Hypertrophic cardiomyopathy (HCM) is a genetic disease characterized by an increased left ventricular wall thickness in the absence of increased afterload conditions. In addition to diastolic dysfunction, obstruction of the left ventricular outflow tract is common in HCM and has an important influence on symptoms and outcome. Over the last five decades or two decades, respectively, surgical myectomy and alcohol septal ablation were the only therapeutic options if standard medical care was not sufficient. Recently, a new option has become available that has the potential to revolutionize the therapeutic strategies for patients with HCM. Mavacamten is a myosin inhibitor that belongs to a completely new drug class and targets the excessive actin–myosin cross-bridging that is the underlying pathology of HCM. By reducing the actin–myosin interactions, mavacamten not only reduces the left ventricular outflow tract (LVOT) obstruction but also seems to have positive effects on the diastolic function, microcirculation, and cardiac structure. This article summarizes the current evidence on alcohol septal ablation and reviews the preclinical and clinical data on mavacamten for the treatment of patients with obstructive HCM. Hypertrophic cardiomyopathy (HCM) is a sarcomeric disease that is characterized by increased left ventricular wall thickness in the absence of any apparent clinical reason for hypertrophy, such as increased afterload due to aortic valve stenosis or arterial hypertension. A pathogenic mutation in the sarcomere protein genes can be identified in up to 60% of HCM cases, and the inheritance is autosomal dominant . The mosThe distribution and severity of hypertrophy can vary greatly. The most common pattern is asymmetrical septum hypertrophy. About 70% of patients have obstructive HCM, with an obstruction of the left ventricular outflow tract (LVOT) or midventricular level at rest or on provocation with the Valsalva manoeuvre or exercise .The clinical presentation of patients with HCM also varies widely, from symptom-free or mildly symptomatic to severely symptomatic. Symptoms include dyspnoea, exercise intolerance, angina, syncope, or even sudden cardiac death, and are generally caused by a combination of diastolic dysfunction, microvascular dysfunction and (in patients with obstructive HCM [oHCM]) are mainly due to an LVOT obstruction with associated mitral regurgitation secondary to the SAM phenomenon. Patients with an LVOT gradient ≥ 50 mmHg at rest or with provocation are likely to be symptomatic.Non-vasodilating β-blockers are currently the first choice of medical therapy for HCM and should be titrated to effectiveness or maximally tolerated dosages. For patients for whom β-blockers are not effective or are poorly tolerated, substitution with a non-dihydropyridine calcium channel blocker  or disopyramide, titrated to the maximum tolerated dose, is recommended ,10. If sOne method of SRT is surgical myectomy, which was first performed early after HCM disease was first described by Morrow  in the 1Alcohol septal ablation (ASA) is another option for septal reduction therapy, which has a class IC recommendation in the 2020 AHA/ACC guidelines for use in symptomatic adults with oHCM with high surgical risk or are inoperable . HoweverThe myosin inhibitor mavacamten now provides a third option to treat LVOT obstruction in patients with HCM. This novel first-in-class drug specifically targets the underlying pathology of HCM by normalizing myosin and actin cross-bridging to decrease contractility. The data from two phase 3 trials support the efficacy and safety of mavacamten for proving durable improvement in LVOT gradients. In 2023, the European Society of Cardiology (ESC) guidelines for the management of cardiomyopathies included cardiac myosin inhibitors as a second-line therapy (level of evidence IIa) when optimal medical therapy with β-blockers, calcium channel blockers, and/or disopyramide is ineffective or poorly tolerated .ASA is a catheter-based method to treat the LVOT obstruction in patients with symptomatic oHCM despite medical therapy. A reduction in the LVOT gradient is achieved by creating a localized septal infarction, which leads to the thinning of the basal septum and the resolution of the SAM phenomenon. Successful use of this procedure in three cases was first reported in 1995 by Ulrich Sigwart . The ratThe key to successful ASA is careful screening to select the appropriate candidates and discuss them with an interdisciplinary heart team. Individuals with oHCM who have a septal bulge and SAM-associated LVOT obstruction at the level of the basal septum seem to be the best candidates. Other concomitant pathologies such as aberrant papillary muscle contributing to the LVOT obstruction, primary mitral valve pathologies, or other severe valve disease should first be excluded because these individuals are better served by cardiac surgery. A septal wall thickness of ≥17 mm is widely accepted as the lower limit to avoid ventricular septal perforation.Coronary angiography can be performed at an earlier time or immediately before ASA to rule out concomitant coronary artery disease and to identify the septal branch suitable for alcohol injection. Femoral or radial access are both feasible and the choice is based on the operator’s preference. The standard Judkins left or extra backup guide catheter is engaged in the left coronary ostium. A right anterior oblique cranial view allows for good visualization of the left descending artery with its septal branches. In the majority of cases, the first septal artery originating from the proximal left anterior descending coronary artery (LAD) is the targeted branch; it is rare that the target branch derives from the first diagonal, circumflex, or right coronary artery. Myocardial contrast echocardiography has made an important contribution to the safety of the ASA procedure and was first described by L. Faber in 2004 . After aThe continuous hemodynamic monitoring of the LVOT pressure gradient via a second arterial access and placement of an end-hole pigtail catheter in the left ventricle might help to guide the procedure but is not compulsory. Myocardial contrast echocardiography guidance as described above is also feasible.Before proceeding to ASA, it is necessary to insert a temporary pacemaker lead into the right ventricle via the femoral or jugular vein; this step can be omitted only in patients with an implanted permanent pacemaker or cardioverter/defibrillator. The risk of a permanent complete atrioventricular (AV) block is about 10% and is caused by injury of the conduction system that is located in the immediate vicinity of the myocardial area perfused by the targeted septal branch. A transient AV block is described in up to 30% of cases; therefore, careful monitoring of the heart rhythm is necessary for at least 48 h after ASA to decide whether a pacemaker is indicated. A post-procedural echocardiogram should ideally show an LVOT gradient of <50%; not all patients show an immediate decrease in the LVOT gradient, but may show a decrease a few months after the remodelling of the infarction area  Figure .There have not yet been any randomized trials comparing the different methods of SRT. Therefore, current therapeutic strategies are based on the data from observational studies, systematic reviews and meta-analyses. ASA has been shown to effectively reduce LVOT obstruction and improve the symptoms, exercise capacity, and oxygen uptake ,24.p < 0.01) and the New York Heart Association (NYHA) functional class from 2.9 ± 0.5 to 1.6 ± 0.7 (p < 0.01). The post-procedural pacemaker implantation rate was 12%. Another key finding was that higher doses of alcohol were slightly more effective in reducing left ventricular (LV) obstruction but were associated with a higher incidence of peri-procedural complete heart block [The EuroASA Registry is the largest registry summarizing the long-term outcome data, and includes 1257 patients who underwent an ASA in Europe [p < 0.01), mainly driven by a higher rate of cardiovascular deaths and pacemaker implantation in the patients treated earlier. Even the rates of residual LVOT gradient >30 mmHg and repeated septal reduction therapy were significantly higher in the first 50 versus subsequent patients [Several sub-studies have been published from this registry. In a secondary analysis, a lower dose of alcohol (<2 mL) was associated with higher rates of reintervention compared with higher alcohol doses, without influencing the pacemaker rate . This anpatients .p = 0.009 and 0.8% vs. 1.5%, p = 0.013, respectively) compared with SM, but higher rates of pacemaker implantations and reintervention ; the long-term all-cause mortality, cardiovascular mortality, and sudden cardiac death rates were similar in both groups [In the absence of randomized trials, several meta-analyses have compared ASA with SM and reported heterogenous results. Osman et al. summarized the data from 4213 patients undergoing ASA and 4240 undergoing SM . ASA wash groups . The mosh groups ,32. A suAfter two decades of use in clinical practice, ASA has been shown to be safe and effective. It has a low complication rate, especially when performed at centers of excellence. However, the weak points are the higher rates of reintervention and pacemaker implantation. Given the patient preference for less invasive procedures, ASA has become the standard procedure for SRT in Europe.Until recently, pharmacotherapy for oHCM was limited to symptom relief via reducing inotropy and the optimizing preload conditions. Non-vasodilating β-blockers were the first-line therapy and, if not tolerated, other pharmacological options were a nondihydropyridine calcium channel blocker or disopyramide (mostly not available in Europe due to a chronic supply shortage).Mavacamten  is a novel first-in-class drug that specifically targets the underlying pathology of HCM by normalizing the myosin and actin cross-bridging to decrease the contractility. It was developed by MyoKardia, Inc. , which is now a wholly owned subsidiary of Bristol Myers Squibb. The US Food and Drug Administration approved mavacamten in April 2022 and in June 2023 it was approved in Europe for the treatment of adults with symptomatic oHCM.Mavacamten is a small molecule with the molecular formula C15H19N3O2 . It is aIn early 2016, mavacamten was tested in in vitro and in vivo mouse models of HCM to explore whether mutations found in HCM increase the power output at the molecular level. Green et al. showed that the administration of this small-molecule inhibitor of the sarcomere power early in the disease course could attenuate the development and progression of the morphologic, histopathologic, and molecular changes that characterize HCM . These fLater in 2016, mavacamten was tested in cats with naturally occurring HCM. The treatment was shown to reduce the contractility, eliminate the systolic anterior motion of the mitral valve, and relieve the LVOT pressure gradient . Prior tp = 0.008) and from 86 mmHg at baseline to 61 mmHg at week 12 in cohort B . Mavacamten was well-tolerated and the adverse events were mostly mild (80%) or moderate (19%) in severity. The most common adverse events definitely or possibly related to mavacamten were a decreased left ventricular ejection fraction (LVEF) at higher plasma concentrations (>1000 ng/mL) and atrial fibrillation. In the patients who experienced a reduction in the LVEF to <50%, the withdrawal of the study drug led to the normalization of the LVEF to the baseline status. PIONEER-OLE, a 120-week open-label trial extension, is ongoing and is anticipated to be completed in November 2023.PIONEER-HCM (NCT02842242) was an open-label, non-randomized, phase 2 trial that included 21 patients with oHCM from five centers in the United States; the aim was to characterize the effect of mavacamten on the LVOT gradient . The pat2) and a reduction in the disease severity of at least one NYHA class, or a ≥3.0 mL/kg/min increase in pVO2 without NYHA class worsening. The proportion of patients achieving the primary endpoint was 37% in the mavacamten group versus 17% in the placebo group (p = 0·0005). Compared with the placebo group, the mavacamten recipients also had a significantly greater reduction in the post-exercise LVOT gradient (from 86 to 38 mmHg versus from 84 to 73 mmHg). A greater increase in the pVO2 and improved symptom scores were also observed in the mavacamten versus the placebo group. A complete response (LVOT gradient <30 mmHg and NYHA class I) was seen in 27% of the patients in the mavacamten group versus 1% in the placebo group. Changes in the baseline systolic function during the treatment with mavacamten were small; the mean reduction in the LVEF was −3.9% versus −0.01% with placebo. The adverse events were generally mild. Serious adverse events were reported by four patients in each treatment group. Two cases of atrial fibrillation and stress cardiomyopathy occurred in the mavacamten group and three cases of atrial fibrillation and one of congestive heart failure occurred in the placebo group. A transient decrease in the LVEF to <50% was observed in seven patients on mavacamten, six of whom had a full recovery after treatment interruption or discontinuation and one patient had partial recovery of the LVEF to 50%. One patient on the placebo experienced sudden cardiac death. The ongoing, long-term extension of this study will provide further evidence for the clinical benefit and safety of mavacamten over 5 years. An interim analysis of 231 patients followed up for 48–84 weeks showed durable improvement in the LVOT gradients, diastolic function, and N-terminal pro B-type natriuretic peptide (NT-proBNP) levels [EXPLORER-HCM (NCT03470545), a multicenter, randomized, double-blinded, placebo-controlled phase 3 trial, was conducted at 68 centers in 13 countries and assessed the efficacy and safety of mavacamten in patients with symptomatic oHCM . A total) levels .2 and lateral E/e′ with change of −3.8, both p < 0.0001) during treatment with mavacamten versus placebo [The analysis of the key echocardiographic parameters assessed by a core laboratory showed significantly improved systolic anterior motion and measures of the diastolic function  had a greater reduction in the mean LV mass index (the primary endpoint) from baseline to week 30 than the patients in the placebo group . Furthermore, mavacamten was associated with a greater reduction in the maximum LV wall thickness, intracellular myocardial mass index, and left atrial volume index compared with placebo. However, no changes were observed in the late gadolinium enhancement or myocardial contractile fraction.The cardiac magnetic resonance Imaging substudy of the EXPOLORER-HCM trial  seems top < 0.001). The difference in the post-exercise LVOT gradient between the mavacamten and placebo groups was −37.2 mm Hg (p < 0.001). A functional improvement by at least 1 NYHA class was documented in 62.5% of the patients in the mavacamten group compared with 21.4% of the patients in the placebo group (p < 0.001). In addition, changes in the Kansas City Cardiomyopathy questionnaire score, NT-proBNP, and troponin I showed significant differences favouring mavacamten . Mavacamten was well tolerated; only two patients had a LVEF reduction to <50%, which recovered completely after temporary drug discontinuation. No patients had a reduction in the LVEF to <30% necessitating permanent drug discontinuation. Atrial fibrillation occurred in 3.6% of the patients taking mavacamten and no patients on the placebo. After 16 weeks of therapy, the mavacamten group continued therapy for another 16 weeks and the placebo group crossed over to dose-blinded mavacamten for the same period [VALOR-HCM (NCT04349072) was a phase 3 trial that assessed whether mavacamten would improve the patient symptoms or LVOT gradients enough to defer septal reduction therapy . The stue period . There wMavacamten is supplied as gelatin capsules for oral use containing 2.5, 5, 10, or 15 mg of mavacamten per capsule. The bioavailability of mavacamten after oral administration is excellent, at 85%. It is rapidly absorbed with a time to maximum concentration of 1 h. Its distribution is through plasma protein binding, which ranges from 97% to 98%. Mavacamten is eliminated by the liver and metabolization is primarily through CYP2C19 (74%), and to a lesser extent by CYP3A4 (18%) and CYP2C9 (8%). Therefore, mavacamten has a variable terminal half-life that depends mainly on the CYP2C19 metabolic status. In CYP2C19 normal metabolizers, the half-life of mavacamten is 6–9 days, which is prolonged to 23 days in CYP2C19 poor metabolizers. This may lead to drug accumulation with increased plasma levels and an increased risk of adverse events . The conThe inducers and inhibitors of CYP2C19 and moderate to strong inhibitors or inducers of CYP3A4 may affect the systemic exposure to mavacamten. Therefore, the concomitant use of mavacamten with moderate to strong CYP2C19 inhibitors or strong CYP3A4 inhibitors, or with moderate to strong CYP2C19 inducers or moderate to strong CYP3A4 inducers, is contraindicated. Dosage adjustments may be required when mavacamten is given with concomitant administration of weak CYP2C19 inhibitors  or moderate CYP3A4 inhibitors .No dosage adjustment is required in patients with mild (Child–Pugh A) to moderate (Child–Pugh B) hepatic impairment, although increased drug exposure is possible. The effects of mavacamten administration in individuals with Child–Pugh C hepatic impairment is unknown.Due to these variances in the metabolism and variable plasma level, the prescription of mavacamten is strictly regulated. In the United States, it is only available through the Risk Evaluation and Mitigation Strategy (REMS) program with three main aspects (physician–pharmacist–patient) to ensure the safe and monitored administration of the drug. The physician needs to be REMS-certified to be able to prescribe mavacamten, which includes a responsibility to provide accurate counselling. Mavacamten can only be dispensed from certified pharmacies participating in the program and the pharmacist is responsible for reviewing the medication regimens and reinforcing drug interactions and avoidances. Lastly, the patient must commit to regular screening echocardiograms during mavacamten therapy. In Europe/Germany, a further safety aspect has been added before prescribing mavacamten: patients need to be genetically tested for their CYP2C19 metabolism. In CYP2C19 poor metabolizers, the mavacamten starting dosage is 2.5 mg/day and the maximum dosage is 5 mg/day. For all other phenotypes of CYP2C19 metabolizers , the starting dosage is 5 mg/day and maximum dosage is 15 mg/day. Regular echocardiographic screening is compulsory, with assessment of the LVOT gradient and LVEF every 4 weeks in the first 12 months and every 12 months thereafter. Algorithms for initiation and maintenance dosing, patient monitoring schedules, and guidance for treatment interruption or discontinuation are provided in the prescribing information and should be followed.Since the US and European approvals of mavacamten in April 2022 and June 2023, respectively, there is still a need to define the application of this drug in clinical practice. Neither the European nor the American guidelines for the management of HCM include cardiac myosin inhibitors in the treatment algorithm for HCM. Given that most patients in the randomized trials remained on their background medication with β-blockers or non-dihydropyridine calcium channel blockers, these agents will probably remain the first-line therapy in the near future.Initially, the prescription of mavacamten is more complicated than prescribing β-blockers due to the need to consider the CYP2C19 metabolic status and have regular echocardiographic follow-up every 4 weeks initially and every 12 weeks thereafter. The annual drug cost for mavacamten was calculated as $US75,000 and the additional cost for regular check-ups may cause issues with the cost effectiveness of this treatment strategy for the health care system. According to the Institute for Clinical and Economic Review, septal reduction procedures dominate mavacamten with respect to cost effectiveness .Furthermore, caution is necessary during treatment with mavacamten because of drug interactions. Regular checks of the medication regimen will be essential to optimize the safety and efficacy during long-term treatment. The concomitant use of strong or moderate CYP2C19 inhibitors with mavacamten is contraindicated because it increases the blood level of mavacamten and may increase the risk of systolic dysfunction and heart failure.Both phase 3 trials of mavacamten had a follow-up duration of <1 year, therefore the long-term safety of chronic mavacamten therapy, especially regarding the LVEF reduction, is unknown. The data from the long-term extension studies of the randomized trials and long-term post marketing registries will provide more information on this. However, mavacamten is the first drug to show a favourable impact on cardiac remodelling in HCM. Whether it will halt disease progression or even reverse hypertrophy and fibrosis in HCM hearts and its impact on prognosis, long-term outcomes, and mortality remain to be determined.The question of whether mavacamten will replace surgical myectomy or ASA is not quite clear yet. The VALOR-HCM trial showed that mavacamten was effective enough to allow SRT to be deferred. Notably, after the primary study duration of 16 weeks, the vast majority of patients (95%), including 93% of those in the placebo group, chose to continue in this active phase of the trial up to 32 weeks rather than proceed with SRT. This suggests that patients prefer medical therapy over invasive procedures. Clinical experience confirms patients’ preference for medical therapy when they are adequately and extensively informed. However, there were a few patients in all the mavacamten trials who did not respond adequately to the treatment or had side effects that prevented further treatment. Reasons for an unsatisfactory response need to be investigated further . These patients will probably still be candidates for SRT. Patients with dual-chamber implantable cardioverter-defibrillators implanted for primary prevention of sudden cardiac death might also be candidates for primary ASA because the major risk of an AV block is already addressed, and other risks of the ASA procedure are negligible. Moreover, it is important to note that SRT is a one-time procedure with a low risk when performed at centers of excellence and is supported by good clinical evidence to eliminate the LVOT gradient and the need for long-term medical therapy. Nevertheless, patients without a suitable septal branch for ASA or who experience reobstruction of the LVOT after any type of SRT now have an alternative medical strategy with mavacamten . WhetherBoth patients and their physicians had high expectations regarding mavacamten. However, the true long-term clinical benefits and safety are still to be determined. The long-term extension trials of PIONEER-HCM and EXPLORER-HCM and post-marketing registries should provide important information that will help define the value of mavacamten for the treatment of oHCM.Several trials are currently investigating mavacamten in a variety of populations and broader clinical indications. A prospective registry  is assessing the real-world patient characteristics, treatment patterns, and longitudinal outcomes in patients with oHCM treated with mavacamten and other treatments in the US. Another phase 3 trial  is underway in Japan to evaluate the effectiveness, safety, and tolerability of a 30-week course and the long-term effects of mavacamten in Japanese patients with symptomatic oHCM. ODYSSEY-HCM is a randomized, double-blind, placebo-controlled, phase 3 trial (NCT05582395) that is evaluating mavacamten in patients with symptomatic non-obstructive hypertrophic cardiomyopathy. Mavacamten is also being investigated in patients who have heart failure with a preserved ejection fraction (HFpEF). EMBARK-HFpEF (NCT04766892) is a phase 2a proof-of-concept study that is assessing the safety, tolerability, and preliminary efficacy of mavacamten treatment on the biomarker levels in patients with HFpEF.Nevertheless, there are still a lot of unanswered questions. Primarily, mavacamten was not developed to treat LVOT obstruction, but to optimize energetics at the sarcomeric level. Whether it has the potential to limit or even reverse the disease progression, whether it can stop the disease expression in genotype-positive carriers without the HCM phenotype, and whether there are differences in the response depending on the type of genetic mutation, are all unclear and need to be investigated.Finally, aficamten, a next-in-class cardiac myosin inhibitor has completed a phase 1 assessment, and the data from a phase 2 study showed that treatment with aficamten improved the LVOT gradient, heart failure symptoms and biomarkers, and was well-tolerated in patients with oHCM .HCM with an LVOT obstruction is associated with increased morbidity and mortality. When standard medication with β-blockers or calcium channel blockers is not sufficient, the next option is SRT. As the first cardiac myosin inhibitor, mavacamten represents a major milestone in the treatment of symptomatic patients with oHCM. The data from phase 2 and 3 trials showed that mavacamten significantly improved the LVOT gradients, biomarkers, and symptoms. Transient reductions in the LVEF were associated with higher plasma drug levels and responded well to dose adjustments. Mavacamten was well-tolerated and the rate of adverse events were low. The data on the long-term safety and efficacy is necessary to determine its place in the treatment algorithm for oHCM alongside the established methods of SRT. Further indications for treatment such as paediatric HCM, non-obstructive HCM, and HFpEF are currently being investigated.


Skin evolves essential appendages with adaptive patterns that synergistically insulate the body from environmental insults. How similar appendages in different animals generate diversely-sized appendages remain elusive. Here we used hedgehog spine follicles and mouse hair follicles as models to investigate how similar follicles form in different sizes postnatally. Histology and immunostaining show that the spine follicles have a significantly greater size than the hair follicles. By RNA-sequencing analysis, we found that ATP synthases are highly expressed in hedgehog skin compared to mouse skin. Inhibition of ATP synthase resulted in smaller spine follicle formation during regeneration. We also identified that the mitochondrial gene COX2 functions upstream of ATP synthase that influences energy metabolism and cell proliferation to control the size of the spine follicles. Our study identified molecules that function differently in forming diversely-sized skin appendages across different animals, allowing them to adapt to the living environment and benefit from self-protection. In the history of evolution, animals have evolved with unique characteristics to adapt to the ever-changing environment. Different adaptive patterns in animals may play different yet important roles in survival, reproduction, defense, food-hunting, etc. Locating on the surface of the body, skin generates obvious and diverse patterned appendages including follicles , glands , and keratinized structures  that function importantly for animals, such as barrier and protection, thermoregulation, communication, hunting, defense, lighting, and secretion The control of organ size is fundamental in living life Mitochondrially encoded cytochrome c oxidase II (COX2) is a subunit of cytochrome c oxidase (complex IV), an enzyme that drives oxidative phosphorylation in the mitochondrial electron transport chain. Cytochrome c oxidase is at the end of the cytochrome system in cellular respiration. It transfers electrons from NADH and succinic acid to oxygen, and produces an electrochemical gradient on the intima. The electrochemical gradient drives transmembrane transport and ATP synthesis. Energy metabolism in mitochondria is crucial for hair follicle development In this study, we study how hedgehog forms bigger spine follicles in size with mouse hair follicles as a comparison. We first characterized the epithelial and mesenchymal components in the hedgehog spine follicle. By RNA-seq analysis of growth phase follicles in hedgehogs and mice, we identified genes including ATP synthase that are specifically expressed in the hedgehog spine follicles. Functional perturbation of ATP synthase resulted in smaller spine follicle formation. Then we investigated the upstream regulator of ATP synthase with a focus on a mitochondrial gene COX2, inhibition of which also led to a dramatically decreased size of spine follicle at the full growth phase. Through comparison between spine follicles in hedgehogs and hair follicles in mice, our study gains new insight into understanding the adaptative mechanism by which the diversity in skin appendages forms between different species in nature.C75BL/6J mice were used in this study. Adult mice were used to study hair follicles and 45-day hedgehogs (Atelerix Albiventris) purchased from Jianghu Pet Corporation  were used to study spine follicles.To observe the differences between hairs in mice and spines in hedgehogs, their back skins were directly dissected and photographed with a camera (Canon) or stereoscopic microscope.The spines and hairs were plucked out from the skin to measure the length and width. The microstructure of the spine and hair was observed with a microscope and photographed with NScope 2.0. The length and width of the spine follicles and hair follicles at full growth phase were photographed with a stereoscopic microscope, and measured by Adobe Photoshop CC 2019.For pulse labeling, BrdU  was administered by injecting intraperitoneally at 50 mg per kg (body weight). The injection was done 4 hours before collecting spine and hair follicles samples.Dorsal skin samples were fixed for 48 hours in 4% paraformaldehyde (PFA) at 4°C, washed with PBS, and dehydrated with gradient alcohol  and xylene. After mounting in paraffin, samples were sectioned at 8 µm in thickness.For H&E staining, samples were stained with hematoxylin for 3 minutes and eosin for 1 minute at room temperature, then dehydrated and mounted with resinene.oC for 2 hours. Primary antibodies were diluted at 1:100 and secondary antibodies were diluted at 1:500 or 1:50 in PBS. The samples were counterstained with propidium iodide (PI) or 4'6'-diamidino-2- phenylindole (DAPI) to label the nuclei.For immunohistochemistry, the slides were immersed in 10 mM sodium citrate buffer (pH 6.0) at 100°C. Then slides were blocked with 1% bovine serum albumin (BSA) buffer at 37oC for 1h, incubated with primary antibodies  at 4°C overnight, followed by incubation with secondary antibodies  at 37ATPase inhibitor  and COX2 inhibitor  were injected into the bottom of the spine follicle at the early growth phase using a microinjector, with DMSO as a control. Spine follicles were injected for two consecutive days and collected on the third and ninth days to observe their phenotypes using HE and immunofluorescence staining.Two available hedgehog skin transcriptome data were downloaded from Hui-Ming Li, 2020 The genes only expressed in hedgehog were analyzed and expression value > 1 and standard deviation < 100 were set as the threshold. Expression value means read count. The higher the read count, the higher RNA in abundance. The standard deviation is used to judge the statistical dispersion of gene expression levels in the replicates of RNA-seq data. STRING We used statistical software R (version 4.0.3) for visualization. R-package “ggplot2” and “ggbreak” were used to analyze and show Boxplot and Volcano map. Heatmap, GO and KEGG enrichment results were plotted by bioinformatics The counting tool in Photoshop is used to label each proliferating cell and the number of proliferating cells is obtained.All experiments were repeated at least six times. Statistical significance was evaluated using the Student's t-test. P values of less than 0.05 were used as the threshold to determine the significance.To show the differences between hedgehog spine and mouse hair on the dorsal back skin, we compared the length and diameter of the spine and hair at the full-growth phase, when they reach their biggest size Figure A. The heSpines and hairs are produced hair their follicles embedded in the skin. We then compared their follicle morphology and size at the full-growth phase. HE staining showed that the spine follicle and hair follicle have similar structures mainly including the epithelial and mesenchymal components, which were verified by immunostaining for K14 and Vimentin, respectively Figure B-C. TogeTo explore if the formation of large spine follicle formation is due to increased cell proliferation, we examined the number and distribution of proliferating cells in spine follicles and hair follicles at the growth phase using BrdU labeling and PCNA immunofluorescence staining. The majority of proliferating cells are distributed in the bulb region in both hair and spine follicles Figure A. Spine To identify the molecular difference between spine follicles and hair follicles that may contribute to their different sizes, we analyzed the transcriptome by RNA-sequencing of growth phase follicles Figure A. Venn dBiological processes of gene ontology (GO) revealed that 9 terms were related to mitochondria and energy metabolism Figure A. These To determine if ATPase affects the size of the spine follicle, we intracutaneously injected Blebbistatin (an ATP synthase inhibitor) However, the spine in the experimental group was significantly shorter and thinner than the control group when the follicle entered the full growth phase at D9. The average length and diameter of the spine have reached 7.52 ± 0.66 mm and 0.99 ± 0.07 mm (P = 0.003), respectively, while the average length and diameter of the experimental group were 5.32 ± 1.86 mm and 0.73 ± 0.21 mm (P = 0.002), respectively Figure A-B. ThesWe then used PCNA immunostaining to determine if ATP synthase influences spine follicle size by regulating cell proliferation. Interestingly, while the spine follicle didn't change in size at D3, the number of PCNA+ cells in the blebbistatin-treated follicle 1040 ± 17) is significantly decreased than that in the control group 1175 ± 25), particularly in the bulb region (Figure 0 ± 17 is ± 25, paThe role of ATP synthase is to synthesize ATP and generate energy in mitochondria. And ATP synthase upstream pathway is the respiratory chain in mitochondria, which transfers electrons to generate oxygen into the water. Therefore, the respiratory chain plays an important role in regulating ATP synthase. The last enzyme in the respiratory chain is cytochrome c oxidase. And previous study shows that COX2 can regulate ATP synthesis in rat liver mitochondria In contrast to the control group, the spine of the experimental group became significantly smaller in size after NS-398 treatment on D9 Figure A. The avWe also used PCNA immunostaining to examine if COX2 influences spine follicle size by regulating cell proliferation. The number of PCNA+ cells in the NS-398-treated follicle is significantly decreased than that in the control group, particularly in the bulb region at D3 and D9 Figure C-F. ThisDiverse skin appendages such as scales, feathers, and hairs are generated in amniotes to form the boundary between the animal and the environment. Interestingly, novel functions evolve in structurally-similar appendages in different animals which enable organisms to adapt to terrestrial conditions. The hair follicle in mice and spine follicles in hedgehogs have similar follicular morphology but with different sizes that allow them to adapt to the environment differently, for example, keeping warm for mice and defensive response for hedgehogs. In this study, we studied the mechanism by which the hedgehogs form bigger spine follicles with mouse hair follicles as a comparison. We unveiled that the COX2-ATP synthase axis controls the size of spine follicles by regulating cell proliferation in hedgehogs.The control of organ size is a central question in biology The present study showed that, although hair follicles in mice and spine follicles in hedgehogs are similar skin appendages in morphology, their sizes can be prominently different. At the full growth phase, the spine follicle is 4 times longer than the hair follicle in length and the diameter is more than 10 times in width. We show that it is because of a significant increase in cell proliferation number but not proliferation rate that leads to enlarged spine follicles in hedgehogs, compared to the hair follicles in mice. Our study identified how cell proliferation controls differently-sized organ formation in different animals. Indeed, this is common in living animals. For example, with the decline of cell proliferation, the liver volume of mice and rats tends to be stable and maintains a specific size. The proliferation in the liver of mice and rats leads to a huge difference in the number of cells, which leads to a difference in liver size Although the size of organs in an individual is constant in adulthood, the size of organs in different individuals of the same species often varies, and this is affected by many factors such as age, sex, height, and body weight. In humans, axial eye length is positively correlated with height and weight Hair follicles in humans, mice, goats, and birds have similar morphological structures and share similarities in the growth cycle. With the interaction between mesenchymal and epithelial cells, they all undergo cyclic regeneration regulated by various factors. Current research indicates that the signals that regulate growth and regeneration are similar, including Wnt, SHH, and BMP, but the regulatory mechanisms of these signals vary among different species and can lead to the formation of different morphological architectures. The hedgehog spine follicle is also one of the skin appendages, and the morphological regulation during the regeneration process of the spine follicle may also be controlled by the same signaling pathways. Thus, it is intriguing to study the specific controlling mechanism in hedgehogs or other animals with skin appendages in future studies.While the size of hair follicles has been studied, we investigated the mechanism by which size control is regulated in spine follicles in the present study. The bioinformatic analysis and functional experiments in our study indicate that ATP synthase and COX2 have a more specific expression in spine follicles compared to the hair follicles, and inhibition of them resulted in a smaller spine follicle and spine in size. However, inhibitors cannot accurately target the location of the spine follicle and may cause off-target effects. While the transgenic technology for hedgehogs is difficult to achieve, we may use lentivirus with specific promoters to activate or silence gene expression in future studies. ATP synthase is the key enzyme for energy metabolism that participates in oxidative phosphorylation COX2 inhibitors can inhibit ATP synthesis in rat liver mitochondria ATP synthase can provide energy for the sodium-potassium pump and power for K+ and Na+ to enter and exit cell, which is crucial for stem cell differentiation and organogenesis in multicellular organisms In conclusion, the present study reveals a novel mechanism by which the mitochondrial COX2 regulates ATP synthase to control spine follicle formation by regulating cell proliferation. Our study also suggests that, in addition to forming the adaptive pattern to accommodate the extrinsically ever-changing environment, the animals also evolve different intrinsic molecular regulatory systems that create biodiversity, including but not limited to the size control of the skin appendages.Supplementary figures.Click here for additional data file.


Health anxiety (HA) by proxy is described as parents’ obsessive worries that their child is severely ill although this is not medically confirmed. Research on HA by proxy suggests that it is highly distressing for the parent and that the child may be at risk of developing maladaptive symptom coping strategies. No targeted treatment for this group exists. We developed PROXY, an 8-week psychological internet-delivered treatment for parents with HA by proxy. The treatment components of PROXY are informed by cognitive behavioral therapy as well as acceptance and commitment therapy, and it was developed in collaboration with parents experiencing HA by proxy and clinical experts.This paper describes the protocol for a study investigating the potential effects of PROXY on parents’ worries about their children’s health using a single-case experimental design (SCED).Five parents clinically evaluated as experiencing HA by proxy will be included. A replicated randomized SCED study will be conducted in which each participant will be randomized to receive treatment after a baseline period of between 7 and 26 days (phase A). The treatment phase duration is 8 weeks for all participants (phase B), followed by a follow-up phase lasting between 14 and 33 days (phase C), ensuring that all participants remain in the study for 96 days. Participants will report daily anxiety level by SMS text message throughout the study. They will also answer self-report questionnaires, including questions on HA by proxy and own HA, 4 times during the study. Data will be submitted to structured visual analysis to inspect anxiety level, the variability of anxiety, trends, the overlap of data points among phases, when effects occur, and the consistency of data patterns across participants. Furthermore, randomization tests will be conducted for each participant to test the null hypothesis that PROXY will have no effect on participants’ anxiety.The recruitment of parents began in June 2022. As of March 2, 2023, a total of 4 parents have been included in the study. Data collection was expected to cease in April 2023.To the best of our knowledge, this protocol describes the only study of treatment for HA by proxy. As the prevalence of this condition is still unknown, a SCED was chosen because this method allows the inclusion of very few participants while still providing information on effects and treatment courses. Conducting the study with a replicated randomized phase design enables methodologically sound testing despite the inclusion of very few participants. The results will inform researchers on individual treatment courses and effects, which may direct future research in terms of the possible mechanisms of change, ideas for how to refine the treatment content, and how the treatment may be offered to patients in the future.ClinicalTrials.gov NCT04830605; https://clinicaltrials.gov/study/NCT04830605DERR1-10.2196/46927 Health anxiety (HA) by proxy is defined as parents’ excessive fear that their child is experiencing a serious illness although this is not confirmed by a medical professional . It is cA recent qualitative study investigating the lived experiences of parents with HA by proxy found that these parents experience significant distress as a result of their anxiety, and they struggle with finding the right balance in worrying about their child’s health . FurtherHA by proxy may also have consequences for the child. Evidence suggests that HA symptoms in parents with health worries and increased focus on somatic concerns play a significant role in HA symptoms in children . FurtherThe members of the research team behind this study have previously investigated the assessment of HA by proxy by developing the Health Anxiety by Proxy Scale (HAPYS) with promising results . Now thaPROXY is inspired by the existing iACT program for HA and uses the same web platform . The treNext, the treatment manual went through several rounds of feedback from the project group. Further written feedback on the text manual was provided by 2 (67%) of the aforementioned 3 parents with individual comments in Microsoft Word document format. These 2 parents also participated in patient videos about living with HA by proxy, how to involve relatives, and how to speak to their children about their anxiety. Finally, the content was programmed on a web platform with close collaboration between the project group and web developers.while using the web-based program [The thinking-aloud method was used to test the usability of specific selected parts of PROXY. The method involved participants verbalizing their thoughts  program ,19. A maPROXY is an 8-week therapist-supported internet-delivered program containing written psychoeducation, audio files, behavioral exposure exercises, homework, and videos distributed in 8 modules . The theWeek 1Gain knowledge about health anxiety (HA) by proxy (module 1)Helper: knowledgeAim: for the parent to obtain knowledge about having HA on behalf of their child and explore how they can begin to understand their own anxietyExercises: (1) write own story of anxiety and (2) fill in the anxiety spiralVideos and audio: (1) parent videos about having HA by proxy, (2) psychoeducation about HA by proxy, and (3) introduction video to the treatmentHomework: pay attention to when and where anxiety arisesLearn about values (module 2)Helper: valuesAim: to help the parent identify their own values as a parent and motivate them to start coping differently with their anxietyExercises: (1) consequences of the anxiety, (2) reflections on being a good parent, and (3) own values as a parentVideos and audio: audio with focus on identifying valuesHomework: practice putting off worries and set aside a specific time of day to worryWeek 2Look at the anxiety from the outside (module 3)Helper: defusionAim: introduction to anxiety-related control and avoidance strategies, followed by exercise to investigate own behaviors; learning how to defuse from thoughts and feelings; the process of coping differently with the anxiety is initiatedExercises: (1) “Don’t think about the ice cream,” (2) behavior analysis, and (3) defusion exerciseVideos and audio: audio with defusion exerciseHomework: practice putting off control and avoidance behaviorWeek 3Practice acceptance (module 4)Helper: acceptanceAim: to practice accepting and holding the anxiety as a new way of copingExercises: (1) stop, breathe, observe inner states, and prioritizeVideos and audio: (1) audio with acceptance exercise and (2) audio with body scanHomework: practice having more breaks during the day, and practice acceptance with mindfulness audio filesWeek 4Challenge the anxiety (module 5)Helper: exposureAim: to motivate parents to gradually expose themselves to their anxiety; focus on letting personal values direct choices instead of control and avoidance strategiesExercises: (1) exposure exercise with pictures of sick children, (2) drafting their anxiety hierarchy, and (3) exposure work sheetVideos and audio: noneHomework: practice exposureExtra: a module for relatives is introduced with the aim of providing information on HA by proxy and how to be supportive during treatment, including exposure exercisesWeek 5Practice self-compassion (module 6)Helper: self-compassionAim: show the connection among one’s expectations, stress, and anxiety, along with how one can be more kind to oneself and practice self-compassionExercises: (1) “How would you treat a friend?” (2) working with own conditional assumptionsVideos and audio: audio on self-compassionHomework: do something nice for oneself and practice exposureWeek 6Use your network (module 7)Helper: parents’ networkAim: to help parents use their close network for support and provide tools to talk about their anxiety with others, including their childExercises: (1) How is the anxiety affecting own close relations? (2) talk about the anxiety with a friend and partner, and (3) talk about the anxiety with child and or write a letter to childVideos and audio: (1) parent videos about talking to their children about their anxiety and (2) parent video about family supportHomework: talk to child about anxiety and practice exposureWeeks 7 and 8Your path further (module 8)Aim: to recap the treatment content, emphasize why it is important to continue working with acceptance, values and exposure, and how to prevent relapse; furthermore, advice about how to handle future child symptoms and physician visits is providedExercises: (1) continued work and insights from the treatment and (2) strategies to apply in the event of setbacksVideos and audio: noneThe content framework is based on a combination of ACT  and CBT helper, which is a treatment component  that will help the patient move in a value-based direction in life instead of being trapped trying to control and avoid anxiety  while measuring the dependent variable  repetitively and often . SCEDs sThe project was registered on ClinicalTrials.gov on April 2, 2021 (NCT04830605).This study will apply a replicated randomized single-case AB phase design for testing the treatment effects. AB designs are particularly useful when testing a psychological treatment that cannot be subjected to withdrawal or to repeated reversal between treatment and baseline. By adding a randomization feature and a replication feature to this basic design, both the internal and external validity are strengthened -29; for Specifically for this study, the intervention is randomly introduced after 7 to 26 days in the baseline phase (phase A). After the intervention (phase B), a follow-up phase (phase C) lasting between 14 and 33 days, depending on when the intervention was introduced, ensures that every participant has the same number of daily measurement points throughout the study , or autism spectrum disorderSuicidal riskRecently started taking psychotropic drug therapy (within the last 2 mo)Child with severe health problems requiring care in hospital settingParticipants are recruited in two ways: (1) participants self-refer to the project through the secure email system at the Research Clinic for Functional Disorders and Psychosomatics (hereinafter the Research Clinic), Aarhus University Hospital, and complete the project questionnaire  in ReseaParticipants are assessed using a short standardized diagnostic interview based on schedules for clinical assessment in neuropsychiatry (SCAN) ,33 and sAll participants receive written and verbal information about the project before signing the consent to participate. The project has been approved by the Danish research ethics committee (1-10-72-296-20) and registered with the Danish data protection agency (1-16-02-921-17).The primary outcome measures of HA by proxy are answered through a link in an SMS text message sent to the participants every day during the study period. The measures contain 3 items selected from the HAPYS  (1-3), 1“On a scale ranging from 1 to 10, how well do the following statements describe your day? 1=‘does not describe it at all’ and 10=‘describes it very well.’”“I have had persistent worries about my child’s health.”“I have been worried that my child suffers from a serious physical illness.”“I have had the need to reassure myself by seeking a physician, examining my child, Googling symptoms, or something else.”“My anxiety for my child’s health has influenced my time together with my child.”“I have done something in the past 24 hours that was important to me in spite of my anxiety.”The secondary outcome measures are full questionnaires answered 4 times during the study period. An overview of measures and data collection is presented in The HAPYS is a 26-item self-report questionnaire assessing HA by proxy, including an impact section with an additional 6 items [abdominal pain with feels unwell. The monitor subscale was translated into Danish for this study following World Health Organization (WHO) guidelines [The Adult Response to Children’s Symptoms scale measures parental behavior in relation to the child having abdominal pain and consists of 4 subscales: protect, monitor, minimize, and distract . For thiidelines . Both scidelines -40.The Pain Catastrophizing Scale–Parent version consists of 13 items assessing parents’ thoughts and feelings when their child has pain symptoms  . PsychomHA and illness worries are assessed using the Whiteley Index ,43. In tParents’ levels of distress, anxiety, and depression are screened by the Symptom Checklist-12, which are subscales from the Symptom Checklist-90-Revised  that havThe WHO-5 Well-Being Index is a 5-item rating scale measuring subjective well-being on a scale ranging from 0 to 5. The raw score ranging from 0 to 25 is multiplied by 4, providing a final score ranging from 0 to 100, where a lower score represents worse well-being . This scThe Experience of Service Questionnaire measures parents’ experience with the treatment they have received ,50. In tThe Negative Effects Questionnaire-20 measures 6 factors related to the negative effects of psychological treatment: encompassing symptoms, hopelessness, failure, stigma, dependency, and quality . Each neThe Internet Evaluation and Utility Questionnaire will be used to evaluate the experience of receiving internet-delivered treatment. Using 15 items, this instrument measures the usability and utility of an internet-delivered treatment program with questions answered on a 5-point rating scale .Three questions evaluating SMS text message data collection will inquire about how the participants experienced answering an SMS text message every day, whether it had any influence on their daily life, and whether their answers were affected by the fact that they had to do this every day. The questions are answered in free writing and will inform the researchers about the experience of this type of self-monitoring.After each module, the participants are asked about the content of the modules answered in free writing.First, the data collected daily will be submitted to visual analysis, with the scores of the dependent variable on the y-axis and the measurement times on the x-axis. Six main features are visually examined using the guidelines provided in the study by Lane and Gast : anxietyRandomization tests will be conducted for each participant to test the null hypothesis that PROXY will have no effect on the participants’ anxiety -59. A raPrimary test statistic: a mean comparison of phase A and BC will be carried out to investigate mean anxiety levels before and after treatment entry (mean A − mean BC = test statistic). As the starting point of phase B (intervention) is randomly determined and the follow-up phase does not include any new intervention methods, phases B and C are combined. As a mean comparison does not take into account the expected gradual and delayed effect of treatment, we also use a secondary test statistic.Secondary test statistic: response functions  will be As the exact course of treatment effect and the exact scores of anxiety are not known a priori, a multiverse approach will be followed exploring different response functions . These dP value of the randomization test [P values are combined using the additive method formulated by Onghena and Edgington [We will calculate the primary test statistic as well as the secondary test statistic for each possible randomization scenario of the treatment start using the obtained data. The proportion of test statistic values that are as extreme as, or more extreme than, the test statistic of the true treatment starting point is the ion test ,57-59. Tdgington .U for SCED studies. Tau-U is particularly good if data show a baseline trend [In addition to the randomization tests, effect size measures will be calculated for each participant. An effect size measure is a standardized indicator that can be compared across studies and is not used as a test statistic in this study. Parker et al  recommenne trend ,66. In aThe secondary outcome measures will be analyzed using descriptive statistics because data from only 5 participants will be included.The recruitment of parents began in June 2022. As of March 2, 2023, a total of 4 parents have been included in the study. Data collection was expected to cease in April 2023. The project was funded in September 2019. Results are expected to be published in 2024.HA by proxy is a novel research area where much still remains to be investigated. Recent studies suggest that parents experiencing this type of anxiety struggle with how to cope with their health-related worries about their child . When a In this study protocol, we described the treatment components of PROXY and how these components were established in the web-based treatment program. It is a collaboratively developed treatment program that involved clinical experts, web developers, and users in the process. PROXY received positive feedback from patients during development, and it seems to be a usable treatment program. Conducting this first testing and having patients receive treatment in the program may shed light on how to further improve the treatment content of PROXY.The SCED approach was chosen for the first testing of PROXY because it is particularly useful when testing novel treatments for smaller patient groups . It enabThe randomization and replication across participants are important methodological strengths of this SCED study. By using randomization and randomization tests, confounding factors that are time related are statistically controlled for -29. The The selected test statistics for the randomization test are mean comparison and predicted response functions. Using test statistics based on predicted response functions for SCED studies is a newly described framework with the purpose of offering statistical analysis for data with nonlinearity and delayed or gradual changes to the primary outcome measure, as is often seen in psychological treatment . The advAs the treatment for patients with HA by proxy has not been previously examined, this study is particularly important in terms of describing the treatment development and content as well as the research protocol for using a SCED for the first testing of PROXY. Applying a SCED provides us with the opportunity to investigate the effect of the treatment in a small sample and explore individual treatment courses in detail. Furthermore, the detailed visual analysis provides implicit tests of treatment mechanisms. This has relevance in clinical practice and can potentially be used to inform larger studies on the mechanisms of change and when and how the treatment may be offered to patients in the future . In addi


Loa loa microfilaraemia in Gabon.There is a lack of systematic evidence for strategies to control loiasis transmission in highly endemic regions. Here we assessed albendazole and ivermectin based treatment regimens to reduce L. loa microfilaraemia between 5,000 and 50,000 microfilariae/ml were randomized to either a control or one of three intervention groups  consisting of three-week twice daily 400mg oral albendazole followed by 1) no treatment, 2) two further weeks of twice daily 400mg albendazole, or 3) a single dose of ivermectin in this open label randomized assessor blinded controlled clinical trial. The primary outcome was the proportion of participants with L. loa microfilaraemia ≤ 100 mf/ml at Day 168.Eligible adult patients with In the efficacy-population of 42 patients 0 , 1 , 5  and 2  participants met the primary outcome of microfilaraemia below 100/ml at day 168. A 80–90% reduction of microfilaraemia was observed in the active treatment groups.The 5-week regimen of albendazole or a 3-week regimen of albendazole followed by ivermectin were most efficacious to reduce microfilaraemia. All therapeutic regimens were well tolerated and safe.PACTR201807197019027.Trial registered at the Pan-African Clinical Trials Registry:  Loa loa transmission in highly endemic regions. This study assessed the parasitological efficacy of albendazole alone (three or five weeks regimen) or albendazole (three weeks regimen) with a single dose of ivermectin for the reduction of microfilaraemia in adult patients in Gabon. This proof of concept study showed highest efficacy of about 90% reduction of microfilaraemia when albendazole was given twice daily for five weeks or albendazole was administered twice daily for three weeks followed by single dose ivermectin. As there were no safety concerns these findings indicate that albendazole based treatment regimens may in principle be useful for implementation in population-based control programs. To overcome the problem associated with prolonged oral treatment regimens new injectable drug formulations with extended release of active substances may be considered to facilitate implementation of such control programs.To date there is a lack of safe treatment options to be used for the control of  Loa loa typically manifesting as a migrating eye worm under the bulbar conjunctiva or transient peripheral oedemas. L. loa is endemic in West and Central Africa [Loiasis is a highly neglected infectious disease caused by the filarial nematode l Africa , 2 with l Africa . For a ll Africa , 5. Howel Africa , 7. Due l Africa .Chrysops silacea and C. dimidiata. There is however a lack of evidence for appropriate drug regimens to be used to treat patients in endemic regions. Diethylcarbamazine (DEC) is known to be active against microfilariae and adult worms [Onchocerca volvulus when suffering from ocular onchocerciasis [Currently there are no control programs for this filarial disease in any of the endemic countries. To effectively reduce onwards transmission of this chronic infection, clearance of microfilaraemia is necessary to reduce infectiousness to the intermediate arthropod hosts lt worms  but its erciasis , 9.L. loa. Its use in highly microfilaraemic individuals  was shown to be associated with the risk for severe neurological adverse reactions with potentially fatal outcome [Ivermectin is one of the most widely used antiparasitic drugs  and high outcome , 7. It w outcome  and is tL. loa and is perceived to gradually kill adult worms. Due to its rather slow onset of action, it is commonly used over a 21 day period to safely reduce L. loa microfilaraemia before initiation of treatment with a rapidly acting anthelminthic drug [Albendazole has been demonstrated to exert a slow but reliable activity against microfilariae of hic drug –15.L. loa infection in a highly endemic region in Gabon and to evaluate the safety and tolerability of the respective regimens.The clinical evaluation of treatment approaches suitable for mass drug administration programs are necessary to ensure that these regimens prove safe and effective in the control of loiasis. As DEC and ivermectin cannot be used without special precaution due to safety concerns in highly microfilaraemic individuals, the use of albendazole as initial drug constitutes currently the only viable treatment option to safely reduce microfilaraemia to a level that allows safe use of sequential therapy with another drug. The aim of this clinical trial was therefore to evaluate three alternative fixed treatment regimens for their efficacy to reduce microfilaraemia of The study was conducted according to the Declaration of Helsinki, the International Council for Harmonisation for Good Clinical Practice and the applicable laws and regulations in Gabon. The study was approved by the institutional ethics committee of the Centre de Recherches Médicales de Lambaréné . Study participants provided written informed consent before any study procedure was performed.L. loa microfilaraemia in a highly endemic region in Gabon. The trial was conducted according to the study protocol  and no changes to the trial protocol or statistical analysis plan were made after trial commencement.This study was designed as a randomized controlled assessor-blinded clinical trial evaluating the efficacy and safety of three albendazole-based regimens versus a control group in adults with proven L. loa and a range of other parasitic infectious diseases [L. loa microfilaraemia between 5,000 and 50,000 mf/ml in peripheral blood during daytime sampling (10 a.m.– 3 p.m.) assessed microscopically by thick blood smears of capillary blood, residence in the study region, willingness to participate in this clinical trial for the entire 6-month follow-up period as evidenced by written informed consent. Exclusion criteria were reported intake of albendazole, ivermectin or DEC within 4 weeks prior to recruitment; intolerance or allergy to study drugs; pregnancy or lactation, known active viral hepatitis or other liver disease, known HIV infection or presence of an immunosuppressive disease, history of epilepsy, encephalitis, meningitis or encephalopathy, or any other acute or chronic medical condition judged by the investigator to put the potential participant at more than acceptable risk or to significantly affect the results of the study.The study was conducted at the Centre de Recherches Médicales de Lambaréné (CERMEL), in the regions of Tsamba-Magotsi and Lambaréné, Gabon, from May 9 2018 to April 16 2020. The study region is highly endemic for diseases . ResidenL. loa microfilarial load was below 4000 mf/ml to minimize potential risks associated with the use of ivermectin in microfilaremic individuals. This conservative approach was taken to minimize study related risks. In case of microfilaraemia above this threshold, participants were transferred to the ALB + ALB treatment group.Eligible patients were randomized by an independent investigator to the inactive control group (CONTROL), or one of the following three albendazole-based fixed sequential treatment regimens: ALB, ALB-ALB, or ALB-IVM. Computer generated block randomization was used to generate the allocation list with a block size of 7 and an allocation ratio of 1:2:2:2. Once seven participants were judged eligible, the randomization procedure was initiated. Based on a sequential list of eligible patients, individuals dropping out before the end of the treatment period were replaced in the same treatment group with the next eligible participant. Participants in the control group (CONTROL) received symptomatic treatment with the antihistaminic drug loratadine 10 mg  oral once daily, intake under supervision, for seven days. All participants enrolled in one of the three intervention groups received twice daily oral therapy with 400mg albendazole  after food intake for 21 days. Supervision of drug intake was daily during the first week of treatment. For the remaining treatment duration directly observed treatment was done every second day and tablets for the unsupervised drug intakes were provided each time only for the following day. At each time point information on compliance and safety was provided. Participants randomized to the ALB-ALB group received an additional 14-day treatment course of twice daily 400 mg albendazole with the same supervision of drug intake as mentioned above. Participants allocated to the ALB-IVM group received supervised treatment with a single dose of 150 μg/kg ivermectin  on day 23. Ivermectin was administered only after assurance that Participants underwent a physical examination with demographic data and medical history recorded at the baseline visit. Blood samples were taken for haematology , biochemistry  and viral serology (hepatitis B and C). A urine pregnancy test (SAS Ultimate hCG—SA Scientific Ltd.) for women under the age of 55 was performed to rule out pregnancy and counselling to avoid conception during the study period was provided. Assessments of treatment adherence, clinical status, adverse events, and concomitant medications were recorded at each visit during the treatment and follow-up period. In addition, participants were advised to contact the study health centre in case of occurrence of any adverse event. Follow-up visits were conducted by a trained nurse and a fieldworker. Any potential signs or symptoms recorded by them were discussed with the study doctor for further assessment or necessary action. Tolerability and safety assessments were performed until study completion at Day-168 including the recording of adverse events and clinical laboratory test results. A further urine pregnancy test was done for female participants <55 years of age at the end of the study follow-up period. All participants randomized in the control group were offered a 28 days albendazole treatment regimen at the end of 6 months follow up period.Loa loa microfilarial density was expressed as the number of microfilaria per ml of blood by multiplying the observed microfilariae in 20μl of blood with the factor 50 [Mansonella perstans microfilaraemia, which is a common co-endemic filarial pathogen in this region [Two thick smears of 20μl venous or capillary blood were prepared at baseline, before study drug administration and on days 1, 3, 7, 10, 14, 17, 21, 23, 24, 26, 28, 30, 33, 35, 37, 42, 49, 56, 70, 84, 98, 112, 126, 140, 154 and 168. Blood smears were dried and stained with 20% Giemsa stain for 20 minutes according standard protocols . Stainedactor 50 . A bloodThis clinical trial was a proof-of-concept study without predefined statistical hypothesis testing between groups. A total of 42 patients were planned to be recruited to ensure adequate precision of the primary outcome, i.e. microfilarial density reduction, based on the natural variability of microfilaraemia. A total of 12 evaluable patients were planned for each of the three intervention groups, in addition to 6 patients in the control group, thus resulting in an overall treatment allocation ratio of 1:2:2:2. Assuming a 50% proportion of patients achieving a microfilaria reduction below 100/ml, this sample size allowed a precision of the point estimate with a 95% confidence interval ranging from 25–75%.L. loa microfilarial density ≤100 mf/ml within 6 months.Microfilarial density thresholds, below which onwards transmission by the Chrysops vector cannot be sustained, are currently unknown but may depend on several factors including the life span of the insect vector . HoweverL. loa microfilaraemia at each month after onset of treatment and the median time from initiation of treatment to the individual nadir (lowest level of microfilaraemia) of L. loa microfilaraemia.Secondary efficacy outcomes were the proportion of individuals in each treatment group being amicrofilaraemic at the last observation on day 168; the median reduction of Principal safety outcomes were defined as the occurrence of any related or unrelated grade 3 adverse event or serious adverse event (SAE) and occurrence of all adverse events at least possibly related to study medication assessed after the initiation of study treatment. Adverse event type, severity, seriousness and relationship to study drugs were assessed by the investigator. Seriousness was determined based on the serious adverse event definition in the ICH guidelines.L. loa microfilaraemia ≤ 100mf/ml were presented by study group using a frequency table with a 95% confidence interval. The albendazole group was chosen as reference to compute odds ratios for achieving the primary endpoint. The reduction in microfilaraemia per group was calculated as the median of the relative reduction in microfilaraemia per each participant. Area under the curve of log-10 transformed L. loa microfilaraemia from baseline until D168 by the trapezoidal rule has been calculated with 95% confidence intervals. The number and percentage of participants who experienced adverse events in the safety population consisting of all patients receiving study treatment were tabulated by study group.All data were collected on paper record forms that were entered into an electronic database  . StatistL. loa microfilariae were screened for eligibility into the study. 49 eligible patients were randomized into the different study groups of whom three dropped out prior to treatment initiation because of withdrawal of consent and 4 patients in the ALB+ALB group discontinued treatment (2 patients moved out of the study area and 2 participants refused to continue the study due to repeated blood sampling during the treatment period). These 7 individuals were replaced by the next available eligible patient. Overall, 36 randomized patients (12 in each of the intervention groups) completed their first 21 days of ALB treatment. Two patients randomized to the ALB+IVM group received follow-up treatment with an additional 14 days of albendazole, instead of a dose of ivermectin, because their microfilaraemia was still above the protocol-specified threshold of 4,000/ml , as specified by the study protocol. These patients were allocated to the ALB+ALB group for statistical analysis. Moreover, one patient randomized in the ALB+ALB group was inadvertedly treated with ivermectin after the initial 21 days of albendazole and was therefore pragmatically considered for statistical analysis in the ALB+IVM group. Six patients in the control group received loratadine for 7 days. The 40 patients who received at least one dose of albendazole, plus the 6 patients in the control group, together constitute the safety population for the safety analysis. One in the ALB group was lost to follow-up after Day 154 because of migration out of the study region and two patients in the ALB+IVM group were lost to follow-up after visits on Day 37 and Day 154 due to withdrawal of consent and migration out of the study region. All other patients were followed-up for 168 days. The per-protocol population for the primary efficacy analysis therefore consists of the 39 patients who completed one of the treatment groups as well as the full 168-day follow-up period. Details of participants’ flow are depicted in the From May 2018 to April 2020 a total of 78 patients positive for As shown in A total of 39 participants were included in this analysis contributing data until the end of follow-up on Day 168. The highest proportion of individuals with blood microfilariae loads below 100/ml were seen in the ALB+ALB group  followed by ALB+IVM group . None of the patients in the control group had a microfilarial load below 100mf/ml .While the median microfilarial load for all treatment groups at baseline was around 10,000 per ml as shown in To compare the activity of each treatment regimen on microfilarial load for each respective group, the relative reduction in median microfilarial load (IQR) was computed. The relative reduction from baseline was >90% in the ALB+ALB and the ALB+IVM groups starting from around one-month post-treatment initiation with some small fluctuations during the 6-month follow-up. The ALB group showed a slightly lower relative reduction of about 80% from one-month post-treatment—also observed at the end of the 6-month follow-up period—while there were also considerable inter-individual variations throughout the follow-up period. The control group did not show any clinically significant relative reduction in median microfilarial load throughout the follow-up period. Details are shown in Area under the curve (AUC) of microfilaraemia was calculated from baseline until D168 in the respective treatment groups as in Of the 46 patients included in the safety population that either received one or more doses of albendazole (40 patients) or were enrolled in the control group (6 patients) a total of 15 (32.6%) reported one or more adverse events at least possibly related to study drugs, including just one patient in the control group, six in the ALB group and four each in the ALB+ALB and the ALB+IVM groups. The most commonly reported at least possibly study drug related adverse events occurred within the first 3 weeks of treatment and were headache, dizziness and fatigue, followed by pruritus, rash and nausea as shown in L. loa micofilaraemia of 10,050 mf/ml. Baseline haematology and biochemistry parameters were normal, except for gamma glutamine transferase at 1.5 time upper normal limit. One day before the end of the three weeks albendazole drug intake, the patient became asthenic with psychomotor retardation without fever. Five days later the decision was taken to hospitalize the patient due to a new onset of vertigo and aggravation of asthenia. At hospital admission, the patient was afebrile, Glasgow 5/5 and asthenic. Neurological examination was normal. Haematology and biochemistry assessments were unremarkable, except for C-reactive protein and gamma glutamine transferase which were 3 and 2 times upper normal limit, respectively. L. loa microfilaria count was 1,400mf/ml. Symptomatic supportive treatment was given and the patient was discharged four days later in full remission with the suspicion of alcohol withdrawal syndrome as the underlying cause of the symptoms. This serious adverse event was classified as unlikely related to study treatment.Among all patients in the safety analysis there were no AEs rated at grade 3 or higher except for one patient with a serious adverse event. This 73-years-old man with a history of alcohol abuse was randomized to the ALB group with an initial The evolution of haematological parameters showed a consistent trend in all active treatment groups with a slight decrease of haemoglobin values from baseline to Day-21 which resolved by day 168. At the same time white blood cell counts increased during the active treatment period before reaching initial levels at the end of follow-up. Importantly, relative eosinophil count increased in all treatment groups before decreasing well below the initial value at the end of the observation period. All these changes were similar in the active treatment groups and were not observed in the CONTROL group. A slight increase in liver transaminases (ALT and AST) was observed following 21 days of albendazole intake in all treatment groups, but not in the control group. Nevertheless, no further increase was observed after an additional course of 14 days of albendazole in the ALB+ALB group, nor after administration of a single dose of ivermectin in ALB+IVM. .Safe and effective treatment regimens that can be used without specific biological monitoring of patients are necessary for the development of drug-based control programs for loiasis. Currently albendazole based drug regimens that are known to be relatively safe in gradually reducing microfilarial load are the only viable options due to safety concerns for the use of DEC and ivermectin when used without detailed biological screening and close follow-up in patients with loiasis –25.L. loa microfilaraemia when compared to an inactive control group. Albendazole given for 5 weeks and albendazole for three weeks followed by a single dose ivermectin treatment appeared to be the most efficacious treatment regimens with 39% and 22% reaching the primary outcome measure of microfilaraemia below 100mf/ml at 6-months. Concordantly, the AUC of microfilaraemia indicated that the ALB+ALB and the ALB+IVM regimens led to the most consistent reduction of microfilaraemia over time. Albendazole is thought not to exert a significant direct activity on L. loa microfilariae, but rather on the female adult worms by inhibiting the reproductive process and direct adulticidal activity in prolonged regimens. Interestingly, the three-week albendazole treatment regimen alone led to a similar decrease during the active treatment period followed by a subsequent rebound in microfilaraemia. This loss of suppressive action in the ALB group was not reported in other retrospective cohorts in Gabon but may be explained by the above-mentioned mode of action of albendazole by insufficient or transient sterilisation of female worms, potential reinfections during the study period, incomplete adherence to the drug regimen, or the high level of transmission of loiasis in the study region. The addition of a single dose of ivermectin has a similar effect as prolongation of albendazole therapy alone for another 2 weeks and was similarly not associated with complete suppression of microfilaraemia. Importantly, adding a single dose of invermectin constitutes a logistical advantage compared to a further two weeks of albendazole intake in terms of treatment adherence and treatment costs. Contrarily to this finding a cohort of patients with imported loiasis in Italy, where reinfection of individuals was no longer possible, showed complete cure with an albendazole-ivermectin based regimen [L. loa filariae are available. Whereas albendazole is thought to act first on the fecundity of female adult worms, an adulticidal effect has been postulated for longer and high dose regimens. Contrarily, ivermectin is thought to primarily act by supressing the microfilarial load without importantly affecting the adult worms. [This clinical trial systematically assessed the parasitological outcomes of three different albendazole-based regimens on  regimen . Whereast worms. , 21, 22Overall all treatment regimens proved safe and well tolerated in this relatively small clinical trial. One SAE was retrospectively judged to be associated with an undisclosed alcohol withdrawal syndrome and no other significant safety findings where observed. Various manifestations have been attributed to loiasis including and a probable case of encephalopathy due to loiasis in the absence of any treatment , 26, 27.The transient increase in liver transaminases and increases in relative eosinophil counts paralleled by a modest reduction of haemoglobin during the active treatment period are well-described reversible side effects of high dose albendazole therapy , 28, 29.L. loa microfilaraemia from both venous and peripheral blood samples is another limitation of this study. However, based on the natural diurnal variation of microfilarial load and the consistent blood drawing schedule between treatment groups, no systematic confounding was introduced between treatment regimens. Finally, the evaluation of treatment response from a patient’s perspective should be performed in future evaluations of individual treatment outcomes.It is remarkable that the study population was rather old with a median age above 60 years. This finding is at least partly explained by the fact that older individuals are overrepresented in rural Gabonese villages compared to the national average and that the prevalence of loiasis and to a certain extent the level of microfilaraemia increases with age in our setting. In addition, the professional activities of rural populations may have discouraged participation in this clinical trial due to a demanding study schedule and a prolonged follow up period. The main limitation of this study is its descriptive study design with the limited number of participants included precluding formal statistical comparison between treatment groups in this proof of concept trial. Whereas this study systematically describes the effect of the treatment regimens over a 6-month period, no further conclusions may be drawn for a longer duration of the effect beyond the 6-month follow-up period. Similarly, only a fraction of participants has reached the pre-specified threshold of 100mf/ml, which was set as measure to reduce onwards transmission of loiasis. Further epidemiological and experimental data on the infectivity of loiasis at respective microfilarial loads are required to corroborate robust endpoints for clinical trials aiming at reducing loiasis transmission in highly endemic regions . The assL. loa endemic area. Given that clinical trials on loiasis treatment are scare, adding systematic information on the safety and efficacy of existing drugs in an endemic area is useful and may help standardizing treatment regimens for the control of L. loa transmission.In summary, the efficacy of albendazole based regimens in clearing microfilaraemia seems to be limited to an approximate 90% reduction in this high transmission area. Its slow onset of action over several weeks makes it suitable for the safe and gradual reduction of microfilaraemia. However, at the same time the requirement for prolonged treatment regimens over 3–5 weeks is an important limitation for its large-scale use in population-based treatment programmes. Alternative drugs or drug formulations are therefore needed to simplify treatment regimens and to provide the tools for community-based control programmes in S1 Consort Checklist(DOC)Click here for additional data file.S1 Data(XLSX)Click here for additional data file.S1 Study Protocol(DOCX)Click here for additional data file.


This study compares the effects on peripheral vision and image quality of four myopia control interventions: a) Perifocal spectacles/ArtOptica, b) Stellest spectacles/Essilor), c) MiyoSmart spectacles/Hoya and d) MiSight contact lenses/CooperVision. Five subjects participated with habitual or no correction as reference. Three techniques were used: 1) Hartmann-Shack sensors for wavefront errors, 2) double-pass imaging system for point-spread-functions (PSF), and 3) peripheral acuity evaluation. The results show that multiple evaluation methods are needed to fully quantify the optical effects of these myopia control interventions. Perifocal was found to make the relative peripheral refraction (RPR) more myopic in all subjects and to interact with the natural optical errors of the eye, hence showing larger variations in the effect on peripheral vision. MiSight had a smaller effect on RPR, but large effect on peripheral vision. Stellest and MiyoSmart also showed small effects on RPR but had broader double-pass PSFs for all participants, indicating reduced retinal contrast. Reduction in peripheral retinal contrast might thereby play a role in slowing myopia progression even when the peripheral refraction does not turn more myopic. YoungerStudies on animal models  have shown that not only the foveal, but also the peripheral optical errors can affect eye growth . Even inCurrently, a number of optical myopia control interventions aim to impose defocus on the peripheral retina while central vision is corrected for distance. These myopia control treatments can be in the form of contact lenses  ; spectac2.a)Perifocal Ms/Ps spectacles from ArtOptica , which hb)Stellest spectacles from Essilor , which ic)MiyoSmart spectacles from Hoya , which hThree optical myopia control spectacles were investigated in experiments 1 and 2: The spectacles were custom-ordered for each of the subjects that participated in the study, to ensure that refraction was correct and matched that of the habitual spectacle correction. Throughout the experiments and before every measurement, the participants verified whether their central visual field was in the clear zone and their peripheral visual field was within the treatment zone of the spectacles. Three different techniques were used for the evaluation of the spectacles: 1) Hartmann-Shack wavefront sensors, 2) double-pass point-spread-function (PSF) imaging system, and 3) peripheral acuity evaluation. The study was approved by the Swedish Ethical Review Authority and conformed to the tenets of the Declaration of Helsinki. After the procedures were explained, written consent was obtained from all participants.2.1In the first experiment, peripheral image quality and vision in the 30° nasal visual field were investigated. Five healthy adult subjects were fitted with Perifocal, Stellest, and MiyoSmart spectacles. Subjects 1 (43 years old) and 2 (32 years old) were myopic with tested eye refractions -1.75 DS/-0.5 DC x 140° and -2.75 DS/-0.25 DC x 175° respectively, and subjects 3,4 and 5  were emmetropic. The subjects were looking straight through the spectacles while the image quality and vision were investigated in the nasal visual field of the right eye. For reference, habitual single vision spectacles were used for the myopes, and no spectacles were used for the emmetropes. The duration of experiment 1 was approximately 2 hours per subject.2.1.1Central and peripheral  wavefront errors of the right eye of all subjects were recorded simultaneously for 10 seconds using an experimental dual-angle open-field Hartmann-Shack wavefront sensor (HSWS) . The acqThe wavefront measurements were reconstructed with Zernike coefficients up to the 7th order. Refractive errors  to avoid any neural preferences due to the meridional effect . The sub2.1.3.1Psychophysical measurements were performed on all five subjects for all spectacles. The fixation target was the same Maltese cross used in the objective measurements; this time situated at 2.95 m away from the eye due to room constraints. Resolution cut-off tests at 100% contrast were performed on the right eye at 30° nasal visual field. The left eye was occluded for the psychophysical tests. To avoid fatigue bias, the spectacles were changed between each measurement.2.2The effect of the MiSight multifocal contact lenses on low-contrast (10%) peripheral vision was studied earlier by Papadogiannis et al.  The MiSiThe purpose of the second experiment was to repeat the measurements under the same conditions as the aforementioned study for the three myopia control spectacles and compare the results.Two of the subjects that participated in the study by Papadogiannis et al.  were recPeripheral vision evaluation with low-contrast (10%) grating resolution acuity measurements at 20° nasal visual field were performed monocularly for all spectacles . The subject was fixating on a Maltese cross mounted 2.6 m away, and thresholds were obtained for the left or right eye while the fellow eye was occluded. The duration of experiment 2 was approximately 1.5 hours per subject.3.3.13.1.1The results of the RPR calculations from the wavefront sensors can be found in The calculated values of peripheral astigmatism (J0) from the wavefront sensors can be seen in 3.1.2Peripheral PSFs from the two separate techniques in the 30° nasal visual field are shown and compared in In the double-pass images (DP technique 2), the PSFs are broader for Stellest and MiyoSmart compared to the reference for all subjects. However, this scattering effect is not seen in the wavefront PSFs (HSWS technique 1).Compared to reference, Perifocal makes the PSF smaller for subject 1, more elongated for subject 2, and much broader for subject 3. These effects are seen both in the wavefront images and double-pass images and are due to Perifocal’s progressive design; the location of the -0.35 D PSF will shift relative to the interval of Sturm depending on the subject’s baseline RPR and astigmatism.The through-focus PSFs from the double-pass instrument agree with the previous results in that a shift towards more myopic RPR compared to the reference is only seen with Perifocal. This is consistent for all three subjects and 3.1.3Data from the 100% contrast psychophysical measurements can be found in 3.2The effects on peripheral vision of the three spectacles for myopia control  were evaluated and compared to those of MiSight contact lenses and single-vision spectacles .4.The aim of this study was to identify possible treatment mechanisms in the off-axis performance of four optical myopia control interventions: spectacles with horizontal progressive addition ; spectacles with highly aspherical lenslets (Stellest); spectacles with defocusing lenslets (MiyoSmart); and multifocal contact lenses (MiSight). To tackle the challenges posed by the large optical errors of the peripheral eye together with the myopia control interventions, in-depth measurements were performed utilizing wavefront aberrations (measured by a Hartmann-Shack sensor), through-focus double-pass point-spread-functions (obtained from a double-pass imaging system), and peripheral acuity evaluation  on a few subjects fitted with all four different designs. In short, the results show that none of the interventions improve the peripheral image quality compared to habitual standard spectacle correction and that the shift towards a more myopic peripheral refraction, if any, is small.All four interventions incorporate treatment zones with more positive power than the distance correction, which can be expected to induce peripheral myopic defocus. However, RPR is difficult to measure both because the location of the best peripheral image is poorly defined due to the large depth-of-focus and because it requires simultaneous control of the central refractive state. Therefore, the current study incorporates wavefront sensing simultaneously for the peripheral and the central visual field, as well as through-focus double-pass measurements. Both measurement techniques showed that only Perifocal with progressive addition produced a clear shift of around 1 D to a more myopic peripheral refraction compared to the reference case for all three subjects. The other three designs produced small  is more difficult to assess with the wavefront sensor compared to double-pass and vision evaluations. This might be because the scattered light from the lenslets is not registered by the wavefront sensor, since we do not have enough resolution for measuring as high order scattering as the double-pass system. Similarly, modal wavefront reconstruction and analyses of individual Zernike polynomials is less efficient for such highly irregular wavefronts. For example, the Zernike coefficients for off-axis astigmatism shows a large increase with all four myopia control interventions when assessed with the wavefront sensor for subject 1, which cannot be seen in the computed PSF nor in the through-focus double-pass measurements. However, the double-pass technique also pose challenges in that the light passes twice through the eye, which amplifies scattering and reduces asymmetric features in the PSF (even though the pupil size for the incoming and the outgoing light is not the same ). PeriphWe have not been able to find studies from other authors that have measured the image quality on the peripheral retina through these optical myopia control interventions. However, theoretical and optical bench testing studies agree with our results in that the best peripheral image becomes less well-defined with the studied interventions –37. For The optical design of a myopia control intervention interacts with the optical properties of the natural eye, which leads to individual variations in the image quality on the retina. This can also be seen in the results of this study and may explain some of the variation in treatment effect between children. The optical outcome of some designs, such as those of Perifocal and MiSight, depend on and interact more with the optical errors of the individual eye than the spectacle designs with lenslets. One hypothesis is that when the more subject-depending designs match with the individual eye they offer more effective treatment by alternating the RPR, whereas the lenslet-designs produce a lower but less subject-dependent treatment mainly through contrast reduction. To verify or reject this hypothesis, longitudinal studies on children with peripheral optical evaluations through the myopia control intervention are needed.