilitre . Drug-rellilitre . M. tuberculosis detection in sputum as 43 CFU/mL; however, future work of this method includes sensitivity investigation [Investigators are overcoming the challenge of genotyping drug-resistant strains of the pathogen directly from sputum. Researchers detected and genotyped RIF and INH resistance by creating a closed system composed of a microfluidic amplification microarray . Likewistigation .M. tuberculosis complex, RIF and INH resistance, and NTM in less than 3 h [More importantly, it is vital to consider the translation to commercialisation of microfluidic diagnostic devices. Suitability, such as user-friendliness, portability, and economic feasibility, should be addressed at the basic research level. Alternative methods to fabricate microfluidic devices are by printed circuit board (PCB) technology. A lab-on-a-printed circuit board (LoPCB) was integrated with a biosensor system able to detect INFy for diagnosing TB ,145. Thethan 3 h ,147. Thethan 3 h . It can M. tuberculosis, peripheral blood mononuclear cells, and type I collagen) was achieved for the pharmacokinetic modelling of antibiotics. After establishing the 3D granuloma microsphere model, the fluidic system was used to mimic the pharmacokinetics of that seen in vivo by altering various concentrations of RIF over time. They observed fluctuations in killing over time compared with fixed antibiotic concentrations [Microfluidic technologies have been developed for portable and disposable TB diagnostics, but more recently there have been attempts to bridge these microfluidic techniques with conventional antibiotic drug discovery for TB. A microfluidic system in combination with microspheres  studied heterogeneity between mycobacterial cell growth rates. Using a microfluidic chamber with live-cell imaging, they measured the elongation rates of single cells and concluded that due to the unipolar manner of bacteria growth, this causes heterogeneity in elongation growth rate. They selected the microfluidic chamber as it advantageously allows single cells to grow in the shallow chamber with fresh nutrient media diffusing across the channel. This permitted the ideal imaging of five generations of bacteria for growth studies . A subseat birth . Additiosistence . M. smegmatis growth dynamics in real time. The visualisation of chromosome and replisome tracking were studied using time-lapse microfluidic microscopy (TLMM) [M. smegmatis to be measured in real time [Time-lapse microscopy combined with microfluidics is proving useful to study population heterogeneity in bacteria strains. A group who are investigating a microfluidic application for tuberculosis previously used a microfluidic chamber to monitor y (TLMM) ,154,155.eal time . Drug meeal time .In addition to the previously mentioned imaging/microfluidic approaches, microfluidic live-cell imaging was combined with time-lapse microscopy to investigate the antimicrobial activity of peptoids (oligo-N-substituted glycines). The study concluded that the investigative molecules’ mechanism of action disrupted the cell membrane shown by the increased rate of uptake of propidium iodide . M. tuberculosis has been immobilised in an agarose matrix and introduced to antibiotics which diffuse into the agarose on a microfluidic chip [Drug susceptibility testing utilising microfluidics has been attempted, which is faster than conventional approaches. dic chip . The agadic chip . Performmsm2570::Tn mutants of M. smegmatis. Single cells were studied using microfluidics and time-lapse microscopy and provided evidence that the mutant strain was more tolerant to INH compared to the wild-type strain. This current example is proof that microfluidics can achieve an improved understanding of resistance mechanisms of mycobacteria to antibiotics and aid the discovery of new antimicrobials [Investigating the mechanism of antibiotic tolerance was demonstrated using wild-type and crobials .Escherichia coli and Mycobacterium smegmatis by utilising optical sensor nanoparticles. The nanoparticles had a phosphorescent indicator dye embedded in poly (styrene-blockvinylpyrrolidone) nanobeads and were easily integrated into a droplet device [The advancement of 3D models such as organ-on-a-chip has provided researchers the tools to study drug actions with more efficient in vitro models. This could reduce the use of animal models. For example, a lung-on-a-chip model was used to mimic the alveolar lung environment of early TB infection. A porous membrane creates an air–liquid interface of alveolar and vascular compartments and was then used to study the effect of pulmonary surfactant on mycobacteria infection of alveolar epithelial cells and macrophages. Utilising time-lapse microscopy, the model not only mimicked the host–pathogen interaction but also found that pulmonary surfactants had a protective role against TB, as shown by increased intracellular bacterial growth when cells lacked surfactants .This lunt device . MonitorM. bovis BCG and M. smegmatis were encapsulated in gel microdroplets, with a mean diameter of 25 μm, along with flow cytometry as a model system to investigate the efficacy of encapsulation and the detection of clonal growth by flow cytometry [The feasibility of using encapsulation technology to rapidly detect and enumerate individual microorganisms in patient samples has been demonstrated . Effectiytometry . The chaE. coli cells have been encapsulated in agarose monodisperse microparticles, approximately 30 μm diameter, using a flow-focusing microfluidic chip. Both the MIC for RIF and the sorting of spontaneous mutants by fluorescence-activated cell sorting (FACS) was demonstrated and characterised by DNA sequencing [Staphylococcus aureus is co-cultured with a recombinant host—Saccharomyces cerevisiae or E. coli, which are capable of secreting biocatalytic antibiotics and/or secondary metabolites from a metagenomic library [One advantage of droplet microfluidics is the approach of “stochastic confinement” . When siquencing . Buildin library . The gel library . −7–1 × 10−9, respectively. Hence, to obtain a reasonable number of mutants (10–100 for DNA sequencing), at least 109 bacteria would have to be screened. A label-free high-throughput method was previously reported for screening up to 1 × 109 bacteria for AMR in water-in-oil picolitre-volume droplets (picodroplets); using Poisson statistics, the occupancy per picodroplet was 100 bacteria (E. coli HS151) [Lactococcus lactis cells producing antimicrobial lanthipeptides with approximately 150 sensor strain cells, Micrococcus flavus, then back to the hydrophilic phase for the activation of lanthipeptide production, and back to the hydrophobic phase for incubation and to prevent lanthipeptide crosstalk between the microdroplets [M. tuberculosis at the single cell level.The frequency of resistance (FOR) of INH- and EMB-resistant mutants has been measured to be in the order of 1 × 10i HS151) . From rodroplets . FinallyMicrofluidics is gradually transforming TB research. The application of fluidic devices in TB diagnostics, microbiological research, and drug discovery has proven the power of this technique in improving throughput and sensitivity and reducing dependence on animal models. Combining microfluidics with diagnostics and detection is ahead of the field compared to drug discovery. Chips were used in combination with PCR, biosensors, and microscopy techniques for detecting TB infection and resistant strains. We can now easily observe mycobacterial cell morphology when coupling microfluidics with imaging techniques which could aid our understanding of mycobacteria heterogeneity. Overall, it was found that the materials and methods used to fabricate microfluidic chips for TB investigation were biocompatible and cheap, making them suitable for long culture times and use in low-resource settings. Droplet fluidics is still rapidly evolving and will continue to grow with the success of its applications. There is a potential to incorporate novel droplet systems for studying biological processes for TB, such as cultivation vessels, investigating environmental stimuli, and the killing activity of antibacterials due to the ability of droplet sorting, injection, incubation, and mixing . DropletFuture research should focus on translating these laboratory platforms into commercial application for industry and clinical practice. Commercial products of single-cell droplet platforms have been successful, such as inDrop, Drop-seq, and 10× Genomics . InteresIt is encouraging to highlight the breadth of research utilising microfluidics and multidisciplinary collaboration for furthering our understanding of TB, its diagnosis, and how best to manage it.


Objective: This retrospective, case–control study was executed to assess the effects of digoxin (DGX) use approaches [continuous use of DGX (cDGX) vs. intermittent use of DGX (iDGX)] on the long-term prognosis in rheumatic heart disease (RHD) patients with heart failure (HF).Methods: A total of 642 RHD patients were enrolled to this study after propensity matching. The associations of DGX application approaches with the risks of all-cause mortality, cardiovascular death (CVD), HF re-hospitalization , and new-onset atrial fibrillation (AF) were analyzed by multivariate Cox proportional hazards or binary logistic regression models, respectively.Results: cDGX was associated with increased risks of all-cause mortality  and CVD  in RHD patients with HF compared to iDGX. With exception of 1-year HF re-hospitalization risk, cDGX was associated with increased HF re-hospitalization risk of 3-year  and 5-year  as well as new-onset AF .Conclusion: cDGX was significantly associated with increased risks of all-cause mortality, CVD, medium-/long-term HF re-hospitalization, and new-onset AF in RHD patients with HF. It remains one of the most major causes of mortality in low- and middle-income countries. Heart failure (HF) is an independent predictor of death in RHD patients . Congestwith DGX . Meanwhiwith DGX . These fOne overlooked reason of this phenomenon is possibly related to the administration strategies of DGX, including continuous use of DGX (cDGX) and intermittent use of DGX (iDGX). According to the congestion status of HF patients, cDGX is defined as continuous use of DGX even after congestion is eliminated. iDGX is defined as adopting de-escalating therapy after the congestion is eliminated, which the DGX dose is gradually reduced and eventually discontinued. Observational and RCT studies showed an opposite association between cDGX and mortality as mentioned above. On the other hand, the Prospective Randomized Study of Ventricular Failure and the Efficacy of Digoxin (PROVED)  and Randvia 2-year follow-up. However, this retrospective study did not distinguish the effect the administration approaches of DGX on prognosis in RHD patients with HF according to the congestive status. Whether the long-term prognosis of RHD patients with HF can be improved by the de-escalation therapy of DGX, it still lacks conclusive evidence from a large well-designed RCT with “hard” clinical endpoints. Hence, the aim of this study is to access the associations between DGX use approaches (cDGX vs. iDGX) and long-term clinical prognosis in RHD patients with HF.However, the influence of this strategy on the clinical prognosis in HF patients is still unclear, especially in valvular HF. Current guidelines still recommend DGX for the treatment of RHD patients with congestive HF, but there is no evidence that it helps to improve long-term prognostic progress in RHD patients with HF. In fact, prescription of DGX is still subjective and unproven to a large extent in RHD patients with congestive HF. The Rheumatic Heart Disease Global Registry (REMEDY) study , as the N = 386) and iDGX (N = 348). The cDGX group of participants receives continued DGX therapy regardless of whether the congestion is eliminated. The iDGX group of participants only receives DGX therapy when congestion occurs. In particular, surgical intervention was defined as valve replacement or repair surgery via surgical or percutaneous intervention for any affected valve(s) with tissue or mechanical prosthesis referring to recommendations. Coexisting medical conditions, including AF, hypertension (HT), coronary heart disease (CHD), stroke, and type 2 diabetes (T2D), were also diagnosed referring to relevant recommendations. Blood sample analyses were executed by standard lab protocols. Echocardiography was carried out according to our previous protocol /American Heart Association (AHA) guidelines. Congestion in HF was evaluated referring to European Society of Cardiology (ESC) guidelines (protocol . The datAll-cause death and cardiovascular death (CVD) were the primary outcomes. The all-cause death was defined as death due to any causes. The CVD was defined as death due to any cardio-/cerebrovascular diseases, such as CHD, HF, AF, stroke, and/or other cardio-/cerebrovascular causes. HF re-hospitalization and new-onset AF were the secondary outcomes. After first discharge for HF, HF re-hospitalization was defined as hospital re-hospitalization with HF treatment. The new-onset AF was defined as AF that first occurred during follow-up period.2 test and independent-sample t-test, respectively. The multivariate Cox proportional hazards regression model was carried out to access the association [hazard ratio (HR) and 95% confidence interval (CI)] of different DGX use methods with all-cause death and CVD, adjusting by baseline characteristics. Binary logistic regression modeling was carried out to evaluate the association [odds ratio (OR) and 95% CI] of different DGX use methods with 1-, 3-, and 5-year HF readmission as well as new-onset AF. A P-value of 0.05 or less was statistically significant (two-tailed).The 1:1 propensity score matching (PSM) and statistical analyses were executed by SPSS version 24 . The 1:1 PSM was executed referring to our previous methods  by baselP = 0.025), DBP (P = 0.004), Hr (P = 0.042), the constituent ratio of mitral stenosis , surgical intervention for valve repair (P = 0.014), NYHA (P < 0.001), coexisting disorder , drug use , and echocardiographic index for right ventricular end-diastolic diameter , left atrial end-systolic diameter , and LVEF (P = 0.009), as shown in P = 0.001) between the two groups.Before PSM, a total of 734 patients with RHD were enrolled to this study, the median follow-up time was 5.8-years among those participants, and there were obvious differences between cDGX and iDGX in RHD patients on levels of SBP P = 0.05, DBP (PP < 0.001) and CVD  in RHD patients with HF compared to those with iDGX. Adjusting for confounding covariates, cDGX was still correlated with increased risks of all-cause death  and CVD  in RHD patients with HF, as shown in After PSM, cDGX was associated with elevated risks of all-cause death , 3-year , and 5-year  in RHD patients with HF compared to those with iDGX. Adjusting for confounding covariates, cDGX was still correlated with increased HF readmission risk of 3-year  and 5-year , as shown in After PSM, cDGX was associated with elevated HF re-hospitalization risks of 1-year  compared to those with iDGX. Adjusting for confounding covariates, cDGX was still correlated with elevated new-onset AF risk , as shown in After PSM, cDGX was associated with elevated new-onset AF risks  in HF patients (https://doi.org/10.1161/circ.142.suppl_3.16085), and lower SDC (0.5–0.9 ng/ml) may be good for improving the overall risk of death and hospitalization. Under conditions of the two DGX use approaches, however, the effect of different SDC on all-cause mortality and CVD is still unclear. In this study, four issues were worth paying attention to as follows: Firstly, RHD patients with DGX treatment at SDC ≥ 1.0 ng/ml had relatively high risk of all-cause death while they had relatively low risk of CVD, and this phenomenon was observed in both DGX use approaches . The posMany studies have shown that DGX increases predisposition to AF. In AF patients after electrical cardioversion, recurrence risk of AF in those patients receiving DGX increased by 2- to 3-fold, and the duration of AF lasted longer . In thispost-hoc analysis of the DIG trial found that the substantial risk of DGX toxicity had nothing to do with metabolic status among HF patients . One of the important mechanisms on coronary microvascular dysfunction (CMD) mainly involves the abnormal production and release of nitric oxide (NO), which is mainly synthesized by endothelial nitric oxide synthase (eNOS) in the cardiovascular system. Under the circumstances of RHD, there is a potential association among DGX use, CMD, and arrhythmia based on NO as follows: Firstly, RHD is an autoimmune disease, with inflammatory factors upregulated, resulting in the inhibition of eNOS activity and then contributing to the reduction of NO bioavailability, which affected the function of coronary microvascular tissue . Secondlium ions , furtherTheoretically, valvular AF  is associated with increased risk of stroke, even cerebrovascular death. In this study, the two DGX use approaches were not associated with the risks of new-onset stroke  and cereThis research has produced some relief, but it still has some shortcomings. The main limitation is that this present study was only a retrospective investigation, and participants did not randomly receive DGX application strategies, which may lead to bias . The neurohumoral condition in RHD patients with iDGX may be better than those with cDGX, which may be linked to better outcomes. This study can only carry out correlation analysis and cannot confirm that DGX application was the reason for increasing poor prognosis. The PSM was performed to eliminate potential bias, but it may also be possible to amplify hidden confounding variables. In addition, because there is still no consensus about the optimal therapy strategy for RHD patients with HF, the evidence-based drugs for HF treatment  in this retrospective study were not adequate. Hence, the results must be interpreted carefully.cDGX was correlated with elevated risk of all-cause death, CVD, medium-/long-term HF readmission, and new-onset AF in RHD patients with HF. In contrast, DGX de-escalation approaches  were related to a better prognosis in RHD patients with HF, suggesting that DGX should be adjusted with a de-escalation therapy as soon as possible after the congestion state of RHD patients with HF is relieved and the clinical condition is stable. It is uncertain whether this relationship observed is due to unnecessary continuous treatment with DGX or simply reflects the inevitable link between more severe HF and more frequent DGX treatment. These substitute hypotheses could be studied in further large well-designed RCTs.The raw data supporting the conclusions of this article will be made available by the authors, without undue reservation.The study received ethics approval from the Institutional Review Board of Guangzhou First People's Hospital, South China University of Technology (K-2018-136-1). Written informed consent for participation was not required for this study in accordance with the national legislation and the institutional requirements.CL: literature search, study format, writing protocol, collecting data, processing data, data interpretation, analyzing data, and writing manuscript. TG and QZ: processing data, data interpretation, and analyzing data. JP, SZ, YL, and DeW: recruiting patients, following up patients, and collecting data. DaW: echocardiography testing. All authors read and approved the final manuscript.This study was funded by the National Natural Science Foundation of China (81100235), the Guangdong Natural Science Foundation of China (S2011040004458), the Guangdong Science and Technology Planning Project of China (2014A020212372), and the Guangzhou Science and Technology Project of China (2012J4100035 and 201804010214).The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. The reviewer RZ declared a shared affiliation, with no collaboration, with the authors to the handling editor at the time of the review.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.


The association between aortic stenosis (AS) and cardiac amyloidosis (CA) is more frequent than expected. Albeit rare, CA, particularly the transthyretin (ATTR) form, is commonly found in elderly people. ATTR-CA is also the most prevalent form in patients with AS. These conditions share pathophysiological, clinical and imaging findings, making the diagnostic process very challenging. To date, a multiparametric evaluation is suggested in order to detect patients with both AS and CA and choose the best therapeutic option. Given the accuracy of modern non-invasive techniques , early diagnosis of CA is possible. Flow-charts with the main CA findings which may help clinicians in the diagnostic process have been proposed. The prognostic impact of the combination of AS and CA is not fully known; however, new available specific treatments of ATTR-CA have changed the natural history of the disease and have some impact on the decision-making process for the management of AS. Hence the relevance of detecting these two conditions when simultaneously present. The specific features helping the detection of AS-CA association are discussed in this review, focusing on the shared pathophysiological characteristics and the common clinical and imaging hallmarks. The coexistence of aortic valve stenosis (AS) and cardiac amyloidosis (CA) is not an uncommon finding in routine clinical practice. AS is the most common valvular heart disease and affects more than 4% of people aged 75 years or older . SimilarAmyloidosis is a rare systemic disorder caused by deposition of amyloid fibrils in different organs including the heart . The twoDespite that, the prognostic impact of CA on AS patients is poorly studied, with the few published studies reporting contrasting results. Whether the diagnosis of CA involvement in patients with AS can influence the therapeutic strategies is still a matter of debate, while the newly identified specific pharmacological therapy for ATTR-CA is finding its application in patients with early disease .The aim of this review is to assess the main specifics  of AS-CA association, which should attract clinicians attention to suspect their coexistence and choose the best therapeutic options. Several retrospective or prospective studies have described the presence of ATTR-CA in AS patients , with a Transthyretin is synthesized in the liver and has a role as a transporter of thyroxin and retinol-binding proteins. Its main function is the control of behavior, cognition, nerve regeneration and axonal growth . The twoThe pathogenetic mechanism of the two types of CA is similar. Over time, the amyloidogenic process results in amyloid fibrils aggregation and precipitation in the extracellular space and consequently causes its expansion. In the heart, this expansion causes increased biventricular wall thickness, myocardial stiffening and restrictive physiology of the left and right ventricles. In addition, amyloid fibrils may exhibit direct toxic effects on myocardial cells, impairing systolic left ventricular function . While tAS is the consequence of inflammatory process caused by endothelial damage due to mechanical stress and lipid penetration, eventually leading to fibrosis, leaflet thickening, sclerosis and calcification . As a reCurrently, the causative link between AS and ATTR-CA has not been demonstrated. However, there is increasing evidence supporting a central role of oxidative stress, inflammation and extracellular remodeling in the ATTR amyloidogenic process . Some auDiagnosis of ATTR-CA in AS patients is challenging, as clinical and imaging features can overlap. Moreover, comorbidities such as coronary artery disease and hypertension are frequently present in the older population, making the diagnostic process, based on any risk score, even more difficult . Researchers have assessed the clinical and imaging features of patients with concomitant AS and ATTR-CA . FirstlyAnother clue is the so-called “natural cure” of hypertension with the need for down-titration or discontinuation of antihypertensive medications. This reflects the presence of an autonomic dysfunction. Moreover, the presence of unexplained peripheral or autonomic neuropathy suggests the possibility of hereditary ATTR amyloidosis but can occur in AL and occasionally in ATTRwt amyloidosis . Some laboratory parameters are suggestive of CA including disproportionate N-terminal pro-brain natriuretic peptide (NT-proBNP) elevation with AS severity and in the absence of chronic renal failure or elevated troponin levels without significant coronary artery disease or renal failure . Unlike Patients with ATTR-CA rarely have normal electrocardiography (ECG). Two criteria are suggestive of CA: pseudo-infarction pattern (mainly in anterior leads) and low-voltage QRS complex. The former is common and has been observed in approximately 60% of patients with ATTR-CA . ConversAs the deposition of amyloid fibrils progresses over time, direct involvement of the sinoatrial node, atrioventricular node and bundle branches can manifest in the form of various degrees of conduction abnormalities and arrhythmias, such as atrial fibrillation. These abnormalities tend to become more frequent with increasing disease severity . Atrial Transthoracic echocardiography is essential in the diagnostic process of both AS and CA. The assessment of aortic valve disease should follow current guidelines. The critical point is that both pathologies share some characteristics, such as LV wall thickening, impaired diastolic function (as reflected on filling pattern), and/or LV systolic dysfunction . An enlaSome Doppler echocardiographic features are mainly observed in CA patients: concomitant left and right ventricular wall thickening, bi-atrial dilation, mild pericardial effusion, thickening of atrioventricular valves and atrial septum, and restrictive left ventricular filling pattern . About 7The analysis of myocardial deformation by tissue Doppler and speckle-tracking echocardiography has a crucial role in the detection of CA . ReducedCardiac imaging using magnetic resonance (CMR) has an important diagnostic role in CA. It carries additional diagnostic value because of the better myocardial tissue characterization with late gadolinium enhancement (LGE), native T1 mapping, and extracellular volume (ECV) sequences . HoweverOnce ATTR-CA is suspected, the next step is to confirm the diagnosis. The 2019 expert consensus for the suspicion and diagnosis of ATTR-CA allows to avoid endomyocardial or extra-cardiac biopsy (fat pad biopsy) if bone scintigraphy is available . FurtherPerforming bone scintigraphy with 99m technetium-labeled bisphosphonates and excluding AL by search of monoclonal light chain in blood and urine is now sufficient to confirm the suspicion of ATTR-CA . In partAlthough endomyocardial biopsy and confirmation using mass spectrometry remain the gold standard for diagnosing ATTR-CA, these procedures often delay diagnosis and may not be appropriate in frail elderly adults, including those referring for TAVR. In the absence of specific CA features on imaging, a positive extracardiac biopsy is not sufficient to confirm the diagnosis. In contrast, a negative extracardiac biopsy may not exclude CA in patients with typical CA features on imaging . A diagnTo date, there is no recommendation on whether patients with AS should be systematically screened for CA . HoweverECG is another important marker of cardiac structure and function in CA and AS. Patients with concomitant ATTR-CA and AS usually exhibit more pronounced ECG abnormalities than those with solely AS. They tend to have broader QRS complex and higher prevalence of right bundle branch block B ,22. Bothp < 0.0001, compared to the rest of the echocardiographic variables. Indeed, a cut-off value of S’ < 6 cm/s had 100% sensitivity (with a 57% specificity) in predicting a positive 99mTc-PYP (99mTc-labeled pyrophosphate) amyloid scan [Once raised a suspicion, echocardiography with myocardial deformation analysis may add discriminatory power for the presence of CA, as initial imaging technique. Interestingly, Nitsche and colleagues showed that apical sparing was a powerful marker for diagnosing AS-CA . AS-CA poid scan . Thus, Soid scan . The higoid scan . In thisoid scan .Cardiac magnetic resonance (CMR) has demonstrated promising value for disease detection, following disease progression or monitoring response to therapy in patients with CA. However, Nitsche et al. confirmed the formerly reported low sensitivity of distinctive LGE patterns. In this study, CMR was diagnostic in only 25% of CA-AS patients . Given tIn a recent study, Nitsche and colleagues created and validated in two cohorts a scoring system for discrimination of lone AS versus AS-CA. The remodeling, age, injury, system and electrical (RAISE) score includes 5 domains: remodeling (LV hypertrophy and/or diastolic dysfunction), age, injury (hs-cTn), systemic  and electrical (right bundle branch block or low voltages). Scores ≥2 and ≥3 points had high sensitivity (93.6% and 72.3%), with adequate specificity (52.1% and 83.6%) for the presence of AS-CA, suggesting a potential additional valid screening tool [Combination of AS with CA is prognostically important. Currently, only few studies have investigated the outcome of AS-CA patients and have shown worse prognosis. Treibel and colleagues have followed 146 patients with severe AS who underwent surgical AVR for a median of 2.3 years. Of all variables assessed, the presence of ATTR-CA had the highest hazard ratio for all-cause mortality . SimilarMost studies assessing patients following surgical aortic valve replacement showed a high risk of mortality and modest improvement in functional class ,21,59,602) and low-gradient AS [In view of the above, individual cases should be discussed by a multidisciplinary team in order to choose the best treatment options  . Depressdient AS ,18 have dient AS . Selectidient AS . The nondient AS . In factdient AS . Thus, sThe combination of AS and CA, especially ATTR, is an important clinical problem. Its already high prevalence is destined to grow because of the aging population. However, current literature shows that CA is often underdiagnosed in old adults, resulting in underestimation of the AS-CA combination. Such clinical issue has its impact on screening patients with AS for the presence of CA and the choice of best treatment strategy. We highlighted some clinical and imaging features which should help raise the suspicion of cardiac amyloid involvement, followed by diagnosis confirmation with bone scintigraphy. They should be followed by individual case multidisciplinary assessment to evaluate the best suitable approach. Based on the available uncertainty of the clinical outcome of combined AS and CA, prospective multicenter studies in large cohorts are necessary to suggest an optimum road map for managing such patients.