Citrus reticulata Blanco and its cultivars, is an important traditional Chinese medicine for the treatment of spleen deficiency-related diseases. To date, the mechanism of CRP alleviating spleen deficiency has not been well investigated. This study aimed to explore corresponding mechanisms with integrating pharmacology and gut microbiota analysis. Firstly, the therapeutic effects of CRP against spleen deficiency were evaluated in reserpine-treated rats. CRP was found to effectively relieve the typical symptoms of spleen deficiency, including poor digestion and absorption capacity, and disorder in gastrointestinal hormones, immune cytokines and oxidative stress. Secondly, high throughput 16S rRNA gene sequencing revealed that CRP could not only up-regulate some short-chain fatty acids producing and anti-inflammatory bacteria but also down-regulate certain spleen deficiency aggravated related bacteria, eventually led to the rebalance of gut microbiota in spleen deficiency rats. In addition, a total of 49 compounds derived from CRP were identified in rat urine using ultra-high performance liquid chromatography-quadrupole- time of flight tandem mass spectrometry. Network pharmacology analysis showed that apigenin, luteolin, naringenin, hesperidin, hesperetin, homoeriodictyol, dihydroxy-tetramethoxyflavone, and monohydroxy-tetramethoxyflavone were the core bioactive components for CRP against spleen deficiency. Further Gene Ontology analysis and pathway enrichment suggested that therapeutic effects of CRP against spleen deficiency involved multiple pathways such as tumor necrosis factor signaling, hypoxia-inducible factor-1 signaling and Toll-like receptor signaling pathway. These results would help to understand the mechanism of CRP alleviating spleen deficiency and provide a reference for further studies.Citri Reticulatae Pericarpium (CRP), dried peels of  Modern researches show that the spleen in TCM theory not only refers to the anatomical spleen, but also includes some functions of the pancreas and lymphatic system , spleen deficiency is a common clinical syndrome and described as symptoms such as emaciation, inappetence, epigastralgia, flatulence, lassitude, wilted complexion, loose stool, c system . Spleen c system . Moreovec system . Gut micc system , antibioc system  and othec system . In turnc system . Recentlc system .Citrus reticulata Blanco and its cultivars (Citri Reticulatae Pericarpium (CRP), commonly referred to as Chenpi in Chinese, is derived from the pericarp of mature fruits of ultivars . It is aultivars . Long-teultivars . Howeverultivars , it is mIn this study, reserpine-induced spleen deficiency rat model was established and used to evaluate the therapeutic effects of CRP. Reserpine, which was once used in the treatment of hypertension, could inhibit the vesicular monoamine transporter and deplete the brain monoamines such as 5-hydroxytryptamine by interfering with storage capacity, and ultimately lead to similar syndromes of spleen deficiency . MeanwhiHereon, the therapeutic efficacy of CRP against spleen deficiency was evaluated in reserpine treated rats based on body signs and biochemical indexes including digestion, absorption, gastrointestinal hormones, immune regulation and oxidative stress. The gut microbiota modulatory properties of CRP in spleen deficiency rats were investigated with high throughput 16S rRNA gene sequencing. Moreover, CRP derived metabolites were identified using ultra-high performance liquid chromatography-quadrupole-time of flight tandem mass spectrometry (UHPLC-Q-TOF-MS/MS) and were further employed in network pharmacology analysis to capture the bioactive components and potential targets of CRP in treating spleen deficiency. This study would be helpful for further understanding of the pharmacological effects and therapeutic benefits of CRP against spleen deficiency related diseases.CRP samples (Batch number: 201712) were acquired from Xinhui Hele Tea Art Co. Ltd. , and were authenticated by Prof. Wenbo Liao from Sun Yat-sen University. Corresponding voucher specimens were kept in our laboratory. Reserpine injection was purchased from Guangdong Bangmin Pharmaceutical Co., Ltd. . The reference standards of hesperidin, naringin, neohesperidin and rutin were obtained from National Institute for Control of Biological and Pharmaceutical Products of China . Hesperetin, naringenin, nobiletin and mass spectrometry (MS) grade formic acid were purchased from Sigma-Aldrich . MS grade acetonitrile was purchased from Fisher Scientific . All water used was distilled and further purified by a Milli-Q system . Other reagents used in the experiment were of analytical grade.−1.The CRP sample (20 g) was cut into small pieces, and soaked in boiled distilled water for three times . After filtration, the whole extracts were evaporated to 500 ml with a rotary evaporator  at 60°C, to obtain the CRP extract with a concentration of 0.04 g mLad libitum. All experimental procedures were approved by the Animal Ethics Committee of the School of Life Sciences in Sun Yat-sen University and carried out in accordance with the National Institutes of Health guide for the care and use of Laboratory animals .Thirty male Sprague-Dawley rats (weighting 180–220 g) were purchased from Guangdong Medical Laboratory Animal Center, and maintained in standard temperature conditions (20–23°C) and a 12/12-h light-dark cycle, with food and water supplied −1 d−1 for 14 days to induce spleen deficiency  for 14 days. Rats in control and model groups received distilled water.Rats were randomly divided into three groups . Rats in model and CRP group were injected subcutaneously with reserpine injection at 0.5 mg kgficiency . Rats in−1) to collect blood samples from abdominal artery. Serum was obtained after centrifugation. In addition, feces samples for gut microbiota analysis were collected from the rectum, transferred into sterile conical tubes, and then immediately frozen in liquid nitrogen. Obtained samples were stored at −80°C until analysis.After the last administration, rats were housed individually in metabolic cages , with fasting and free access to water. Urine samples were collected within 12 h post-dose for metabolite identification. Subsequently, about 1 ml blood was sampled from retro orbital plexus, and then 5 ml 3% D-xylose solution was intragastrically administered to rats. Exactly 1 h later, rats were anesthetized with 10% chloral hydrate (3 ml kgThe activities of superoxide dismutase (SOD) and amylase in serum, as well as the concentrations of D-xylose and malondialdehyde, were determined following protocols of corresponding Kits . The concentrations of gastrin, motilin, cholecystokinin-8 (CCK-8), interleukin 6 (IL-6) and tumor necrosis factor-alpha (TNF-α) in serum were detected using commercial ELISA kits  according to the manufacturer’s instructions.www.biocloud.net). The Spearman’s rho non-parametric correlations between gut microbiota (the top 60 genera in relative abundance) and spleen deficiency related indexes were then calculated by using SPSS software (version 22.0) and displayed by R software with a pheatmap package.Total bacterial DNA were extracted and amplified as previously described . After PTo identify CRP derived metabolites, an aliquot of 100 μL urine sample was vortex-mixed with 200 μL volume acetonitrile for 3 min, and centrifuged at 15,000 × g for 30 min at 25°C. Finally, 10 μL supernatant was subject to UHPLC-Q-TOF-MS/MS analysis.18 column  and maintained at 40°C. The mobile phase consisted of acetonitrile (A) and water containing 0.1% aqueous formic acid (v/v) (B). The elution was carried out following the program: 5–100% A (0–30 min), 100% A (30–34 min) with the flow rate kept at 0.3 ml/min. The mass spectrometry detector was equipped with an electrospray ionization source and operated under the same parameters with our reported studies  coupled with quadrupole/time-of-flight mass spectrometry . Gradient chromatographic separation was performed on a Kinetex C studies .Data acquisition was carried out using Analyst® TF 1.6 software  in information-dependent acquisition mode. Metabolite identification was based on chromatographic elution time, chemical composition, MS/MS fragmentation pattern, and comparisons with available standards and references.With the UHPLC-Q-TOF-MS/MS system, a total of 49 metabolites were characterized in rat urine. The associated proteins of CRP were searched in Comparative Toxicogenomics Database , EncycloAs there are no therapeutic targets about spleen deficiency that were available directly in the databases, targets of diseases that have similar pathological features with spleen deficiency were collected as alternatives. Diarrhea, dyspepsia, enteritis and gastritis were chosen out, and the corresponding targets were searched in DisGeNET .Protein-protein interaction (PPI) data was constructed by inputting official gene symbol to the “Multiple Proteins” search on String website , with orThe Database for Annotation, Visualization and Integrated Discovery was applied for Gene Ontology (GO) enrichment and pathway analysis . The spen = 10). The significant differences between the groups were assessed by ANOVA test in SPSS software (version 22.0), and p values less than 0.05 or 0.01 were considered as significant difference. Figures were plotted with GraphPad Prism (Version 7.00).Data were expressed as mean ± standard deviation (p < 0.01). With the administration of CRP for 14 days, above-mentioned symptoms of spleen deficiency in rats were significantly improved. Rat body weights of CRP group were higher than that of model group, although there was no significant difference. These results preliminarily suggested that CRP administration could relieve the symptoms of spleen deficiency induced by reserpine.TCM clinical syndromes are often difficult to quantify with modern analysis methods. How to use animal models to characterize the syndromes is one of the difficulties in the modern researches on TCM . In thisp < 0.01), suggesting that reserpine treatment could significantly reduce the digestion and absorption in rats while CRP administration could effectively reverse these changes.Subsequently, we further investigated the effects of CRP on biochemical indexes in reserpine-induced spleen deficiency rats, including the parameters used to characterize digestion, absorption, gastrointestinal hormones, immune regulation and oxidative stress. Obtained results were illustrated in p < 0.01), showing that reserpine treatment led to the disorder of gastrointestinal hormones secretion in rats. Changes in these three gastrointestinal hormones caused by reserpine treatment were up to normal after the administration of CRP for 14 days, revealing the efficiency of CRP in improving digestive dysfunction through regulating neuroendocrine.Gastrointestinal hormones take an important role in regulating secretory and motor functions of the digestive tract . It has p < 0.01). After the intervention with CRP, the level of TNF-α and IL-6 restored to normal, suggesting that CRP could help maintain the balance of Th1 and Th2 immune response.The dynamic balance between Th1 and Th2 immune response is important for the immune system . Th1 cytp < 0.05) than control group, indicating that reserpine treatment resulted in free radical disorder in spleen deficiency rats. CRP administration could improve the activity of antioxidant enzyme and reduce lipid peroxidation damage (p < 0.05).In addition, the balance between oxidants and antioxidants is necessary in maintaining health, and corresponding imbalance may result in oxidative stress causing functional disorders and certain diseases . In thisIn summary, CRP administration could effectively alleviate the syndromes of spleen deficiency induced by reserpine treatment in rats, including poor digestion and absorption capacity, and disorder in gastrointestinal hormones, immune cytokines and oxidative stress.p < 0.01), which suggested that reserpine treatment disturbed the balance of gut microbiota. By contrast, supplement with CRP gave rise to a higher community richness than that of the model group (p < 0.01), indicating that CRP is beneficial to the growth of gut microbiota. Beta diversity analysis among experimental groups was performed with PCoA. As shown in Gut microbiota plays a vital role in maintaining normal intestinal functions, such as food digestion, nutrient absorption, integrity of epithelial barrier, and development of mucosal immunity . RecentlBifidobacterium, Lactobacillus, Allobaculum, Psychrobacter, Prevotellaceae_Ga6A1_group, [Eubacterium]_coprostanoligenes_group, Parasutterella, etc. SCFAs , the end products of gut microbial fermentation of indigestible dietary components, appeared to enhance epithelial barrier function, improve gut permeability, inhibit the inflammation. Among them, Bifidobacterium and Lactobacillus are the most widely used probiotics with many health-promoting properties, such as prevention of enteropathogen colonization (barrier effects)  and spleen deficiency-related indexes were calculated using Spearman’s rho non-parametric correlation analysis . The heaeffects) , anti-ineffects) . There heffects) . Allobacdiabetes . Eubacteeduction  In additbutyrate . Studiesbutyrate .Alistipes, Anaerotruncus, Desulfovibrio, Oscillibacter, Ruminiclostridium_9, Ruminococcaceae_UCG-003, Parabacteroides, Ruminiclostridium_5, etc. Among them, Desulfovibrio is the predominant bacteria in human colon sulfate-reducing bacteria, which can reduce sulfate to produce H2S. Since endogenous H2S can poison intestinal epithelial cells, clinical studies have inferred that Desulfovibrio was associated with intestinal diseases  less than protonated nobiletin. Herein compound 26, 27, 28 were tentatively assigned as the mono-demethylated metabolite of nobiletin. Using in vitro incubation methods, Koga et al.  were m/z 151.0043, 119.0508, 107.0154, and 93.0381. Among them, product ions m/z 151.0043  and 119.0508  were presumed to be produced by retro Diels-Alder reaction on chemical bond 1 and 3, while m/z 107.0154 was derived from 1, 3A− with the loss of CO2 (44 Da). The signal at m/z 93.0381 was presumed to be yielded by the breaking of chemical bond 5. Above fragmentation schemes were proposed in As presented in Oral ingestion is the most common mode of administration used in herbal medicine. Generally speaking, exogenous compounds could only exert its pharmacological activity if it is absorbed into the blood and maintains a certain concentration. Urine is filtered from the blood and accumulated all changes in the body. It contains all metabolites in the blood, and thus comprehensively reflects the metabolic information. In this work, the concentration of prototype compounds detected in urine was much lower than that of the corresponding metabolites, suggesting that flavonoid metabolites may be the primary substance for pharmacological activity of CRP, rather than its prototype compounds. Therefore, identified metabolites were employed in subsequent network pharmacology analysis so as to discover the bioactive components and potential targets of CRP against spleen deficiency.On the basis of identified metabolites, a total of 583 potential targets for CRP’s efficacy were defined. Meanwhile, a total of 520 spleen deficiency-related disease targets were acquired by integrating data on diarrhea, dyspepsia, enteritis and gastritis from disease databases. Detailed information was presented in p < 0.05. Top 5 enriched terms of BP, MF, CC categories in the GO analysis were presented in P-Value , cell component (CC), and molecular function (MF) terms. In GO analysis, 330 of 426 BPs, 58 of 72 MFs, and 26 of 33 CCs enriched for these targets were recognized as  P-Value , and thediseases . Among mdiseases . HIF-1 sdiseases . Toll-lidiseases . HoweverClostridium subcluster XIVa in feces was significantly reduced, while the cecal SCFA pool was noticeably increased. Therefore, in the follow-up study to clarify the mechanism of action, we should not only pay attention to identified targets and pathways but also focus on gut microbiota.As shown in Bifidobacterium, Lactobacillus, Allobaculum, Psychrobacter, [Eubacterium]_ coprostanoligenes_group, Prevotellaceae_Ga6A1_group, Parasutterella were up-regulated, while some spleen deficiency aggravated related bacteria including Alistipes, Anaerotruncus, Desulfovibrio, Oscillibacter, Ruminiclostridium_9, Ruminococcaceae_UCG-003, Ruminiclostridium_5, Parabacteroides were down-regulated in CRP group rats. Further Spearman's correlation analysis indicated that there existed a close correlation between the profiles of gut microbiota and the spleen-deficiency related biochemical indexes. In addition, a total of 26 prototype compounds and 23 metabolites were detected in rat urine after the ingestion of CRP. Through network pharmacology analysis, apigenin, luteolin, naringenin, hesperidin, hesperetin, dihydroxy-tetramethoxyflavone, monohydroxy-tetramethoxyflavone and homoeriodictyol were assigned as the core bioactive components, while STAT3, IL6, TNF, JUN, AKT1, TP53, MAPK1, MMP9, PIK3R1, PTGS2, VEGFA, EGFR, IL1B, CXCL8, IL4, CCL2, IL10, and FOS were defined as the major potential targets. Further GO analysis and pathway enrichment suggested that therapeutic effects of CRP against spleen deficiency involved multiple BPs, including inflammatory responses, immune system and oxidative stress such as TNF signaling pathway, HIF-1 signaling pathway, and Toll-like receptor signaling pathway. Besides these targets and pathways, identified compounds could also interact with gut microbiota after oral administration, which probably contribute to the overall therapeutic effects of CRP. This work provided systematic insights to understand the mechanism of CRP alleviating spleen deficiency-related diseases.In this study, the efficacy against spleen deficiency and gut microbiota modulatory properties of CRP were investigated using reserpine treated rats as the animal model. CRP derived metabolites were identified in rat urine and further applied to explore the core bioactive components and potential targets through network pharmacology analysis. As a result, CRP administration could effectively alleviate the typical symptoms of spleen deficiency induced by reserpine treatment, including poor digestion and absorption capacity, and disorder in gastrointestinal hormones, immune cytokines and oxidative stress. Meanwhile, CRP was found to improve the diversity and structure of the gut microbiota in spleen deficiency rats. Compared with model group, some SCFAs producing and anti-inflammatory bacteria including The datasets generated for this study can be found in GenBank accession numbers (MW015148–MW015742).The animal study was reviewed and approved by Animal Ethics Committee of the School of Life Sciences in Sun Yat-sen University.YZ, WP, and WS conceived and designed the study. YZ, XZ, and TC carried out the experiments. YZ, XZ, and PC analyzed the research data and wrote the manuscript.This research was supported by the Science and Technology Planning Project of Guangdong Province in China (2019B090905002).The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Electronic health  has the potential to improve health outcomes. However, eHealth systems need to match the eHealth literacy needs of users to be equitably adopted. Socially disadvantaged groups have lower access and skills to use technologies and are at risk of being digitally marginalized, leading to the potential widening of health disparities.This systematic review aims to explore the role of eHealth literacy and user involvement in developing eHealth interventions targeted at socially disadvantaged groups.A systematic search was conducted across 10 databases for eHealth interventions targeted at older adults, ethnic minority groups, low-income groups, low-literacy groups, and rural communities. The eHealth Literacy Framework was used to examine the eHealth literacy components of reviewed interventions. The results were analyzed using narrative synthesis.A total of 51 studies reporting on the results of 48 interventions were evaluated. Most studies were targeted at older adults and ethnic minorities, with only 2 studies focusing on low-literacy groups. eHealth literacy was not considered in the development of any of the studies, and no eHealth literacy assessment was conducted. User involvement in designing interventions was limited, and eHealth intervention developmental frameworks were rarely used. Strategies to assist users in engaging with technical systems were seldom included in the interventions, and accessibility features were limited. The results of the included studies also provided inconclusive evidence on the effectiveness of eHealth interventions.The findings highlight that eHealth literacy is generally overlooked in developing eHealth interventions targeted at socially disadvantaged groups, whereas evidence about the effectiveness of such interventions is limited. To ensure equal access and inclusiveness in the age of eHealth, eHealth literacy of disadvantaged groups needs to be addressed to help avoid a digital divide. This will assist the realization of recent technological advancements and, importantly, improve health equity. Electronic health , “the use of information and communications technology (ICT) in support of health and health-related fields” , is incrdigital divide .-87.36-87Domain 4 Feel safe and in control. Providing information in users’ preferred language for interventions that target ethnic minorities will be a component that matches Domain 7 Digital services that suit individual needs. The classification of intervention components was initially undertaken by one author (CC), followed by discussion and review with one of the eHLF developers (RO) and among the other authors. The details of the intervention components relating to the eHealth literacy domains are presented in Owing to the heterogeneity of study designs and outcome measures among the included studies, a narrative synthesis was used to answer the research questions. For the research question relating to whether eHealth literacy domains were likely addressed in interventions, a directed content analysis approach was adopted. This approach allows researchers to use an existing theory or framework as coding categories, to develop operational definitions for each category as determined by the theory or framework, and to analyze the content accordingly . For thiFor the research question regarding the effectiveness of interventions, the overall effect size could not be determined because of the diversity of outcome measures and data analysis methods. Therefore, effectiveness was estimated by reporting statistically significant improvement between intervention and control groups or between before and after intervention for one-group pretest-posttest for the outcome measures stipulated. If more than 1 primary outcome measure was stated, only the first 3 were included.The search resulted in 2640 studies; after removing 820 duplicates and an additional 25 records identified through other sources, a total of 1845 records were screened. Following a review of titles and abstracts, 75 studies were retrieved for full text review. A total of 24 studies were excluded for reasons including non–target groups, non–health outcomes, and not eHealth as defined by the inclusion criteria and 2 pilot studies of included studies, resulting in 51 studies reporting on the findings of 48 interventions .Among the 51 studies, 11 were pilot studies, 43 were RCTs . A total of 7 studies used the one-group pretest-posttest design, and 1 was a case study. Among the 43 RCTs, 42 control groups received no intervention, waitlist, standard care, usual care, or in-person education, whereas 1 study did not describe this. The sample size ranged from 1 to 755 .Apart from the postintervention assessment, 13 studies also conducted follow-up assessments, ranging from 1 week to 12 months. Clinical health outcomes were reported in 22 studies, whereas 28 studies measured health-related outcomes such as attitude, behaviors, or knowledge, and 1 study measured both behavior and clinical outcomes. For the quality rating, 2 studies were rated as strong, 24 studies were rated as moderate, and 25 studies were of weak quality. Among the quality rating criteria, 46 studies received a weak rating for selection bias because of their recruitment strategy or fewer than 60% of eligible participants taking part. Only 1 study received a strong rating for blinding, whereas the remaining studies either indicated blinding was not possible or did not report on blinding .Among the 48 interventions, 32 were from the United States, 4 from Australia, 3 from Canada, 2 from the Netherlands and South Africa, and 1 each from China, Malawi, Pakistan, and Taiwan, whereas 1 intervention was undertaken across 3 South American countries, namely, Argentina, Guatemala, and Peru. Low-income groups were the most common target group (n=20), followed by ethnic minorities (n=18), older adults (n=10), and rural communities (n=8). Low-literacy groups were targeted in 2 interventions ,73. A wiWebsites were the most commonly used platforms, with 10 interventions using websites only and 12 interventions combining websites with other platforms such as email or text messaging. A total of 11 studies used text messaging alone, and 4 combined this with other platforms. A total of 10 interventions employed mobile apps. Facebook was used in 2 interventions, and WhatsApp was used in 1 intervention. Mobile phones were the most popular device, being used in 26 interventions, followed by the computer in 22 interventions. Tablets were used in 6 interventions.Among the 48 interventions, 37 were interactive, providing information, tailored content, and/or health-engaging activities, and 11 were noninteractive, providing information or reminder text messages only. The duration of interventions ranged from one 30-min session to a 13-month program, with 3 months being the most common duration.No interventions explicitly reported that eHealth literacy needs were considered during the development, and no assessment of eHealth literacy was undertaken. In fact, eHealth literacy was only mentioned in a study by Carroll et al , which iResearch-based Web Design and Usability Guidelines developed by the US Department of Health and Human Services (UDHHS) [Theoretical frameworks were the most used guidelines for developing interventions, with social cognitive theories (n=15) and the transtheoretical model (n=6) most commonly used. A total of 7 interventions were adaptations or modifications of previous programs, whereas 13 interventions did not provide any details about their theoretical frameworks or developmental frameworks. Only 1 intervention was reported on using the  (UDHHS)  to infor (UDHHS) .User-centered principles were not discussed in any of the papers. The development of interventions in the included studies was mostly expert driven. A total of 22 interventions reported on involving users during development, with needs assessments using focus groups or interviews in 8 interventions, usability or pilot testing in 15 interventions, and 2 interventions mentioning participatory formative research with no details provided. Only Agyapong et al  reportedMost reviewed interventions did not address all eHealth literacy domains. The number of domains addressed ranged from 1 to 7, with only 1 study containing components addressing all 7 eHealth literacy domains . A total1. Using technology to process health information and 2.Understanding of health concepts and language, were both identified in 45 interventions. The domain 5. Motivated to engage with digital services was addressed in 37 interventions through different strategies to encourage users to engage with interventions. A total of 26 interventions provided access to hardware, data plans, or technical support to address the domain 6. Access to digital services that work, whereas 23 interventions supported the domain 4. Feel safe and in control by requiring personal log-in or other forms of privacy measures. The 2 most overlooked domains were 3. Ability to actively engage with digital services and 7. Digital services that suit individual needs; both were identified in less than half of the 48 interventions. Of the 19 interventions containing components of 3. Ability to actively engage with digital services, 15 provided training or instructions on using the intervention, whereas only 6 featured an easy-to-use navigation interface. Among the 18 programs addressing the domain 7. Digital services that suit individual needs, the main strategy was to provide the preferred language of users. Accessibility features catering to individual capability or providing a user interface that suited individual needs such as large fonts or icons or audio options were only identified in 6 interventions [The 2 most addressed eHealth literacy domains, ventions ,73-75,82Although no study explicitly considered or assessed eHealth literacy, the effectiveness of eHealth interventions was nevertheless examined, and the results were mixed. Approximately one-fourth of the reviewed interventions (n=13) did not find statistically significant improvements in their primary outcomes. Although 19 studies reported significant improvements in their primary outcomes, another 12 studies found significant improvements in some primary outcomes, but not all. In addition, 4 studies found improvements but did not report whether such differences were significant. The results of long-term effectiveness also produced mixed evidence. Among the 13 studies that conducted follow-up assessments, 8 found the effects sustainable up to a period of 12 months. A total of 3 studies found effects were not sustainable, whereas 2 did not report on the significance .1. Using technology to process health information could be identified within the intervention components.Among the reported effective 19 studies, there were no consistent patterns of intervention characteristics or eHealth literacy domains likely addressed. These interventions could be interactive or noninteractive, although platforms and devices also varied. The number of eHealth literacy domains likely addressed ranged from 1 to 7. Although a study by King et al  likely a3. Ability to actively engage with digital services) but lack motivation , features to address motivation should be a prominent feature of the intervention. However, if target users demonstrated limited ability to use technologies, interventions such as simple unidirectional text messages, rather than interactive mobile apps, are likely to be more suitable for the target users. Hence, the eHLF and eHLQ will have the potential to advance the field of eHealth literacy and strengthen the reach and impact of digital health interventions [Although the concept of eHealth literacy was introduced more than a decade ago , this reventions .Research-based Web Design and Usability Guidelines by the UDHHS to inform intervention development. However, the guideline authors specifically indicate that they may not be applicable to all audiences, such as people with low literacy who may have different reading and layout needs [There is growing concern that frameworks or guidelines informing the development of eHealth interventions so that they meet users’ needs are lacking , and thiut needs . In addiut needs . Such prut needs . Althougut needs ,21,26. Eut needs ,99,100. 1. Using technology to process health information, 2. Understanding of health concepts and language, and 5. Motivated to engage with digital services. Strategies to assist users in using or engaging with technology and accessibility features of systems that are tailored and responsive to an individual’s ability and capability are generally overlooked. These findings resonate with those from a systematic review of diabetes apps targeted at older adults that there is a limited variety of accessibility features [Although eHealth literacy needs may not be explicitly considered when developing eHealth programs, this review still finds that interventions generally have features that may meet eHealth literacy needs based on the eHLF. However, the common focus is on providing information or features that address the domains of features . This isfeatures ,103 or pfeatures . In addifeatures  noted thfeatures  also poifeatures .In addition to exploring the role of eHealth literacy and eHealth intervention development, this review also examined the effectiveness of eHealth interventions targeted at socially disadvantaged groups and found inconclusive evidence. Although significant improvements were found in 19 studies, these findings should be interpreted with caution, as 10 studies are of weak quality and 7 studies are of moderate quality. Although 3 studies reported a large effect size, they had smaller sample sizes and were of moderate or weak quality ,65,83. TSeveral limitations of this review need to be acknowledged. Only peer-reviewed journals were included for this review, and there may be other studies that were not accounted for. The search was conducted by one researcher, which may have led to potential bias. The findings of this review may not reflect all details of the actual intervention, as authors generally only briefly describe their intervention development processes , and fewThe WHO recognizes health literacy as a critical determinant of health that has the potential to empower individuals and bring about health equity . HoweverDespite the concept of eHealth literacy being introduced in 2006, its potential role in empowering individuals has not been realized. Without meeting the eHealth literacy needs of disadvantaged groups, adoption of eHealth interventions is likely to be low, resulting in ineffective interventions ,100,110.


To evaluate screening efficiency and suggest cut-offs for parent and child Mood and Feelings Questionnaire (MFQ) and the short version (SMFQ) in unselected help seeking child- and adolescent psychiatric outpatients for subgroups of 6–12 versus 13–17 year olds and boys versus girls.Eligible for inclusion were newly admitted outpatients age 6–17 years (n = 5908) in four Swedish child- and adolescent psychiatry clinics. They were prompted consecutively and n = 307 accepted a specific day for assessment until time slots randomly were filled. We prospectively validated the MFQ (33 items) and SMFQ (13 items) in patients (n = 186) using receiver operating characteristics against a reference test of Longitudinal Expert All Data DSM-IV depression based on a Kiddie-Schedule for Affective Disorders and Schizophrenia and 1.2 (sd .6) years of follow-up.A depressive disorder was confirmed in 59 (31.7%) patients ranging from 14.0% for girls 6–12 years to 53.3% for girls 13–17 years. SMFQ performed roughly equivalent to MFQ. Adolescent score on SMFQ discriminated fairly for boys with Area Under Curve .77  and good  for girls and parent ratings for adolescent girls , but not for boys. Depression in children below age 13 could not be discriminated by MFQ or SMFQ whether filled in by child and mostly also when filled in by parent. Favouring maximum kappa value, the optimal cut-off was for MFQ self-report girls ≥32 versus boys ≥11 and for SMFQ self-report girls ≥17 versus boys ≥ 6. Suggested clinical SMFQ cut-offs for girls were ≥12 and for boys ≥ 6.MFQ and SMFQ can, with gender-based cut-offs, be used for screening in clinical populations of adolescents but not in children. Parent MFQ and SMFQ can be used for adolescent girls but not boys. SMFQ is sufficient for screening. Depression is a common and increasing burden on adolescents , 2. DeprThe Mood and Feelings Questionnaire (MFQ) was developed to screen for depression in epidemiological samples . It has The Short Mood and Feelings Questionnaire (SMFQ) was developed to enhance clinical and epidemiological use . ValidatA clinical Swedish study of the ten-item Montgomery Åsberg Depression Rating Scale-Self report versus a semi structured Schedule for Affective Disorders and Schizophrenia for School-Age Children (K-SADS) clinician rated diagnosis of depression revealed significant and intriguing gender differences with girls needing a higher cut-off score than boys (16 versus 11) for maximum kappa separation in adolescents with mean age 15 years  and contTo sum up, MFQ-child and MFQ-parent has repeatedly been shown to discriminate a depressive disorder most often in the good range but somewhat worse in a clinical setting or for MFQ-child with externalizing disorders. SMFQ-child and SMFQ-parent has correlated strongly to MFQ and has discriminated as MFQ in adolescents but just fair in younger samples. MFQ and SMFQ have usually been validated in epidemiological, high risk or among already diagnosed and clinical samples but not in unselected help seeking child psychiatric outpatients except the MFQ Arabic version in a small study of mostly adolescents . AnotherThe aims were: a/ to assess criterion validity of MFQ and SMFQ from self and parent ratings compared to Longitudinal Expert All Data (LEAD) depression diagnoses as the criterion measure in an unselected clinical sample with comparisons for gender and age and b/ to arrive at suggested cut-offs for use in clinical samples.d ≈ 0.3, p< 0.001) and in externalizing symptoms .There were 307 patients assessed in a diagnostic study from a consecutive sample of 5908 outpatients of ages 6–17 years, who sought treatment at four CAP clinics in Sweden from January 2010 to March 2013. All new referrals were routinely interviewed by phone and 5553 (94%) consented to participate in a diagnostic study. The parents, who had given consent, were then in a consecutive manner asked to participate at the available time slots. If the suggested date was not suitable, the next consecutive patient at the site was asked. In order to arrive at roughly equal numbers between children and adolescents and between boys and girls, about 15 (6%) children were at later stages of recruitment actively selected in order to include more pre-pubertal girls. Forty cases were discarded due to protocol violations in the K-SADS as all assessments from two clinicians were incomplete. The included sample was, compared to the entire outpatient sample, more affected by internalizing symptoms (Cohen’s d about 0.3) and externalizing symptoms (Cohen’s d about 0.5) as assessed by parental report. A more detailed description of the sample and recruitment procedures can be found at previous publications . The samParents of new referrals to outpatient CAP clinics were interviewed in a standard intake procedure by phone for a baseline assessment and triaging. At the end of the phone interview, the parent was informed about the diagnostic study. Within six weeks the participants, who had given oral consent, were offered a day of assessments including the MFQ, various other scales and the semi-structured K-SADS- Present and Lifetime version (K-SADS-PL) until time slots randomly were filled. Patients and parents were interviewed separately. Adolescents started with K-SADS-PL while parents were interviewed afterwards while adolescents filled in the MFQ. Children, on the other hand, started with the MFQ as, per suggestion, parents are to be interviewed before prepubertal children in the K-SADS-PL. The clinician performing the K-SADS-PL and subsequent clinicians were blind to the MFQ. The K-SADS-PL interviews yielded clinician established DSM-IV diagnoses, which were added to the clinical records. Later on, “Longitudinal Expert All Data Diagnoses” (LEAD) diagnoses were arrived at by two senior consultants, who reviewed the K-SADS-PL and all subsequent clinical information after a mean follow-up of 1.2  years .The Ethical Review Board at Lund University approved the study. Patients aged 15 years and above and parents consented to the study in writing.LEAD diagnoses were based on the K-SADS-PL 2009 version and further clinical work-up and follow-up. A blinded reliability test of 30 cases of the LEAD procedure was performed between the two senior authors (HJ and TI) using randomly chosen cases. Kappa values for spectrum diagnoses were excellent with ADHD, behaviour, and anxiety disorders in full agreement and for depressive disorders at kappa 0.92. A comprehensive description of measures and procedures can be found in a previous report on the KSADS using the same sample –25.The MFQ is a questionnaire assesses depressive symptoms over the previous two weeks. Responses are rated on a three-point scale . Total score ranges from 0–68 with a suggested cut-off usually ≥29. MFQ was developed to assess depressive symptoms in youth aged 8–18 years. There are matching versions for self (with 33 items) and parent report (with 34 items)  and a shA Chi-square test was used to analyse gender differences in prevalence of disorders. Internal consistency reliability of the MFQ and SMFQ was assessed with the Cronbach´s alpha, which is a measure of correlations between the items in a scale. Alpha 0.90 or above is considered excellent and .85–.89 as good . T-testsTests were two-tailed. Alpha was set to .05. SPSS version 24 was used for analyses and MedCalc statistical software for ROC analyses. Data are available in a supplementary file.Depressive disorders were diagnosed in 59 (31.7%) with major depression in 35 (18.8%), dysthymia in 7 (3.8%) and depression not otherwise specified in 17 (9.1%) patients. In the four subgroups, depression was diagnosed in 8 of 57 (14.0%) girls 6–12 years, in 19 of 97 (19.6%) boys 6–12 years, in 21 of 53 (39.6%) boys 13–17 and in 32 of 60 (53.3%) girls 13–17 years old. Further, attention-deficit/hyperactivity disorder (ADHD) was diagnosed in 114 (61.3%), anxiety disorder  in 64 (34.4%), disruptive disorder in 60 (32.3%) and autism spectrum disorder in 20 (10.8%).2 = 12.1, p = .001) but not for depression or other disorders. See also Gender difference across disorders was seen for ADHD  and SMFQ good (α = 0.87) Cronbach´s alpha for parents (n = 232) and patients (n = 186).MFQ scores for girls were higher than for parents (p< 0.001) and ratings between girls and their parents correlated , and most so for adolescent girls . The MFQ score levels for boys and their parents did not differ (p = 0.115) but did not significantly correlate .Patients with depression had significantly higher scores than non-depressed for both child and parent ratings and with both MFQ and SMFQ .There were marked age and gender differences in MFQ scorings. Teen-girls scored the highest while preadolescent boys scored the lowest. Differences were not as marked in parent ratings, but parents scored higher for adolescents in both MFQ and SMFQ .Adolescent’s score on MFQ and SMFQ predicted a diagnosis of depressive disorder with fair to good AUC (.77–.82) and moderate kappa (.46–.60). Parent score on MFQ and SMFQ predicted a diagnosis of depressive disorder in adolescent girls only with good AUC (.85) and a good kappa agreement (.60–.63) but not for adolescent boys. Sensitivity when using cut-offs for maximum kappa for adolescent depression ranged from 64% for girls to 100% for boys while specificity ranged from 57% for boys to 96% for girls.Depression in children younger than 13 years could not be predicted by MFQ or SMFQ except for parents predicting depression with a fair accuracy (.70) and a fair kappa (.30) in preadolescent boys .Further analyses are displayed for adolescents only and with just SMFQ as screening efficiencies for 6–12 year olds were poor and in adolescents roughly equal between MFQ and SMFQ. Optimal cut-offs to minimize false-positives and false-negatives equally and focusing on maximizing efficiency but also cut-offs for prioritizing sensitivity or specificity at .80 are displayed in Adjusting the suggested cut-offs for clinical use to arrive at a minimum of about .75 sensitivity resulted in no changes for parents of girls or teenage boys but a lowered cut-off for teenage girls on the SMFQ from 17 to 12. This cut-off conferred still a moderate kappa of .48.MFQ and SMFQ were, in this highly comorbid and unselected clinical sample, unable to predict depression in prepubertal children, which is at odds with previous studies. For adolescents, both the MFQ and SMFQ were adequate. We recommend SMFQ for screening in clinical populations due to brevity but we suggest gender-based cut-offs. Adding a parental SMFQ improves the prediction for girls but not for boys.The sample was highly comorbid and in that way representative of referrals for specialized CAP services. Previous studies have examined patients already diagnosed and often selected from subspecialized units , 16, 17,Levels on MFQ also illustrate disparities in sampling. Our levels on MFQ-patient for depressed parallel those of the diverse American clinical and non-clinical sample of about the same mean age of 13 years  while thSubgrouping of MFQ-patient levels across age and gender have been reported with girls scoring higher than boys but no difference across age , 34 whilA very surprising and intriguing finding is the lack of correlation between boys and their parents’ ratings on MFQ, which is at odds with earlier reports , 14, 16.The MFQ and SMFQ patient versions performed in our study rather well as a screening measure for adolescents albeit slightly below the AUCs from most , 19, 20 Parental MFQ and SMFQ had largely poor or barely fair agreements for preadolescents except for in adolescent girls, were the AUC was good. Again, this is at odds with previous studies where parent MFQ generally had fair , 16, 18 Our statistically optimized cut-offs varied extremely between boys and girls at odds with previous studies who suggest gender-neutral cut-offs but in line with the Swedish MADRS-S study . TeenageFor clinical purposes, we suggest using the SMFQ for adolescents with a cut-off yielding 80% sensitivity. Our adolescent girls had an optimized cut-off of 17, which gave a somewhat low sensitivity of 64%. Raising sensitivity to 80% resulted in a cut-off of 9 e.g. almost halved and severely compromised specificity and the kappa value. Unfortunately, a small sample size probably accounts for this dilemma. A reasonable cut-off is between 9 and 17 and somewhat in line with the most recent study of SMFQ in clinical adolescents  but oppoThe main strength of this study was the unselected sample of participants from a child and adolescent clinical population. All patients were new referrals without prior contact with psychiatric services. Thus, they had not received any prior psychiatric diagnosis, assessment or psychoeducation about depression. This recruitment is an ecologically suitable ground for testing the validity of a screening measure. The criterion or reference test of LEAD diagnoses were of good quality and independent from the MFQ scores as no information from the scales was included in the clinical records.The main limitation is the sample size when analysing subgroups based on age and gender. Preadolescent girls and teenage boys had low numbers conferring a risk of type-II errors. The AUC of parent SMFQ in preadolescent girls was in the lower fair range but still did not reach significance, possibly due to low numbers. Secondly, adolescents filled in the MFQ after a semi-structured K-SADS-PL, which might have given them knowledge of the meaning of screening items while children were first given the MFQ without any preceding clinical interview whatsoever. This could possibly inflate adolescent agreement. Another limitation concerns generalizability as our help seeking population have high rates of ADHD and externalizing disorders, which might not hold true in other societies.MFQ and SMFQ can, with gender-based cut-offs, be used for screening in clinical populations of adolescents but not in children. Parent MFQ and SMFQ can be used for adolescent girls but not boys. SMFQ is sufficient for screening.S1 Table(DOCX)Click here for additional data file.S2 Table(DOCX)Click here for additional data file.S3 Table(DOCX)Click here for additional data file.S4 Table(DOCX)Click here for additional data file.S1 Dataset(SAV)Click here for additional data file. 7 Feb 2020PONE-D-19-26460Screening efficiency of the Mood and Feelings Questionnaire (MFQ) and Short Mood and Feelings Questionnaire (SMFQ) in Swedish help seeking outpatientsPLOS ONEDear Dr. Jarbin,Thank you for submitting your manuscript to PLOS ONE. After careful consideration, we feel that it has merit but does not fully meet PLOS ONE’s publication criteria as it currently stands. 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Any typographical or grammatical errors should be corrected at revision, so please note any specific errors here.Reviewer #1: YesReviewer #2: Yes**********5. Review Comments to the AuthorPlease use the space provided to explain your answers to the questions above. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. Reviewer #1: Dear AuthorsWould you consider comments as follows:IntroductionSince the introduction section describes both children and adolescents psychiatric disorders and instruments for detecting these problems, I’m wondering why authors began the section with depression in adolescents while the target group in the study was 6-17 years.The section is not well organized and should be revised. For example, it is not clear what is the role of K-SADS here. .The aim of the study should be mentioned as separated segments clearly at the end of the introduction.MethodsPage 12, lines 130-131, researchers referred readers to their previous published paper, which may not be accessible.It is suggested to mention the reason of attrition clearly.Since researchers aimed to assess criterion validity of the scales, I’m wondering which scale was used as the criterion in the study. Its psychometric characteristics including validity as well as reliability should be described.Reviewer #2: The study from a scientific point of view seems to be well done and presents good results, from where to derive valid results. The problem discussed is currently valid. The summary sufficiently informs about the content of the paper. I have minor comments:-The questionnaire was well explained and internal validity was reported, what about validity?-The final conclusion of study was missed.-Some of references were before 2000, please update them.**********what does this mean?). If published, this will include your full peer review and any attached files.6. PLOS authors have the option to publish the peer review history of their article  digital diagnostic tool,  3 Mar 2020Reviewer #11. Background on depression in children. Good point which we had omitted. We have now added that prevalence is considerably lower and not increasing in children while we also added that the degree of clinical recognition in children is not known. 2. Role of the K-SADS-PL in the introduction section. We have specified the different criterion test that arrived at opposing cut offs (same for boys or girls versus a lot higher cut off for girls). The criterion was a clinician diagnosis utilising K-SADS-PL in one study while the criterion was just a cut-off on a self report in the other study. Thus, the role of K-SADS as a more reliable diagnostic test than a self report form is explained. 3. Aims of study as clear separate segments. The aims are now separated by a line break and are specified as just two and more clearly phrased as ”to …”4. To describe the reason for attrition more clearly. A more detailed description of the inclusion process are included as well as a specification of the missing 35 parental MFQ was due to administrative problems.5. Criterion scale in the study. Very important point and possible ”reference test” was not a clear description of the role of LEAD in this major design issue. We have now clearly stated in several segments  that a LEAD diagnosis was the criterion in the study. However, it is not possible to describe the validity of the LEAD while the interrater reliability of the LEAD is now described in the methods section.Reviewer #21. Validity of the MFQ and SMFQ in the methods section. As the validity of the scale is the main focus of the paper, we chose to describe and discuss this topic in the introduction. This discussion is the base for the summing up of the background and aims of the study. Therefor, we would still prefer to describe previous research on validity of MFQ in the introduction. 2. A final conclusion (same as in abstract) is added. Thanks for this suggestion!3. References before 2000. We would like to keep these references as the original studies on MFQ were published in the late 90´s. However, we are refering to just about every relevant study MFQ and SMFQ including all with more recent dates.AttachmentResponse to Reviewers.docxSubmitted filename: Click here for additional data file. 5 Mar 2020Screening efficiency of the Mood and Feelings Questionnaire (MFQ) and Short Mood and Feelings Questionnaire (SMFQ) in Swedish help seeking outpatientsPONE-D-19-26460R1Dear Dr. Jarbin,We are pleased to inform you that your manuscript has been judged scientifically suitable for publication and will be formally accepted for publication once it complies with all outstanding technical requirements.Within one week, you will receive an e-mail containing information on the amendments required prior to publication. 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Your manuscript will remain under strict press embargo until 2 pm Eastern Time on the date of publication. For more information, please contact With kind regards,Ali MontazeriAcademic EditorPLOS ONEAdditional Editor Comments :Reviewers' comments: 9 Mar 2020PONE-D-19-26460R1 Screening efficiency of the Mood and Feelings Questionnaire (MFQ) and Short Mood and Feelings Questionnaire (SMFQ) in Swedish help seeking outpatients Dear Dr. Jarbin:I am pleased to inform you that your manuscript has been deemed suitable for publication in PLOS ONE. Congratulations! Your manuscript is now with our production department. onepress@plos.org.If your institution or institutions have a press office, please notify them about your upcoming paper at this point, to enable them to help maximize its impact. If they will be preparing press materials for this manuscript, please inform our press team within the next 48 hours. 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Multiple guidelines on cutaneous melanoma (CM) are available from several consortia and countries. To provide up-to-date guidance in the rapidly changing field of melanoma treatment, guideline developers have to provide regular updates without compromises of quality. We performed a systematic search in guideline databases, Medline and Embase to identify guidelines on CM. The methodological quality of the identified guidelines was independently assessed by five reviewers using the instruments “Appraisal of Guidelines for Research and Evaluation” (AGREE II) and “Recommendation EXcellence” (AGREE-REX). We performed descriptive analysis, explored subgroup differences using the Kruskal–Wallis (H) test and examined the relationship between distinct domains and items of the instruments with Spearman’s correlation. Six guidelines by consortia from Australia, France, Germany, Scotland, Spain and the United States of America were included. The German guideline fulfilled 71%–98% of criteria in AGREE II and 78%–96% for AGREE-REX, obtaining the highest scores. Deficiencies in the domains of “applicability” and “values and preferences” were observed in all guidelines. The German and Spanish guidelines significantly differed from each other in most of the domains. The domains “applicability” and “values and preferences” were identified as methodological weaknesses requiring careful revision and improvement in the future. The incidence of primary cutaneous melanoma (CM) continues to increase steadily each year ,2. In 20The management of melanoma is subject to country-specific health care conditions. Evidence-based clinical guidelines are important tools for clinicians, assisting them in finding the best therapy option for their patients, as well as providing guidance for diagnostic procedures and surveillance. They include statements and recommendations for the optimization and standardization of patient care, which are developed based on a systematic literature review of evidence and an evaluation of the benefits and harms of various care options . A multiNumerous evaluation tools have been developed for assessing the methodological quality of guidelines ,4,5. TheOur initial database search identified 2489 records . Twenty-p = 0.001), as well as the German and the French guideline (p = 0.035), significantly differed from each other in this domain.In this domain, the main objectives of the guidelines and their intended target population were evaluated. Overall, an average score of 5.18 was achieved (± 1.76) . The Gerp = 0.019) in this domain. Additionally, the Spanish and the German guideline significantly differed from each other (p = 0.001).This domain assesses whether appropriate stakeholders were involved in the developing process, and whether the views of the intended guideline users are represented. A mean score of 4.50 (± 1.74) was achieved. The fulfilled values ranged from 16% for the Spanish guideline to 90% for the German guideline. The Scottish guideline was significantly different from the Spanish guideline (p = 0.001) and the French (p = 0.009) ones.This domain focuses on the methodology and assesses whether the evidence for the guideline was gathered in a systematic and transparent search. The mean score was 5.10 (± 1.45). Besides, the German guideline showed the best methodological quality in this domain (95% of items fulfilled), followed by the guidelines from Australia and Scotland with 83% and 82% of items fulfilled, respectively. The other guidelines varied from 33% to 71%. Additionally, the German guideline was significantly different from the Spanish ; the German guideline was rated as the best guideline (97% of fulfilled criteria), followed by the Scottish (87%) and Australian (86%) guideline.The domain “application” focuses on the relevant processes for a successful guideline implementation, including facilitators, barriers and the provision of additional material. It also evaluates whether monitoring and/or auditing criteria are described. Interestingly, this domain had the lowest overall mean score of all domains (3.95 ± 1.75). The Australian guideline was rated as the best compared to the remaining guidelines (75%), while the Spanish guideline achieved the lowest score (23%).This domain investigates funding and the competing interests of the experts who were involved in guideline development. The mean score of this domain was 4.93 (± 1.76). The US guideline fulfilled 83% of the criteria, whereas the Spanish guideline demonstrated 32% of fulfilled editorial independence items.The overall quality and whether the guideline can be recommended for use is evaluated in this final domain. The overall score was 4.90 (± 1.49) and the fulfilled criteria ranged from 20% (Spanish guideline) to 70% (German guideline). Therefore, the guidelines from Australia, Germany, Scotland and the US would be uniformly recommended for use in practice, whereas the two guidelines from Spain and France were recommended with modifications only, or rated not to be recommended at all by the five reviewers.p = 0.002) and the French guidelines in this criterion (p = 0.025).This domain assesses whether the guideline was developed based on evidence, and also evaluates whether the recommendations are applicable for the intended target users setting. The mean score was 5.07 ± 1.23, and the German guideline was rated as the best clinically applicable guideline with 96%, whereas the guideline from Spain was rated lowest with 44% . The Gerp = 0.046) as well as for the Spanish and German guidelines (p = 0.010).This domain evaluates the relative importance that the target guideline developers, users, policy/decision-makers and patients attach to outcomes of interest. Overall, the mean score was 3.92 ± 1.71. The guidelines from Australia and Germany achieved high values with 71% and 78% of items fulfilled. The Spanish guideline yielded the lowest score with 18% of items fulfilled. Significant differences were found between the Spanish and the Australian guidelines (p = 0.010).This domain evaluates whether the recommendations are suitable for the patients, population or the health care systems which they were developed for. It investigates whether the guideline recommendations align with the implementation goals of the guideline, as well as whether the guidelines include advice or tools and resources to facilitate the implementation of the recommendations, since these are easier to adopt in practice. Again, the German and Australian guidelines achieved the highest results with 77% and 78%, respectively. The mean score was 4.73 ± 1.38. Besides, a significant difference was identified between the Spanish and the German guidelines (r = 0.92), “overall assessment” (r = 0.85) and “rigor of development” (r = 0.81). There was also a strong, positive correlation between the domain “stakeholder involvement” and the domains “rigor of development” (r = 0.89), “clinical applicability” (r = 0.85) and “overall assessment” (r = 0.84). In addition, “clinical applicability” was positively associated with “rigor of development” (r = 0.86). Notably, all AGREE-REX domains were positively and statistically significantly correlated with each other.Most of the AGREE II domains were significantly correlated with each other . The domAltogether, the German guideline was evaluated best with both instruments. It appeared to strictly adhere to the AGREE domains and described the guideline developing process in detail. This may be explained by the set of rules that the Association of the Scientific Medical Societies in Germany (AWMF) has established for the creation of guidelines, and to which guideline developers have to adhere. Besides, thorough and continuous methodological support is provided by the German Cancer Society and AWMF, which are both involved in the development of cancer guidelines in Germany. A similarly strict framework is provided by the SIGN consortium responsible for the development of the Scottish guidelines that also achieved high scores in this evaluation ,33. The The field is rapidly evolving, as new treatments and treatment combinations are steadily on the rise, and also since more analyses from clinical studies and long-term follow-up are available ,36,37. IThis study has several limitations. The fact that all reviewers of this study are from Germany might have influenced and positively biased the evaluation of the German guideline, which had achieved the best results among all six guidelines. However, none of the evaluators was part of the guideline committee. Besides, we only included guidelines that had been available in English or in the German language. Hence, the assessment may be biased.For our appraisal, we included national and international guidelines with a focus on the treatment of CM in the English or German languages, as the field in melanoma treatment has been rapidly changing over the previous years. Besides, guideline chapters on treatment, especially covering palliative and (neo-) adjuvant approaches, are often dynamically modified and updated regularly for melanoma. The guidelines had to be published within the last 3 years , as we aimed to appraise the most recent and up-to-date guidelines while simultaneously accounting for a time lag for the implementation of newly approved interventions, which began in 2011 with the approval of ipilimumab by the Food and Drug Administration. Furthermore, the period of validity of guidelines is usually 2–6 years. These aspects suggest that a time frame from 2016–2019 appeared appropriate. Furthermore, we restricted our search to guidelines which were developed based on a systematic literature assessment and a structured consensus process, i.e., evidence- and consensus-based guidelines. Guidelines covering only a specific aspect of melanoma were excluded.We systematically searched for eligible guidelines in several guideline databases, including multidisciplinary and subject-specific guideline providers , MedlineTwo authors  independently collected relevant information on each guideline, including title, national authority/author and country of origin, publication date, methodological approach and its scope. Five independent reviewers assessed the methodological quality of each guideline identified in the search using the evaluation tools AGREE II and AGREE-REX as recently described by the authors .p-values < 0.05 were considered as statistically significant. The interrater agreement of the five reviewers was determined using Fleiss’ Kappa [All domain scores were calculated according to the AGREE II and AGREE-REX instructions ,8. The ts’ Kappa .Altogether, this analysis demonstrates that only a few of the currently available melanoma guidelines are of high methodological quality. The published guideline from Germany showed the best rating, suggesting that it may be used as a template for other guideline projects. The domains “applicability” and “values and preferences” had achieved the lowest scores in our evaluation, which should be taken into consideration in future guideline development processes. A domain that needs to be included in future evaluations is whether these guidelines are up-to-date.


The WHO recommends that those with established cardiovascular disease should be treated with lipid-lowering therapy, but there is no specific guidance regarding lipid monitoring. Unnecessary general practitioner visits may be a burden for patients and increase healthcare costs. A systematic review of the current guidelines was performed to reveal gaps in the evidence base for optimal lipid monitoring approaches.For this systematic review, a search of Medline, Cumulative Index to Nursing and Allied Health Literature and Turning Research Into Practice databases was conducted for relevant guidelines published in the 10 years prior to 31 December 2019. Recommendations surrounding the frequency of testing, lipid-lowering therapies and target cholesterol values were compared qualitatively. Each guideline was assessed using the 2009 Appraisal of Guidelines for Research and Evaluation II tool.Twenty-two guidelines were included. All recommended statins as the primary lipid-lowering therapy, with a high level of supporting evidence. Considerable variation was found in the recommendations for cholesterol targets. Seventeen guidelines provided at least one cholesterol target, which for low-density lipoprotein (LDL) cholesterol ranged between 1.0 and 2.6 mmol/L, although the most frequently recommended was <1.8 mmol/L (n=12). For long-term follow-up, many recommended reviewing patients annually (n=9), although there was some variation in recommendations for the interval of between 3 and 12 months. Supporting evidence for any approach was limited, often being derived from clinical opinion.Further research is required to provide an evidence base for optimal lipid monitoring of the on-statin secondary prevention population. There is a large evidence base, supported by the WHO, for the use of statins as lipid-lowering therapy for the prevention of further cardiovascular events. There is no similar guidance regarding the use of cholesterol targets or long-term follow-up in secondary prevention populations.A formal, systematic comparison of recommendations of target plasma lipid levels and the frequency of lipid monitoring in the secondary prevention population from 22 guidelines.Patients generally prefer to minimise visits to general practitioners, and unnecessary visits and lipid testing also may increase healthcare costs. This work highlights the need for further research to provide an evidence base for optimal lipid monitoring of the secondary prevention population.In 2016, cardiovascular disease (CVD) was the leading cause of death worldwide and was responsible for an estimated 17.9 million deaths, with heart attacks and strokes accounting for 85% of these.The need for risk management in secondary prevention, which encompasses coronary heart disease, stroke and peripheral artery disease, is clear. The rate of further cardiovascular events per annum in unmedicated patients with previous events has been estimated to be around 5.6% and 3.7% depending on whether the previous event was coronary heart disease related, compared with 1.8% in those without.3However, the WHO has offered no specific guidance regarding target plasma lipid levels or the monitoring of these since the publication of their prevention of cardiovascular guidelines in 2007.This systematic review aimed to investigate similarities and differences in clinical guidelines surrounding the recommendations for the therapeutic treatment, targets and monitoring of lipid risk factors in adults who have established CVD. This will help to highlight variation in the guidelines, thereby providing guidance for future research priorities.https://www.crd.york.ac.uk/prospero/) on 19 June 2018 (Ref: CRD42018098582).13A protocol which documented the prespecified analysis and the inclusion criteria for this systematic review was first registered on the International Prospective Register of Systematic Reviews , the National Library for Health Guidelines Finder (UK), the Canadian Medical Association Clinical Practice Guidelines Infobase and Guidelines International Network International Guideline Library. Finally, an additional hand search was performed to identify the most recent versions of the guidelines identified through the systematic search. A copy of the search strategy is included in the 10.1136/openhrt-2020-001396.supp1Supplementary dataPapers were retained if they met the Institute of Medicine’s 2011 definition of a clinical guideline, ‘Clinical Practice Guidelines are statement that include recommendations intended to optimize patient care that are informed by a systematic review of evidence and an assessment of the benefits and harms of alternative care options’.Two reviewers (REB and JL) independently reviewed the titles and abstracts of the results against the eligibility criteria. The same two reviewers also performed the full-text review, where the reason for exclusion of the guidelines was also documented. In both instances, any discrepancies of opinion were resolved through discussion.Data extraction was performed initially by one reviewer (REB) with accuracy checked by a second reviewer (JL). Data extracted included the target population, the publishing society, the country or region the guideline applied to and the year it was published. Recommendations specifically for the secondary prevention population surrounding the frequency that plasma lipid monitoring should be performed, therapies that should be used, and any lipid target values were also extracted if given within the guideline. The strength and the level of evidence of each recommendation were also extracted. Once extracted, the recommendations were compared by all authors.The quality of the development processes of each of these guidelines was then assessed using the 2009 Appraisal of Guidelines for Research and Evaluation (AGREE) II tool by two reviewers (REB and JL). The AGREE II tool consists of 23 questions covering six domains  and an overall assessment of the quality of the guideline. Each of the items, including the overall quality assessment, is scored on a seven-point scale , with scaled domain scores then calculated.The literature search found 6948 results , of whicThe guidelines included are summarised in Recommendations for the use of statin medication, cholesterol targets and the frequency of monitoring are presented in All of the guidelines presented recommendations for the treatment of the secondary prevention population, with all recommending statins as the primary therapy. Only UK/Scottish guidelines suggested the specific drug and dose, namely, atorvastatin 80 mg, with many instead recommending the maximally tolerated high-intensity doses in general, with lower doses considered when contraindications were present, or they were poorly tolerated by the patients. Few caveats were stated regarding the prescription of statins. For example, all stroke guidelines recommended statins only when the cause of the stroke was likely to be atherosclerotic, with the South African Stroke Society additionally only recommending them in the case of total cholesterol >3.5 mmol/L, and the Australia Stroke Society only considering them appropriate when the patient’s life expectancy was considered reasonable. For the secondary prevention population as a whole, the European Society of Cardiology and European Atherosclerosis Society (ESCEAS) tailored their recommendations for patients whose baseline LDL was <1.4 mmol/L at baseline, respectively, with therapy considered rather than offered to these patients. Finally, the 2018 American Consensus (ACD) guidelines stated that the benefit–risk ratio should be considered when offering medication to patients over the age of 75 years.For the guidelines which reported corresponding levels of evidence with their treatment recommendations (n=12), all considered that the level of evidence for statins was high, resulting in strong recommendations for their administration to the secondary prevention population. For situations where lower doses of therapy may be needed, such as in cases of contraindications or lack of tolerance, if specified at all, guidelines often considered the level of evidence supporting these changes to be lower than for the main treatment recommendation. Specifically, the level of evidence was typically assessed to be moderate (rather than high) or such alterations to medications were considered to be only good practice.Besides statins, other lipid-lowering medications were also discussed within the guidelines . The mosSeventeen of the guidelines provided at least one target, with all except one of these providing an LDL cholesterol goal. Target values ranged between 1.0 and 2.6 mmol/L, although the most frequently recommended was <1.8 mmol/L (n=12). Many guidelines additionally suggested that a 50% reduction in LDL could be used as an alternative where this target may be unattainable or for patients whose baseline values were already <3.5 mmol/L (n=7). A non-high-density lipoprotein (HDL) cholesterol target was also common (n=8), with target values ranging from 2.5 to 3.4 mmol/L, with 2.6 mmol/L the most frequent (n=5). These targets were usually given in combination with a target for LDL cholesterol, though in the case of the Japan Atherosclerosis Society, the non-HDL target was considered only relevant when a patient’s triglycerides were elevated. Meanwhile, the National Institute for Health and Clinical Excellence did not provide a numerical target for non-HDL, recommending a 40% reduction from the patient’s baseline only. Only two guidelines referred to other lipid parameters in their recommendations. The Canadian Cardiovascular Society (CCSG) provided an apolipoprotein B target as an alternative for LDL, and the International Diabetes Federation provided additional targets for triglycerides and HDL cholesterol. There were no apparent differences in recommendations for stroke or diabetes-specific guidelines, although some guidelines for all secondary prevention populations provided different targets for those patients with additional comorbidities (n=2), or specific cardiovascular events (n=2).For the majority of the guidelines that provided targets, the recommendations provided either no supporting evidence or graded it as low. Consequently, the associated strength of the recommendations was often either not given or was given as preferences and opinions of those involved in the guideline’s construction. There were few exceptions. Singapore’s (SMH) recommendation of LDL <2.1 mmol/L considered the level of evidence to be high, resulting in a strong recommendation. The target of LDL <1.8 mmol/L, when stated in some guidelines, was also strongly recommended, with the National Lipid Association considering the supporting evidence to be high, while others graded it as only moderate (n=2). CCSG’s target for patients who have not experienced an acute coronary syndrome of LDL <2.0 mmol/L was also given as a strong recommendation, with the guideline viewing the supporting evidence for it as moderate. Its recommendation of LDL <1.8 mmol/L for patients who have experienced an acute coronary syndrome cited no supporting evidence, and consequently was given as a preference among the guideline’s creators.Thirteen guidelines detailed recommendations regarding the ongoing monitoring of this population, with the specifics falling into three categories: monitoring following the initiation of treatment (n=5), monitoring prior to stable lipids (n=2), and long-term follow-up (n=11), with some providing recommendations in more than one of these categories (n=5). Of those who detailed monitoring following statin initiation, all recommended a review of the patient’s lipids within 3 months. Within these, two guidelines recommended also measuring the patient’s alanine aminotransferase at this review,The majority of guidelines considered the evidence behind their frequency of monitoring recommendations to either be low (n=1) or gave no evidence to support them (n=11), sometimes referring to them as good practice points or clinician’s opinions or preferences (n=5). However, the ACD guidelines were the exception to this, which graded the evidence behind their monitoring recommendations of every 3–12 months as strong.This systematic review illustrates the variation in recommendations surrounding optimal on-statin lipid monitoring within secondary prevention. Specifically, there were considerable differences in the recommendations for cholesterol targets (including their use) and the ongoing monitoring of lipid levels over the longer term. These findings reflect the fact that no guideline identified a specifically designed randomised controlled trial to assess either treatment targets or monitoring of therapy. However, such trials are likely to be expensive, although in the future advances with electronic health records may facilitate the evidence base for this. Nonetheless, this systematic review illustrates that better evidence is needed to provide an optimal approach to lipid monitoring in order to balance safety, adherence, cost, and time burden to patients.All guideline committees are likely to be searching a broadly similar evidence base, where the efficacy and safety of statins has been well established in the prevention of further cardiovascular events.The evidence that ‘lower is better’ for LDL and non-HDL cholesterol in terms of secondary CVD prevention is well supported by evidence, and the recent advent of PCSK9 inhibitors further supports this notion.26 40 41There was virtually no evidence to support any recommendations regarding the frequency of ongoing monitoring once lipid-lowering therapies had been commenced. Guidelines that recommended retesting following statin initiation tended to additionally recommend liver function tests were performed as a safety indicator, especially if hepatic symptoms were present, including those produced by the ACC/AHA and the ESC/EAS.To our knowledge, this is the first review to compare guidelines surrounding the management of lipids in the secondary prevention population, as previous research has focused on comparing guidelines for assessing risk and managing it through lifestyle interventions in the primary prevention population only.The safe and optimal treatment of plasma lipids within the secondary prevention population is key to reducing the increasing burden of CVD in society. However, given the paucity of evidence for the frequency of ongoing monitoring, there is a clear need for further research in these two key areas of its management. This will improve patient care while optimising costs in an evidence-based manner.