Leishmania donovani parasites will stay asymptomatic, but some develop VL that, if left untreated, is almost always fatal. This stage of the disease is associated with a profound immunosuppression, characterised by impaired production of Interferonγ (IFNγ), a cytokine that plays a key role in the control of Leishmania parasites, and high expression levels of an inhibitory receptor, programmed cell death 1 (PD1) on CD4+ T cells. Here, we tested the contribution of the interaction between the immune checkpoint PD1 and its ligand PDL-1 on the impaired production of IFNγ in VL patients. Our results show that in the blood of VL patients, not only CD4+, but also CD8+ T cells express high levels of PD1 at the time of VL diagnosis. Next, we identified PDL-1 expression on different monocyte subsets and neutrophils and show that PDL-1 levels were significantly increased in VL patients. PD1/PDL-1 inhibition resulted in significantly increased production of IFNγ, suggesting that therapy using immune checkpoint inhibitors might improve disease control in these patients.Visceral leishmaniasis (VL) is a neglected tropical disease that causes substantial morbidity and mortality and is a growing health problem in Ethiopia, where this study took place. Most individuals infected with  Leishmania, that is transmitted during the blood meal of an insect. When individuals cannot control Leishmania replication, they develop visceral leishmaniasis, that is characterised by enlarged spleen and liver, low blood cell counts and wasting. We have previously shown that lymphocytes from these patients have an impaired ability to produce a soluble mediator, IFNγ, that contribute to the killing of the parasites, but that this was restored after successful anti-leishmanial treatment. Here we identified high expression levels of a marker, PD1, on lymphocyte; that has been associated with dysfunctional lymphocytes. We also identified the ligands of this marker, PDL1, on different blood cells. Furthermore, we showed that blocking the interaction between PD1 and PDL1 resulted in increased levels of IFNγ. These results suggest that treatment that blocks the interaction of PD1 with PDL1 might improve disease management and patient care.Visceral leishmaniasis is a neglected tropical disease, that affects the poorest of the poor in low and middle-income countries. It is caused by a parasite,  Leishmania (L.) donovani and is one of the most significant vector-borne diseases, with over 3.2 million people at risk of infection ) of PD1 on CD4(TIFF)Click here for additional data file.S2 FigAPC, CD16FITC and PDL-1PE and the expression levels (Median Fluorescence Intensity [MFI]) of PDL-1 on the three subsets of monocytes were measured by flow cytometry. A. FSC and SSC of the monocyte gate (P4). B. Different monocyte subsets based on the expression levels of CD14 and CD16: classical (R18), Intermediate (R19) and Non-classical (R20) monocytes. C, D and E. PDL-1 MFI on the Classical (M31), Intermediate (M32) and Non-classical monocytes (M33).PBMCs were purified as described in Materials and Methods. PBMCs were stained with anti-human CD14(TIFF)Click here for additional data file.S3 FigA.FSC and SSC of the neutrophil gate (P3). B. CD15+ neutrophils in P3. C. PDL-1 MFI (M10) on neutrophils in M1.Neutrophils were purified as described in Materials and Methods and the expression of PDL-1 on neutrophils was measured by flow cytometry. (TIFF)Click here for additional data file. 18 Jan 2022Dear Dr. Kropf,Thank you very much for submitting your manuscript "Impaired in vitro Interferon-γ production in patients with visceral leishmaniasis is improved by inhibition of PD1/PDL-1 ligation" for consideration at PLOS Neglected Tropical Diseases. As with all papers reviewed by the journal, your manuscript was reviewed by members of the editorial board and by several independent reviewers. In light of the reviews (below this email), we would like to invite the resubmission of a significantly-revised version that takes into account the reviewers' comments. The reviewers raise a number of methodological issues that must be addressed. The gating strategies for flow cytometry must be provided. The role of neutrophils in PD-1 mediated IFNg suppression should be addressed. Thee effect of PD1 blockade on individual cell populations (intracellular cytokines) should be addressed.We cannot make any decision about publication until we have seen the revised manuscript and your response to the reviewers' comments. Your revised manuscript is also likely to be sent to reviewers for further evaluation.When you are ready to resubmit, please upload the following:[1] A letter containing a detailed list of your responses to the review comments and a description of the changes you have made in the manuscript. Please note while forming your response, if your article is accepted, you may have the opportunity to make the peer review history publicly available. The record will include editor decision letters (with reviews) and your responses to reviewer comments. If eligible, we will contact you to opt in or out.[2] Two versions of the revised manuscript: one with either highlights or tracked changes denoting where the text has been changed; the other a clean version (uploaded as the manuscript file). Important additional instructions are given below your reviewer comments.Please prepare and submit your revised manuscript within 60 days. If you anticipate any delay, please let us know the expected resubmission date by replying to this email. Please note that revised manuscripts received after the 60-day due date may require evaluation and peer review similar to newly submitted manuscripts.Thank you again for your submission. We hope that our editorial process has been constructive so far, and we welcome your feedback at any time. Please don't hesitate to contact us if you have any questions or comments.Sincerely,Peter C. Melby, M.D.Associate EditorPLOS Neglected Tropical DiseasesCharles JaffeDeputy EditorPLOS Neglected Tropical Diseases***********************The reviewers raise a number of methodological issues that must be addressed. The gating strategies for flow cytometry must be provided. The role of neutrophils in PD-1 mediated IFNg suppression should be addressed. Thee effect of PD1 blockade on individual cell populations (intracellular cytokines) should be addressed.Reviewer's Responses to QuestionsKey Review Criteria Required for Acceptance?As you describe the new analyses required for acceptance, please consider the following:Methods-Are the objectives of the study clearly articulated with a clear testable hypothesis stated?-Is the study design appropriate to address the stated objectives?-Is the population clearly described and appropriate for the hypothesis being tested?-Is the sample size sufficient to ensure adequate power to address the hypothesis being tested?-Were correct statistical analysis used to support conclusions?-Are there concerns about ethical or regulatory requirements being met?Reviewer #1: The objectives of the study were clearly described and articulate with the hypothesis of the study. The work was designed in a way to address the objectives and the population was clearly described and is appropriate to the aim of the investigation. The sample size is sufficient to test the hypothesis of the work. The statistical analysis was appropriate. The study protocol was approved by research ethics committees.Reviewer #2: The authors had previously shown that in VL patients, CD4+ T cells from whole blood stimulated with SLA express high levels of PD-1, and that this is associated with a reduced secretion of IFN-gamma.In the current study, the authors aim to determine which cells produce PD-L1, the ligand of PD-1, and if the reduction in IFN-gamma secretion can be abolished by cellular treatment with the PD-1 blocker Nivolumab.Overall, the study provides conclusive results on the stated objectives, and we are aware that the number of experiments that can be conducted with precious patient’s material (8ml of whole blood) is limited. However, the amount of data shown to prove the link between the single observations is a little sparse. With 16 VL patients and 10 control individuals, the sample size seems reasonable to reveal data for answering the main questions. The statistical analysis of the data is appropriate.Reviewer #3: About Figure 1: Are T cells stimulated? Is the difference of PD-1 expression in the presence or absence of SLA?About Figures 2 and 3: are they stimulated with SLA? Are there diferrences?About figure 4: It is missing isotype control. It is missing the production of SLA--------------------Results-Does the analysis presented match the analysis plan?-Are the results clearly and completely presented?-Are the figures  of sufficient quality for clarity?Reviewer #1: The analysis presented by the author match with the analysis plan, and the results are clearly presented. The figures are of sufficient quality.Reviewer #2: The experiments in this study are well chosen to provide information on the objectives.Table 1 summarizes the clinical and haematological parameters of the VL patients and the non-infected control individuals.Could the authors distinguish between healthy individuals (not infected until then) and individuals that might have had an asymptomatic Leishmania infection before?It might be beneficial to graphically depict the parasite grade of the different infected individuals for better insight into the variance in parasite load, which might correlate with the extent of the later analyzed PD-1 and PD-L1 expression as well as the IFN-y secretion.Fig. 1: Besides the already previously published PD-1 expression on CD4+ T cells (A), the authors now additionally determine PD-1 expression on CD8+ T cells (B), which is significantly increased in comparison to the healthy controls. Data in Panel 1a was already published previously, so why show it again, new donors? Please specify. Please show the gating strategy that was applied to measure the PD-1 MFI of the T cell subpopulations. Did the authors set a gate on cell size (FSC/SSC), single cells, did they check for cell viability? Information should be provided in the material and methods section or graphically in the results section. Fig. 2: As the binding of PD-L1 to PD-1 can elicit a T cell exhaustion phenotype which might explain a pronounced disease development. Thus, the authors measured the relative amount of PD-L1 that is expressed on blood monocytes.For all three examined monocyte subtypes, there is a significant difference between PD-L1 expression in healthy controls and VL patients. However, it’s not clearly stated why exactly those three monocyte subtypes were chosen for the analysis. What is their individual role in the recognition of Leishmania parasites or in activation of the adaptive immune system?Can the authors confirm that these blood monocytes were  infected with Leishmania? If not, what would be the trigger to make them express PD-L1. How would they get in contact with the parasites?Would it be an option to additionally investigate PD-L1 expression on macrophages from splenic aspirates, or even more specific from infected macrophages?Figure 2D and 2E are redundant to Fig 2A-C as this is just another combination of the graphs. This is not correct, please change accordingly and put all 6 datasets into one chart in order to show all relevant comparisons and significances.Fig. 3: Here the authors show the PD-L1 expression by neutrophils. As in Fig 2, it is not clear if these neutrophils are  infected with Leishmania in the blood, which would explain their activation. Once recruited to the affected organs  neutrophils might fulfill their function as local drivers of an immune response but they might not get back into the blood stream.The authors should also state in the figure legend or results text which surface markers were used for identifying the cells as neutrophils Fig.4: Treatment of whole blood cells from VL patients with SLA in the presence of the anti-PD-1 mAB Nivolumab increases IFN-gamma secretion as shown by ELISA.How much SLA was used (concentration) and from which strain was it produced, please specify.According to the methods section, cells were treated with 1µg Nivolumab. What was the total volume of the assay and/or the (approx.) number of cells treated?The authors should also provide data on whole blood cells derived from healthy patients in this assay.Reviewer #3: Figure 1: Please include the representative dot plotFigure 2: Please include the representative dot plotWhat is the difference in figure 2D? The median is very similar. Figure 3: Please include the representative dot plotFigure 4: Is there a possibility to do a correlation of expression of PD1 versus IFNg production?What is the level of PD-1 before and after the treatment with anti-PD1.--------------------Conclusions-Are the conclusions supported by the data presented?-Are the limitations of analysis clearly described?-Do the authors discuss how these data can be helpful to advance our understanding of the topic under study?-Is public health relevance addressed?Reviewer #1: The conclusion are supported by the data provided. Yet, study limitations are not so well described. The subject is of highly public health relevance and the authors have clearly emphasized the importance of the current study.Reviewer #2: nonoyesyesReviewer #3: It is missing intracellular citokines IFNg on CD4 and CD8 T cells to see the impact of anti-PD1 in each population.--------------------Editorial and Data Presentation Modifications?Use this section for editorial suggestions as well as relatively minor modifications of existing data that would enhance clarity. If the only modifications needed are minor and/or editorial, you may wish to recommend “Minor Revision” or “Accept”. Reviewer #1: Minor revision.Reviewer #2: -Reviewer #3: Figure 2 and 3 could be combined.--------------------Summary and General CommentsUse this section to provide overall comments, discuss strengths/weaknesses of the study, novelty, significance, general execution and scholarship. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. If requesting major revision, please articulate the new experiments that are needed.Reviewer #1: The authors in this study investigated the expression levels of PD-1 on CD4 and CD8 T cells of visceral leishmaniasis patients, and of its ligand PDL-1 on monocytes and neutrophils. The authors have found that anti-PD-1 treatment of whole blood cells of VL patients increases the production of INFg.General comments:The article requires minor modification that can be addressed through revision. The study addressed the interruption of PD-1/PDL-1 pathway, but do not mention the other ligand PDL-2 which has been shown to contribute as well to the success of anti-PD-1 treatment. It would be interesting to introduce PDL-2 as another ligand of PD-1 and discuss whether PDL-2 has been addressed in VL or not and the consequences.Methods:- How SLA is produced? Could the authors describe on that as well?- What are the clones of the monoclonal antibodies used for FACS? This information should be in the paper.- What is the clone of the commercial anti-PD-1 used for blocking assays? Is this information possible to add in the paper?- The authors inform that acquisition was performed on a BD Accuri B6, how many events were acquired per sample? How was the gating strategy used for the analyses?- Why the authors did not use CD3 or CD45 in the panels to better define the cell types investigated?- The authors should explain why they did not use the same antibody isotype of PD-1 as control for the antibody blocking assay. This is a critical part of the methods used in the paper.Figures:Table 1. should be reformatting.Fig 1. It would be interesting to provide all the gating strategy used to define CD4 and CD8 T cells, and the other cell types. This information could be as supplementary material to the article. Did the authors observed a frequency difference as well? Do the authors imagine that these T cells are exhausted? It would be interesting to discuss this as well.The information on the markers to define each cell type evaluated should be in figure legends as well.Fig 2. Do the authors have any data on the levels of PDL-1 of in situ macrophages from biopsies? It would be interesting for the discussion.Fig 4. Do the authors have an idea if blocking affects the production of other cytokines that could be relevant for VL as well? I understand that the main hypothesis was drafted on the impairment of IFNg production ToD, but it would be scientifically relevant to show the data on other cytokines as well, such as IL-10, IL-4, IL-2 and TNF.Discussion:The authors could at least discuss how the interaction of PD-1/PDL-1 mechanistically contribute to IFNg impairment during VL.Reviewer #2: -Reviewer #3: It is missing important references- PD-1 Blockade Modulates Functional Activities of Exhausted-Like T Cell in Patients With Cutaneous LeishmaniasisDOI: 10.3389/fimmu.2021.632667- Leishmania Parasites Drive PD-L1 Expression in Mice and Human Neutrophils With Suppressor CapacityDOI: 10.3389/fimmu.2021.598943- Immunotherapy using anti-PD-1 and anti-PD-L1 in Leishmania amazonensis-infected BALB/c mice reduce parasite loadDOI: 10.1038/s41598-019-56336-8--------------------what does this mean?). If published, this will include your full peer review and any attached files.PLOS authors have the option to publish the peer review history of their article  digital diagnostic tool, Data Requirements:http://www.plosbiology.org/article/info%3Adoi%2F10.1371%2Fjournal.pbio.1001908#s5.Please note that, as a condition of publication, PLOS' data policy requires that you make available all data used to draw the conclusions outlined in your manuscript. Data must be deposited in an appropriate repository, included within the body of the manuscript, or uploaded as supporting information. This includes all numerical values that were used to generate graphs, histograms etc.. 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Read more information on sharing protocols at  22 Apr 2022AttachmentResponse to reviewers Takeleetal210422.docxSubmitted filename: Click here for additional data file. 30 May 2022Dear Dr. Kropf,We are pleased to inform you that your manuscript 'Impaired in vitro Interferon-γ production in patients with visceral leishmaniasis is improved by inhibition of PD1/PDL-1 ligation' has been provisionally accepted for publication in PLOS Neglected Tropical Diseases.Before your manuscript can be formally accepted you will need to complete some formatting changes, which you will receive in a follow up email. A member of our team will be in touch with a set of requests.Please note that your manuscript will not be scheduled for publication until you have made the required changes, so a swift response is appreciated.IMPORTANT: The editorial review process is now complete. PLOS will only permit corrections to spelling, formatting or significant scientific errors from this point onwards. Requests for major changes, or any which affect the scientific understanding of your work, will cause delays to the publication date of your manuscript.Should you, your institution's press office or the journal office choose to press release your paper, you will automatically be opted out of early publication. We ask that you notify us now if you or your institution is planning to press release the article. All press must be co-ordinated with PLOS.Thank you again for supporting Open Access publishing; we are looking forward to publishing your work in PLOS Neglected Tropical Diseases.Best regards,Peter C. Melby, M.D.Associate EditorPLOS Neglected Tropical DiseasesCharles JaffeDeputy EditorPLOS Neglected Tropical Diseases***********************************************************The authors have sufficiently addressed the concerns of the reviewers <span class="s1"> Reviewer's Responses to Questions </span> Key Review Criteria Required for Acceptance?As you describe the new analyses required for acceptance, please consider the following:Methods-Are the objectives of the study clearly articulated with a clear testable hypothesis stated?-Is the study design appropriate to address the stated objectives?-Is the population clearly described and appropriate for the hypothesis being tested?-Is the sample size sufficient to ensure adequate power to address the hypothesis being tested?-Were correct statistical analysis used to support conclusions?-Are there concerns about ethical or regulatory requirements being met?Reviewer #1: The objectives of the current work articulate with the hypothesis and the authors have chosen the correct tools to address it. In this new version the population studied was appropriately described and the number of samples is enough to draw the conclusions. It seems that proper statistics has been employed and there are no ethical concerns in this study.**********Results-Does the analysis presented match the analysis plan?-Are the results clearly and completely presented?-Are the figures  of sufficient quality for clarity?Reviewer #1: In this work the analysis presented matched with the analysis planned. The results were clearly and completely presented and the figures of sufficient quality.**********Conclusions-Are the conclusions supported by the data presented?-Are the limitations of analysis clearly described?-Do the authors discuss how these data can be helpful to advance our understanding of the topic under study?-Is public health relevance addressed?Reviewer #1: The conclusions are supported by the data presented and the limitations or questions have been clarified in this new version and in the rebuttal letter. The authors discussed how their data can advance the understanding of the role of PD1/PDL1 pathway during visceral leishmaniasis. The topic of this study is of utmost importance in worldwide public health.**********Editorial and Data Presentation Modifications?Use this section for editorial suggestions as well as relatively minor modifications of existing data that would enhance clarity. If the only modifications needed are minor and/or editorial, you may wish to recommend “Minor Revision” or “Accept”.Reviewer #1: Accept.**********Summary and General CommentsUse this section to provide overall comments, discuss strengths/weaknesses of the study, novelty, significance, general execution and scholarship. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. If requesting major revision, please articulate the new experiments that are needed.Reviewer #1: The data presented in this work is really important to understand the role of PD1/PDL1 during T cell disfunction/exhaustion in visceral leishmaniasis. So far, VL is a public health issue in many countries and the current drugs available are limited and the ones available are extremely toxic. I believe the study addresses a new perspective in the field. Yet, it lacks a more comprehensive panel of effector molecules involved in T cell disfunction and how interruption of PD1/PDL1 also impact on the production of other molecules and genes. Even tough the study is extremely significant for the field of parasite immunology and also helps paving the way for new treatments against VL.**********what does this mean?). If published, this will include your full peer review and any attached files.PLOS authors have the option to publish the peer review history of their article  are generated on a different schedule and may not be made available as quickly.Soon after your final files are uploaded, the early version of your manuscript will be published online unless you opted out of this process. The date of the early version will be your article's publication date. The final article will be published to the same URL, and all versions of the paper will be accessible to readers.Thank you again for supporting open-access publishing; we are looking forward to publishing your work in PLOS Neglected Tropical Diseases. Best regards,Shaden Kamhawico-Editor-in-ChiefPLOS Neglected Tropical DiseasesPaul Brindleyco-Editor-in-ChiefPLOS Neglected Tropical Diseases


We performed a retrospective and observational study of patients with psoriasis. The aim of this study was to define the laboratory indicators reflecting the treatment response to tumor necrosis factor (TNF)-α inhibitors and the predictors for the treatment response. From January 2010 to June 2022, 28, 15 and 12 patients with psoriasis were treated with infliximab (IFX), adalimumab (ADA) and certolizumab pegol (CZP), respectively. The values of C-reactive protein (CRP), platelet to lymphocyte ratio (PLR), neutrophil to lymphocyte ratio and monocyte to lymphocyte ratio decreased in parallel with psoriasis area and severity index (PASI) at weeks 12 and 52 of treatment. The percentage reduction of the CRP was correlated with that of the PASI at week 52 in all patients and subgroups treated with IFX. The percentage reduction of the PLR was correlated with that of the PASI at week 52 in all patients. Linear multivariate regression analyses revealed that the presence of scalp lesions was associated with a high percentage reduction of the PASI at week 52 in the ADA subgroup. The CRP and PLR might act as biomarkers reflecting the treatment response to TNF-α inhibitors in patients with psoriasis. The presence of scalp lesions might be a predictive factor for a high treatment response to ADA in patients with psoriasis. Psoriasis is a chronic inflammatory skin disease with enhanced immune axis of tumor necrosis factor (TNF)-α/interleukin (IL)-23/IL-17 [Psoriasis is also known to be a systemic inflammatory disease often affecting internal organs such as joints, circulatory organs, intestines or the liver. The C-reactive protein (CRP), the neutrophil-to-lymphocyte ratio (NLR), the monocyte-to-lymphocyte ratio (MLR) and the platelet-to-lymphocyte ratio (PLR) have been identified as indicators of systemic inflammation ,3,4,5. IWe herein analyze the transition of the NLR, MLR, PLR or CRP during the treatment with TNF-α inhibitors, IFX, ADA and CZP in patients with psoriasis and tried to determine which of the parameters can act as biomarkers reflecting the treatment response. We also examined which of the patients’ background factors, including the baseline values of the above parameters, might predict a high treatment response to TNF-α inhibitors.A total of 55 Japanese patients with plaque-type psoriasis  living in Chiba Prefecture were treated with TNF-α inhibitors  from January 2010 to June 2022. Subcutaneous ADA was initially administered at a dose of 80 mg, then 40 mg at 2 weeks and 40 mg or 80 mg every 2 weeks thereafter. Intravenous IFX was initially administered at a dose of 5 mg/kg at 0, 2 and 6 weeks and every 8 weeks thereafter, and in some patients the dose was increased up to 10 mg/kg. Subcutaneous CZP was administered at 400 mg every 2 weeks. They were analyzed retrospectively using the medical record. This study was conducted based on the Declaration of Helsinki (2004) and was approved by the Ethics Committee of Nippon Medical School Chiba Hokusoh Hospital. The patients provided written informed consent.The diagnosis of psoriasis for the patients was made clinically. Before treatment, we examined the patients’ age, body mass index (BMI), disease duration, presence or absence of bio-switch, past history of tuberculosis, arthritis, scalp, nail and genital lesions, diabetes mellitus, cardiovascular diseases or current smoking status.The values of the PASI, NLR, MLR, PLR and CRP were analyzed at weeks 0, 12 and 52 of treatment. The proportion of patients who had achieved at least a 75% or 90% reduction in the PASI from the baseline  or absolute values of the PASI ≦ 2 was calculated.t-test for normally distributed variables or using the Mann-Whitney U test for variables with non-parametric distribution. Group comparisons for categorical variables were performed using Fisher’s exact test. The correlations of the variables with each other were analyzed by Spearman’s correlation coefficients. Statistical significance was set at p < 0.05.All statistical analyses were performed using the EZR . The Shapiro-Wilk test was used to assess the normality of the data distribution. Results are expressed as the mean ± standard deviation (SD) for variables with a normal distribution or as the median and interquartile range for variables with a non-parametric distribution. Differences between weeks 0, 12 and 52 were analyzed using repeated measures analysis of variance for normally distributed variables or using Friedman’s test for variables with non-parametric distribution. Bonferroni correction was used for post-hoc analysis. The differences between patients’ subgroups treated with IFX, ADA or CZP were analyzed using a one-way analysis of variance using the Tukey-Kramer test for normally distributed variables or using the Kruscal-Wallis with Steel-Dwass test for variables with non-parametric distribution. A comparison between two independent groups was performed using Student’s p value < 0.05 in univariate analyses and was adjusted for age, sex and BMI. Variables with a variance inflation factor >10 were excluded to avoid multicollinearity.A linear multivariate regression analysis was performed to determine the predictive factors for the high percentage reduction of the PASI at week 12 or 52 of treatment with TNF-α inhibitors. The analysis included only the variables with a Background factors of the patients are shown in We first evaluated the achievement rates of PASI 75, 90 and absolute PASI ≤ 2 during the treatment with individual TNF-α inhibitors in patients with psoriasis. During the treatment, until week 24, the magnitude and increasing speed of PASI 75 and 90 achievement rates appeared to be the highest in the IFX subgroup (70.4% of PASI 75 at week 24) followed by the ADA subgroup (53.3%), while those of the CZP subgroup (40%) appeared to be lower a,b. In cIn all patients and the IFX, ADA and CZP subgroups, the PASI scores at weeks 12 and 52 were significantly reduced compared to the baseline levels, while there were no significant differences between the PASI scores at weeks 52 versus 12 a. In allWe next analyzed the correlations between individual parameters during the treatment with TNF-α inhibitors . In all We then analyzed if the percent reduction of the PASI in response to TNF-α inhibitors might be correlated with those of laboratory parameters. In all patients, the percentage reductions of the PLR and CRP were positively correlated with that of the PASI at week 52 , indicatWe then divided the patients into those who achieved PASI 75 (high responders) and those who did not (low responders) at week 12 or 52 and compared the percentage reductions of the NLR, MLR, PNR and CRP between the high and low responders . The perWe then examined the background factors that can predict a high treatment response to TNF-α inhibitors, as evaluated using the percentage reduction of the PASI. We first examined whether age, BMI, disease duration and baseline values of the PASI or laboratory parameters affected the treatment response. In the ADA subgroup, the baseline PASI was positively correlated with the percentage reduction of the PASI at week 52 . In the We next analyzed whether sex, presence or absence of bio-switch, arthritis, scalp, nail or genital lesions, diabetes, current smoking status or history of tuberculosis affected the treatment response to TNF-α inhibitors. Patients with genital lesions had a lower percentage reduction of the PASI at week 52 than those without, in all patients and in the IFX subgroup . PatientThese results indicate that genital lesions may predict a poor response to IFX, while scalp lesions may predict a good response to ADA at week 52. There were no significant associations between the percentage reduction of the PASI at week 52 versus the presence or absence of nail lesions, diabetes, current smoking status or bio-switch . There wThen linear multivariate regression analysis was performed to determine the predictive factors for the treatment response to TNF-α inhibitors, as evaluated using the percentage reduction of the PASI at week 52 . The preIn this study, the values of the CRP, NLR, MLR and PLR as well as the PASI significantly decreased in response to treatment with TNF-α inhibitors. Further, the percentage reductions of the CRP and PLR were correlated with that of the PASI at week 52 in all patients. The patients who achieved PASI 75 at week 52 showed a higher percentage reduction of the CRP compared to those who did not, in all patients and in the ADA subgroup. These results indicate that the CRP and PLR might act as biomarkers reflecting treatment response to TNF-α inhibitors in psoriasis. In particular, the high percentage reduction of the CRP might indicate a high treatment response to TNF-α inhibitors. Tumor necrosis factor-α enhances the secretion of IL-6, which stimulates the production of CRP in hepatocytes . AsahinaMonitoring the percentage reduction of the PLR during treatment with TNF-α inhibitors might complement that of the CRP to evaluate the treatment response. Tumor necrosis factor-α induces megakaryocytic differentiation, promoting platelet production, contributing to the increase of the PLR . Thus, TA recent study revealed that a higher baseline PASI was associated with a higher percentage reduction of the PASI after treatment with biologics if the baseline PASI is ≤ 30 . This phThe presence of scalp lesions might predict a good response to ADA. Though scalp psoriasis belongs to difficult-to-treat types of psoriasis, recent studies revealed that scalp psoriasis showed a good response to ADA ,20. TumoThe baseline CRP was negatively correlated with the percentage reduction of the PASI at week 52 of treatment with CZP. This indicates that high systemic inflammation before treatment might predict a poor response to CZP. Based on the present results, when deciding the type of TNF-α inhibitors to treat psoriasis patients, ADA might be applicable to patients with scalp lesions and/or higher baseline PASI scores; CZP might be applicable to patients with a lower baseline CRP; IFX might be applicable to patients without genital lesions. Whether the treatment choice above may be appropriate should be prospectively examined.The baseline NLR and PLR before treatment were negatively correlated with BMI in this study . The finThe baseline PLR was negatively correlated with age in this study . The resThis study has several limitations. Firstly, this was a retrospective study with a small number of patients. Especially the sample size of each subgroup treated with IFX, ADA or CZP was small. Further large-scale prospective studies are needed to validate our present findings and to clarify the differences between IFX, ADA and CZP. Secondly, medications for comorbidities or previous treatments for psoriasis, which might influence the baseline PASI or laboratory parameters, were not included in the analysis. Thirdly, we used the conventional method for the measurement of the CRP. The high-sensitivity CRP might be preferable.Testing the CRP and PLR during treatment with TNF-a inhibitors in patients with psoriasis is relevant and useful for monitoring treatment responses to these biologics. The presence of scalp lesions might predict a high treatment response to ADA in patients with psoriasis.In conclusion, the NLR, MLR, PLR and CRP values significantly reduced after treatment with TNF-α inhibitors in patients with psoriasis. Percentage reductions of the CRP and PLR were significantly correlated with that of the PASI at week 52 of treatment and a percentage reduction of the CRP was higher in patients with the achievement of PASI 75 than in patients without. The C-reactive protein and PLR might act as biomarkers reflecting a treatment response to TNF-α inhibitors in patients with psoriasis. The presence of scalp lesions might be a predictive factor for a high treatment response to ADA in patients with psoriasis.


Familial hypercholesterolemia (FH) is a common, inherited disease characterized by high levels of low-density lipoprotein Cholesterol (LDL-C) from birth. Any diseases associated with increased LDL-C levels including atherosclerotic cardiovascular diseases (ASCVDs) would be expected to be overrepresented among FH patients. There are several clinical scoring systems aiming to diagnose FH, however; most individuals who meet the clinical criteria for a FH diagnosis do not have a mutation causing FH. In this review, we aim to summarize the literature on the risk for the various forms of ASCVD in subjects with a proven FH-mutation (FH+). We searched for studies on FH+ and cardiovascular diseases and also included our and other groups published papers on FH + on a wide range of cardiovascular and other diseases of the heart and vessels. FH + patients are at a markedly increased risk of a broad range of ASCVD. Acute myocardial infarction (AMI) is the most common in absolute numbers, but also aortic valve stenosis is by far associated with the highest excess risk. Per thousand patients, we observed 3.6 incident AMI per year compared to 1.9 incident aortic valve stenosis, however, standardized incidence ratio (SIR) for incident AMI was 2.3 compared to 7.9 for incident aortic valve stenosis. Further, occurrence of ischemic stroke seems not to be associated with increased risk in FH+. Clinicians should be aware of the excess risk of almost all kind of ASCVD in FH+, and the neutral risk of stroke need to be studied further in FH + patients. Atherosclerotic cardiovascular diseases (ASCVDs) comprising coronary heart disease, cerebrovascular disease, peripheral artery disease and aortic disease are common diseases affecting a huge number of patients worldwide . Low-denA recent editorial by Khera and Hegele asked the seemingly unnecessary question—“What Is Familial Hypercholesterolemia, and Why Does It Matter?”. Different definitions of FH lead to different groups of patients being identified as having FH .There arIn this review, we aim to summarize the literature on the risk for the various forms of ASCVD in subjects with a genetically proven FH-mutation (FH+).In the present European guidelines for management of dyslipidemias, FH individuals are categorized as individuals at high-risk of disease by default, and in primary prevention treatment goals are at least a 50% reduction from baseline in LDL-C down to <1.8 mmol/L or <1.4 mmol/L depending on the global risk burden . HoweverThen we also included our and other groups published papers on FH + on a wide range of cardiovascular and other diseases of the heart and vessels including; coronary heart disease, cerebrovascular disease, peripheral artery disease, aortic disease, ASCVD mortality, heart failure and atrial fibrillation and aortic valve stenosis.We searched PubMed using the search string “[” or “”]. No limitation for search period was used. Date of search was 1 July 2021. We identified 132 papers that were reviewed by two authors (KR and AH) to identify the inclusion criteria which were 1) a confirmed pathogenic FH mutation in more than 90% of the study population, 2) heterozygous FH only, not homozygous, 3) CVD outcomes clearly reported, 4) English language, 5) studies reporting on quantitative data, not qualitative studies, and not single case reports. Review articles and letters to the editor were also excluded. Nine articles on CVD in mutation positive FH were included and discussed below. The references of these articles were then reviewed to identify additional studies and we also performed hand searches of papers in our collections and the reference lists of extensive review articles.LDL-receptor gene. Two mutations in the APOB gene (5.4%) and three mutations in the PCSK9 gene (0.6%) have been found to cause FH.Then we also included our and other groups’ published papers on FH + on a wide range of cardiovascular and other diseases of the heart and vessels including; coronary heart disease, cerebrovascular disease, peripheral artery disease, aortic disease, ASCVD mortality, heart failure and atrial fibrillation and aortic valve stenosis. Lastly, we explore the spectrum of ASCVD in mutations positive FH patients, utilizing data from the Norwegian Register of FH+. In 2021, this register reported to have identified 264 different mutations to cause FH, of which 94.0% is in the  ReceptorWhen FH + patients was compared to unaffected relatives, the LDL receptor mutation type only partially contributed to the risk of future CVD .When genotype-phenotype interactions were studied in FH + children the CVD risk in the parents was dependent on type of mutation .In FH + patients, the presence of tendon xantomas was associated with increased risk of CVD .Small, dense LDL-particles in FH + patients were more prominent in patients with CVD Patients with severe FH + defined as LDL-C >8.0 mmol/L had increased risk of CVD with an adjusted hazard rate of 1.25  comparedANKS1A gene is associated with increase in CVD events in FH + patients  compared to unaffected relatives  found a 3.4-fold increased prevalence of angina pectoris in the FH group . This stIn FH + patients without prior AMI, 99 incident AMI was observed during 2001–2009 in Norway, which is 3.58 AMI per thousand patients per year with an average age of 56.2 (13.4) at the time of the first event . ImportaIn 103 patients with ACS with a mean age of 55 with LDL-C> 4.1 mmol/L, 87% men, and offered these patients genetic testing and found that the prevalence of genetically confirmed FH was 9% in this group . This waBy means of cascade testing FH + patients were identified and offered statin therapy and compared to a matched control group (10:1). The median follow-up time was 21 years, and a coronary event  was found in 23% of the FH patients and in 4% of the control population .FH + patients have increased risk of coronary artery disease, but an unanswered question is whether the excess risk of other types of ASCVD is increased to a similar degree? To compare the risk of various types of CVD it is an advantage to study incidence in the same cohort during the same period. We here present risk of various types of ASCVD studied in all FH + patients registered in Norway either at 31 Dec 2009 or at 31 April 2014 and discuss these data together with updated relevant literature in the field.In 3166 FH + patients studied during more than 18 500 person years there was no increased risk of cerebrovascular disease or ischemic stroke . A recenA Mendelian randomization study has demonstrated that people with lifelong low levels of PCSK9 and LDL-C had lower risk of coronary heart disease but with no effect on ischemic stroke . Taken tIn 3162 FH + patients compared to the general Norwegian population, we found that the risk of peripheral artery disease was nearly tripled in the FH group compared to controls in both women and men .Pereira et al. also demonstrated an increased risk of peripheral artery disease in FH (90% FH+) patients with a mean age of 51 years compared to normolipidemic persons, with a prevalence of peripheral artery disease assessed by ankle-brachial index of 17.3 vs 2.3% respectively, the patients were older in the FH group, and there was more diabetes in the FH group, however there was a higher prevalence of smokers in the control group .In a study investigating presence of comorbidities at time of death among 79 mainly FH + patients, 39% of the patients had peripheral artery disease  at time of death at mean 60 years of age .The SAFEHEART registry with 2752 FH + patients with a mean age of 44 years upon enrollment was compared to 993 unaffected relatives showed 39 cases of any peripheral artery disease in the FH group compared to two cases in the control group, and 14 cases of peripheral artery revascularization in the FH group, compared to none in the control group .Indeed, these studies suggest that the risk of peripheral artery disease is increased in FH, and accordingly, increased LDL-C burden during life should be considered a risk factor for this kind of ASCVD.We have recently demonstrated an increased risk of aortic disease including abdominal aortic aneurisms in FH + men, but not in women . LDL-C iIn a cohort of 4688 FH + patients, risk of CVD death was increased compared to the rest of the Norwegian population in all age groups below 70 years . These dIncreased risk of heart failure was reported in 4273 Norwegian FH + patients compared to the general Norwegian population, and the highest excess risk was observed in the younger age group 25–49 years, and most of the heart failure patients had previous coronary artery disease . We alsoCoronary heart diseases including myocardial infarction is a common disease in patients with familial hypercholesterolemia and therefore the increased risk of heart failure and atrial fibrillation could be expected as coronary heart disease is a common risk factor for both.LDL-C seems to be important in the early phase of aortic valve stenosis build up , howeverIn a study of homozygous FH, Alonso et al. showed that patients with null mutations had higher levels of LDL-C and more aortic valve stenosis than patients with a receptor defective mutation, indicating that the level of LDL-C is important for progression of this disease .The association of increased risk of aortic valve stenosis in FH+ is quite clear despite those previous studies of LDL-C lowering in aortic valve stenosis being negative. Most of the studies on aortic valve stenosis have been performed relatively late in the disease, and early intervention is less studied. However, this could be further explored in FH patients. New treatment principles including PCSK9-inhibitors, inclisiran and evinacumab facilitate LDL-C- lowering in FH patients to even lower levels than we have achieved earlier and might be tested out with respect to aortic valve stenosis progression. Data suggest that high lipoprotein(a) values in particular increase the risk of aortic stenosis . This miRegarding the increased risk of different kinds of ASCVD in patients with FH + there seems to be a wide range in the increased risk induction of the different diseases . As an eFH + patients are at a markedly increased risk of a broad range of ASCVD. AMI is the most common in absolute numbers, however, aortic valve stenosis is by far associated with the highest excess risk. Per thousand patients, we observed 3.6 incident AMI per year compared to 1.9 incident aortic valve stenosis, however, SIR for incident AMI was 2.3 compared to 7.9 for incident aortic valve stenosis. Further, occurrence of ischemic stroke seems not to be associated with increased risk in FH+.It should be mentioned that most of the data on FH + are collected in Caucasian persons, and it remains to be studied whether this applies to all races.Clinicians should be aware of the excess risk of almost all kind of ASCVD in FH+, and the neutral risk of stroke need to be studied further in FH + patients. Clinicians should suspect an FH diagnosis in patients with high levels of LDL-C (>5.0 mmol/L in adults), and a family history of premature ASCVD. Fewer FH patients have xanthoma now compared to earlier times, as many are treated with lipid lowering drugs, however without knowing of the FH diagnosis. Further, as the number of treated FH patients increases, there will be less ASCVD in the parent generation which weakens the accuracy of the clinical scoring systems for FH. In emergency rooms and coronary care units, FH patients may present with early onset coronary artery disease, and an FH diagnosis should be suspected in young patients with high LDL-C. In suspected FH, genetic counselling and testing should then be offered to establish treatment and to perform cascade testing if a mutation is demonstrated. Early detection and correct treatment are paramount in reducing the risk of ASCVD among FH + patients.


The Coronavirus Disease 2019 (COVID-19) has severely challenged healthcare delivery systems worldwide. Healthcare Workers were unable to assess and manage the cases due to limited knowledge of treating the virus and inadequate infrastructure. Digital interventions played a crucial role in the training of healthcare workers to get through the pandemic. Project Extension for Community Healthcare Outcomes (ECHO) initiated the COVID-ECHO telementoring program for strengthening the knowledge and skills of healthcare workers. The study aimed at assessing the effects of the ECHO telementoring model in the capacity building of healthcare workers in the context of COVID-19 in India.p < 0.05. In-depth Interviews were analyzed using Framework Analysis. The evaluation followed the first five levels of Moore’s model.We adopted a mixed-method approach with a parallel combination design. A quantitative survey was used to measure changes in the knowledge and self-efficacy among doctors and nurses. In-depth Interviews were used for qualitative exploration of perceptions and experiences of all the study participants. Student t-test and ANOVA were used to assess significant differences between mean scores across participant characteristics for different themes. Statistical significance was set at The results highlighted the strengthening of knowledge and skills of healthcare workers in the assessment and management of COVID-19 after the ECHO training. Learning and performance ratings were high as 96% reported an increase in knowledge and 98% were able to apply it in their clinical practices. The key challenges identified were technical issues like internet connectivity and lack of interaction due to limited visual connection. The hybrid sessions, use of video camera, feedback mechanism, and inclusion of Continuing Medical Education were recommended by participants to improve the model.The findings of this study are an important addition to the pre-existing literature supporting the replicability of the ECHO model in the upskilling of healthcare professionals working in underserved and remote areas, not only in the context of COVID-19 but also in other public health domains. To enhance the effectiveness of this ECHO model, the study findings may be used to refine the model and improve the areas of concern.The online version contains supplementary material available at 10.1186/s12913-022-08288-5. The novel coronavirus disease COVID-19) outbreak due to severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) was first reported in Wuhan, China, on 31 December 2019. Due to an alarming rise in the spread, World Health Organization (WHO) officially declared COVID-19 as a pandemic on 11 March 2020 9 outbrea. EducatiThe training and capacity building of healthcare workers (HCWs) has been identified as one of the important strategies under the National Patient Safety Implementation Framework (NPSIF) 2018–2025 as well as in the Regional Strategy for Patient Safety (RSPS) in the WHO south-east Asia Region (2016–2025) . LearninDigital interventions can provide many opportunities for strengthening health systems and can be vital resources in the current public health emergency . The appThe Extension for Community Healthcare Outcomes (ECHO) is a model that provides informative training and case discussion by medical experts via telementoring, in urban and rural settings, connecting through the web-based videoconferencing platform. It uses a “hub and spoke” model, in which the ‘hub’ is the central physical location from which a specialist team hosts the clinic and the ‘spokes’ are HCWs who connect remotely from their workplace , 15. In Understand the barriers and facilitators in using the ECHO telementoring model for COVID-19.Examine the healthcare providers’ knowledge, competence, performance, and satisfaction associated with the use of the COVID-ECHO telementoring program.Explore the potential of the ECHO telementoring model for the expansion of clinical services beyond COVID-19.The present study aimed at assessing the effects of the ECHO telementoring model in the capacity building of HCWs in the context of COVID-19 in India. Specific objectives of the study were to:The study involves a mixed-method approach using a parallel combination design , where qThe study was conducted at four ECHO hubs located in different states of India - Punjab, Assam, Kerala, and Tamil Nadu from January 2021 till April 2021. Inclusion criteria for selection of hubs was based on the minimum number of sessions (ten training sessions) conducted and the minimum number of participants (500 participants) trained. The study participants for survey data were doctors and nurses (trainees) who had received training. For qualitative data, we included other stakeholders involved in the implementation of the training program i.e. hub-leaders and trainers.A non-probability, purposive sampling was used to select the universe of the trainees. This was followed by simple random sampling for the quantitative survey. Doctors and nurses who attended at least three or more sessions of this program and agreed to participate in the study were approached for the survey.For qualitative inquiry, we used In-depth Interviews (IDIs). Purposive sampling with maximum variation was used to generate different perspectives from all stakeholders. Interviews were conducted until data saturation was achieved.The data was collected through telephonic (cell phone) interviews to avoid exposure/risk to both data collectors and participants because of the COVID-19 situation. A data collection team that had expertise in telephonic interviews was selected and trained by the research team in a two-day training program to collect the data.For the quantitative study, a structured survey tool was developed for this study. This questionnaire was designed keeping Moore’s Expanded Outcomes Framework for assessing learners and evaluating instructional activities. This tool was developed by adapting instruments used by previous researchers to assess similar ECHO educational projects in low- and middle-income countries –19. We uQualitative data was led by two researchers (NM and SL) who worked closely with the field data collectors. Separate discussion guides were developed for conducting IDIs with different stakeholders. The interviews were audio-recorded after taking participant's consent, each of which lasted for 20-30 minutes. Audio recordings were translated and transcribed verbatim by a professional transcriptionist.To estimate the sample number for the survey, we assumed a 10% improvement in knowledge, learning, and skills; anticipated 8% precision and response rate 60% with a design effect factor of 1.5. The sample estimated was 135 participants from each hub (state). The final sample size for this study was 540 participants. These were selected from 4 hubs that consented to participate in the study.A total of 28 IDIs were conducted with trainees as well as other stakeholders .p < 0.05.Discrete variables are reported as frequency, proportions, and continuous variables as mean± SD. The mean scores were calculated using Likert 5 scale for the different themes. Student t-test and ANOVA were used to assess significant differences between mean scores across participant characteristics for different themes. Subgroups analysis was performed for different subgroups (doctors and nurses). The regression diagnostic and residual plot were used for testing violation of assumptions. Polynomial Linear Regression was performed to identify the factors associated with favourable learning and competence, performance, and satisfaction scores. Age and years of experience variables were used as the polynomial variables in the regression model. All statistical analysis was done using STATA Version 16 software . All tests were two-sided, and statistical significance was set at The qualitative data was analysed by a framework analysis approach . To famiTo check the data credibility in quantitative data, 10% of the data was analyzed by two researchers (KS and RP). The researchers conducted a preliminary descriptive analysis of the 10% data and checked for inconsistencies and missing data. To ensure the accuracy of qualitative data, we reviewed the audio recordings and compared them with transcriptions, and field notes of data collectors.The quantitative and qualitative findings were arranged with Moore’s first five levels of evaluation. In addition, the qualitative findings explored challenges faced during the training as well as understanding the potential and scope of the viability of the ECHO program.Moore’s Model of Outcomes Assessment framework for continuing medical education (CME) focusses on the first five outcome levels - participation, satisfaction, learning, competence, and performance .Table Most of the participant’s (41%) practiced in primary care facilities that included primary health care centre and sub-centres. The doctors (47%) in our study were from tertiary care facilities whereas half of the nurses (49%) were from primary care facilities. One-third of the doctors (39%) were having a MBBS degree while one-fourth of nurses (26%) were having a diploma nursing degree.p=0.002). The doctors who were practicing in private facilities showed greater satisfaction levels (32.9±3.0) when compared to doctors practicing at primary (29.8±3.5), secondary (31.4±3.3), tertiary  facilities (Table Seven questions assessed participants’ satisfaction with the COVID-ECHO telementoring program. Most participants (97%) were satisfied with the ECHO model Figure , with pa“The model was very good, it was very practical, and it met my expectations” “It was a good interaction in the sessions. we got to meet people sitting thousands of kilometres away, (and with) doctors across the globe” The qualitative findings complement the quantitative findings and suggest that participants were satisfied with the model. The findings indicate that the sessions were interactive and participants’ experience with trainers was satisfactory.“It's [medical profession] dynamic, things keep changing, New protocols or new guidelines come, we get to learn and do things now and then. So, this platform helps us to gain knowledge faster and easily” “We need to go someplace to learn [for physical sessions]. For example, if I need to learn something. I need to travel someplace. Once we got it virtually, there is no need to travel, and it is a time-saving factor” (Doctor. ID 22.10)Participants also noted the advantages of the virtual learning platform.The learning and competence among the participants were assessed together in the quantitative survey. Around 96% of participants reported improvement in knowledge and clinical skills for assessing and recognizing COVID-19 symptoms after attending the program . Doctors practicing in private health facilities which include personal clinics reported higher mean score on the knowledge scale (54.6 ±6.0) when compared with doctors practicing in primary (51.3±5.5), secondary (53.9±5.9), and tertiary  facilities The t-test and ANOVA results showed that the dentists (doctors with BDS degree) 57.2 ±4.8) had a higher mean score on the knowledge scale as compared to doctors with MBBS (52.2±5.6) or MD degrees  kits, how to wash hands, how to protect healthcare workers” “So, dealing with dementia cases is also very new to us. It was helpful interacting with people from all over the country with experts from NIMHANS ” A few study participants mentioned improved task performance and skill development.“Some of the people have sent their messages to me that after listening to you on this particular topic I could manage with this thing” “It was really helpful in gaining enough confidence to write on the prescription of psychiatric drugs” The qualitative findings show that the training helped participants gain confidence in their practice.Participants were also asked to rate their self-reported performance on a 5-point Likert scale. Majority of participants (above 95%) have reported that they have implemented the learning in their clinical practice and disseminated the knowledge with colleagues Figure .p=<0.001). The doctors practicing in urban areas had a higher performance mean score (17.9±2.0) compared to doctors in rural areas   “As far as I feel, my patients benefitted too.” The dentists (doctors with BDS degree) showed a higher self-reported performance mean score 18.9±2.0) compared to doctors with MBBS degree 17.3±1.9) or a MD degree Participants also felt that there was a positive change in their behaviour towards patient care after attending the training program.p=0.012) compared to doctors with MBBS degree. The doctors practicing in secondary  and tertiary  care settings had significantly higher learning scores as compared to primary care settings. The doctors practicing in secondary care settings  were more satisfied as compared to doctors in primary care settings. The dentists had a significantly higher self-reported performance score  as compared to MBBS doctors  had a significantly higher learning score  were identified as challenges faced by the participants during these training. A summary of challenges is mentioned in Table .Table 4S“I liked their manner of explaining very much, their method of telling. That is why I would certainly want to attend their next session” “So, beyond COVID-19 and cancer there is the scope of expanding this ECHO session [training in other clinical services]” The majority of participants felt that the training program is effective, and most of the trainees were in the favor of attending future ECHO sessions.Based on their experience, participants also recommended strategies to overcome the challenges and improve the model. We summarized the recommendations under the given themes in Table .Table 5ROur study findings reveal that participants of the COVID-ECHO telementoring program were satisfied with the virtual ECHO platform, the sessions as well as the trainers conducting the sessions. Participants reported improvement in their knowledge and confidence in COVID-19 assessment and management after attending the sessions. These findings are similar to other studies that have used Project ECHO for building capacity in chronic pain , hyperteInterprofessional collaborative practice is considered an ideal method of improving staff satisfaction while benefitting the community , 26. ParOur study findings regarding providers’ level of satisfaction towards the telementoring program, our data is aligned with previous reports by Zhou et al. . The revParticipants also described some of the limitations of the training such as administrative issues like inconvenient schedule of sessions, and technical issues like internet connectivity, and video conferencing. Technical issues, in particular internet connectivity and bandwidth, have been identified as problematic by other studies evaluating online training , 32. By To improve the model few suggestions were put forward by the participants. One of them was to have ECHO sessions not only in the clinical domain but also in other areas like training human resources and strengthening community awareness in the area of COVID-19. The potential of ECHO sessions to go beyond the clinical domain to cover vulnerable populations has been found effective in a study conducted in the USA . To imprThe COVID-19 pandemic has shown how lack of knowledge among HCWs has been a major barrier for managing outbreaks. Training programs like ECHO has the potential to reach and strengthen the healthcare workforce in collaboration with public health universities and government institutions , 35. TheThis study is one of the first to use a mixed-method approach to assess an online model for building the capacity of healthcare providers in the context of the COVID-19 in India. Despite current COVID wave constraints, we were able to collect good quality data from different stakeholders across different states in India. The limitations of the study include the smaller number of qualitative interviews with nurses due to constraints posed by the second wave of COVID. We were unable to validate the quantitative survey tool due to resource constraints. The retrospective design of the study involved data collection after 6 months of the COVID- ECHO training and there could be a potential recall bias by study participants.Digital health innovations are rapidly changing the delivery of healthcare services globally. The results of our study support that the ECHO telementoring model has the potential to bring this transformation through building capacities in HCWs working across different geographies. This model can bridge the knowledge asymmetry and help build capacity in healthcare providers working in inaccessible communities. The findings of this study may be used to improve the model. This will help expand and enhance the effectiveness of the ECHO model.Additional file 1.Additional file 2.Additional file 3.Additional file 4.


Success rate of transcatheter aortic valve replacement (TAVR) in aortic regurgitation (AR) patients is relatively low on account of the absence of calcified anchoring structures. Morphological classification and corresponding TAVR strategies for AR are lacking yet.The AURORA study is a prospective, multicenter, single-arm cohort study to evaluate the safety and efficacy of transfemoral TAVR for severe AR in patients with high or prohibitive risk for surgery. Patients who are ≥ 65 years and diagnosed with severe pure AR as defined by the Echocardiographic Core Laboratory will be consecutively enrolled for further multidetector computed tomography (MDCT) scanning and multiplanar analyses. Based on a new anatomical classification and dual anchoring theory, patients will be classified into 4 types according to the level of the anchoring area. Types 1, 2 and 3 (at least 2 anchoring areas) will undergo the TAVR procedure with a domestic Chinese self-expanding valve , whereas type 4 (0 or 1 anchoring area) patients will be considered unsuitable for TAVR and will receive medical treatment. Our goal is to recruit 100 patients to account for 10% missing data or loss of patients to follow-up. Procedural, 30-day, 6-month and 12-month outcomes will be assessed according to Valve Academic Research Consortium-3 criteria.The AURORA study will establish a new AR anatomical classification based on dual anchoring theory through MDCT multiplanar measurement and assess the safety and efficacy of TAVR guided by this new classification and strategy in AR patients.Trial registration: This Study was registered at Chinses Clinical Trial Registry. The registration number: ChiCTR2200055415; The date of registration: 9, January 2022; The URL of the registration: http://www.chictr.org.cn/showproj.aspx?proj=141209. Transcatheter aortic valve replacement (TAVR) has become the preferred treatment option in patients with severe aortic stenosis (AS) –3. HowevAnatomical classification and dUal anchoRing theory to Optimize the tavR strategy for pure severe Aortic regurgitation (AURORA).The presence of a double or multiple anchoring zones in the multiplane tract (from LVOT to ascending aorta) will significantly improves the stability of the prosthesis. A “TAVR-directed AR morphology classification” was proposed by our group. Based on this new classification and relevant procedure strategies, 32 cases of AR-TAVR using first-generation devices from China  have been performed before March 2021. The procedural success rate was 100% and the permanent pacemaker implantation rate was relatively low. These preliminary results are much better than many results using early-generation device reported by other centers. Therefore, we propose a prospective, multicenter, cohort study to evaluate the safety and efficacy of a novel To construct an accurate morphological classification of aortic root anatomy in patients with severe pure AR to guide TAVR.To establish a novel double-anchoring strategy for transfemoral TAVR based on this new morphological classification.To evaluate the safety and effectiveness of transfemoral TAVR for patients with severe pure AR performed according to the double-anchoring theory and the new aortic root morphological classification.The objectives of the proposed study are as follows:The AURORA study is a prospective, multicenter, single-arm, cohort study that will be conducted in 14 high-volume centers with specified expertise in TAVR. Patients with symptomatic pure severe AR confirmed by an Echocardiographic Core Laboratory who are ≥ 65 years will be consecutively enrolled for multidetector computed tomography (MDCT) scanning. After discussions with the heart team, the patients who are considered to have a prohibitive or high risk for surgery will be classified into 4 types: types 1, 2 and 3 patients who have more than 2 anchoring areas for THV will be treated with the TAVR procedure, whereas patients classified as type 4 who have only 1 area  or no suitable area for anchoring will continue to receive medical treatment. Procedural and 30-day, 3-month, 6-month and 12-month outcomes will be assessed according to the Valve Academic Research Consortium-3 consensus (VARC-3 criteria). Enrollment is expected to be completed within 18 months. Institutional ethical approval will be requested in each center according to local regulations that comply with the principles outlined in the Declaration of Helsinki for research in human subjects. The study flow chart is depicted in Fig. Pure severe AR diagnosed by echocardiography; ≥ 65 years old; high surgical risk or surgical contraindications as discussed by the heart team; MDCT multiplanar with measurements; and a signed informed consent form.Patients with AS diagnosed by echocardiography; left ventricular ejection fraction < 20% and left ventricular end-diastolic diameter > 70 mm; poor medical compliance; participation in other interventional clinical trials within the past 30 days. The details of the inclusive and exclusive criteria are listed in Table The candidates will be screened and selected by our extended heart team, which includes an interventional cardiologist, a heart failure specialist, a cardiac imaging specialist, and a cardiac surgeon. The screening process includes an initial selection based on routine available measurements, dedicated confirmatory assessments (including echocardiography and MDCT), and final review by a central screening committee.All patients with symptoms of heart failure and severe pure AR will be screened for eligibility to participate in the study by the local heart teams. The initial evaluation by the extended heart team is intended to evaluate the surgical risk based on routinely available information and to provide written informed consent to undergo the dedicated screening investigation and the subsequent morphological classification.2; (4) regurgitant volume ≥ 60 ml/beats; and (5) dilation of the left ventricle.The comprehensive transthoracic echocardiography or transesophageal echocardiography  data are uploaded to the Echocardiographic Core Laboratory for confirmation of the echocardiographic eligibility criteria. According to the 2017 American Society of Echocardiography guidelines , pure seSixty-four-slice CT scans will be performed, using 100–120 kV, and tube current is modified according to patient size. All MDCT scans will be electrocardiogram-gated, primarily incorporating retrospective gating to yield a systolic phase for annular measurements. MDCT DICOM data are analyzed prospectively for all patients. Curved multiplanar reconstruction analyses will be performed using software specifically customized for valve analysis .A line is generated through the center point of the AA, aortic valve, annulus, and LVOT. The basal annular plane is defined as defined by the three basal attachment points of the aortic valve leaflets: the left-coronary cusp (LCC), the noncoronary cusp (NCC), and the right coronary cusp (RCC). A tracing of this annular plane provided maximal and minimal annulus diameters, area, and perimeter. LVOT, annulus, sinotubular junction (STJ), AA dimension 40 mm above the annulus, and maximal AA dimensions were measured routinely. Moreover, supra-annular and AA multiplanar dimensions  will be also measured to identify the proper anchor levels for THV  is considered an adequate anchor on the annulus. If the THV can be anchored at more than two of the areas mentioned above with a considerable oversizing index rate, they are considered to have adequate anchoring stability for the TAVR procedure, which is termed the dual-anchoring theoryBased on multiplanar measurements of aortic root anatomy and dual-anchoring theory, patients with AR will be classified into 4 types: Type 1: THV anchoring at the levels of the LVOT, the annulus, and the AA; type 2: THV anchoring at the levels of the annulus and the AA; type 3: THV anchoring at the levels of the annulus and the LVOT; type 4: THV anchoring at 1 level  or no anchoring at all.Patients with AR with types 1, 2 and 3 THV anchoring will be candidates for TAVR, whereas those with type 4 THV anchoring will be assigned to continue optimal guideline-directed medical treatment for heart failure   will be All procedures will be performed with the patients given local or general anesthesia with intubation in the hybrid catheterization laboratory, under fluoroscopic guidance, by the multidisciplinary heart team. The transfemoral procedures will be performed as described previously , 14.The following technical aspects will be investigated: (1) Releasing strategies: Rapid pacing (heart rate 180 beats/min) is recommended to decrease the regurgitation volume and systolic blood pressure. For patients classified as having type 2 anatomy, rapid pacing with 1 fast deployment will be used to improve the stability of the THVs. For patients with types 1 and 3 anatomical configurations, rapid pacing is used until two-thirds of the TAVR frame is released. Once the position is carefully checked with angiography, the THV is completely deployed. (2) Transcatheter aortic valve replacement-in-transcatheter aortic valve replacement technique: In patients in whom there is significant paravalvular regurgitation after the first THV deployment, it is suggested that a second THV should be implanted to reduce paravalvular regurgitation, malposition, or embolization of the first valve diving into the left ventricle.The study end points will be defined according to VARC-3 criteria . The priThe safety end points include the major adverse cardiovascular and cerebrovascular events, which are defined as the composite end points of death, stroke, myocardial infarction, renal failure, arrhythmia, and conduction block occurring after the puncture, and reoperation for device- or procedure-related adverse events. All-cause mortality , cardiovascular mortality , and the post-procedure rehospitalization rate due to heart failure . All primary and secondary end points and the relationship of the event to the device and to the operation will be determined by an independent clinical event committee based on the prespecified definitions after review of the original medical documentation. The details of the study end points are summarized in Table t-test or the Mann–Whitney U test. Categorical variables will be reported as numbers with the relative percentage and compared using the χ2 or the Fisher exact test as appropriate.Data will be expressed as mean ± standard deviation for continuous variables and compared using the unpaired Student A recent meta-analysis  has repoTo the best of our knowledge, this is the first prospective, multicenter cohort study to evaluate the safety and efficacy of TAVR for pure severe AR in a large sample of high-risk surgical patients. Moreover, our study will be the first to systematically analyze the anatomical characteristics of the aortic root in patients with pure native AR by MDCT multiplanar measurements. Based on this approach, we will propose a new anatomical classification and develop a novel dual anchoring theory with specific releasing strategies to improve the device success rate in TAVR, which will lead to significant breakthroughs in screening suitable patients for TAVR and improving the prognosis of patients with AR with a high risk for surgery.Many existing studies have described the anatomical characteristics of the aortic root in patients with AS to guide the TAVR procedure . HoweverDue to the significant differences in aortic root anatomy among the 3 types, we recommend different strategies for selecting the valve oversizing index and for deploying the valve. In patients with type 2, the THV should be released as rapidly as possible under rapid pacing (180 beats/min) until the AA anchoring works. For patients with types 1 and 3, we recommend deploying the THVs at the LVOT–annulus area in 2 stages as described previously. In these latter types, it is essential to ensure adequate anchoring without any significant perivalvular leakage before complete deployment.In addition, we cannot ignore the fact that the large oversizing index may lead to compression of the conduction system by the valve frame and an increased risk of the need to implant a new pacemaker. In this study, we will use the VitaFlow device because this THV has a straight cylindrical shape with an outer skirt that may lead to increased friction between the prosthesis and the surrounding tissues and may help avoid displacement of the device. We speculate that the second-generation device with a recyclable system can be used in the higher releasing position to reduce the occurrence of abnormalities of the conduction system. However, the key breakthrough of TAVR for patients with AR is to design suitable devices based on the morphological classification and the dual anchoring theory.


The strength of the immune response, as measured by antibody concentrations, varies between pneumococcal conjugate vaccines (PCVs). Linking immunogenicity and effectiveness is necessary to assess whether changes in immune response from currently recommended PCVs to next-generation vaccines could impact effectiveness. Simulated reverse cumulative distribution curves were generated using published serotype-specific IgG concentrations with placebo or PCV7. This was combined with the published estimates of serotype-specific vaccine effectiveness of PCV7 against invasive pneumococcal disease to estimate the protective antibody concentration for each serotype in PCV7. Then, based on the published serotype-specific IgG concentrations in PCV13 recipients, reverse cumulative distribution curves were generated for the serotypes shared between PCV13 and PCV7. These estimated protective antibody concentration values were then used to predict the vaccine effectiveness of PCV13. The results were compared to published aggregate values for vaccine effectiveness. The aggregate median predicted vaccine effectiveness values were similar to previously reported observed values for the United Kingdom (93% versus 90%), Australia (71% versus 70%), and Germany (91% versus 90%). These results demonstrate that IgG concentrations of next-generation PCVs can be used to generate reliable estimates of vaccine effectiveness for serotypes shared with established PCVs. Streptococcus pneumoniae is a major cause of meningitis, bacteremia, and pneumonia worldwide and is responsible for over 300,000 deaths annually in children under the age of 51. Although pneumococcal conjugate vaccines (PCVs) have substantially reduced the burden of invasive pneumococcal disease (IPD) in children and adults, breakthrough disease and serotype replacement  are ongoing concerns3.4. Higher valency PCVs are being developed to target an even larger number of serotypes5. Ideally, the efficacy of these new PCVs would be evaluated in randomized controlled trials (RCTs). However, it is not feasible to conduct RCTs for several reasons: if populations are already using PCVs, the number of events would be too small for meaningful evaluations and comparisons between products. Additionally, placebo-controlled trials are not considered ethical when an effective vaccine is available6. Therefore, new PCVs, starting with PCV10, have been evaluated via their immunogenicity and approved by the FDA if they elicit an immune response that is non-inferior to existing PCVs. For pediatric vaccines, the primary outcome used for evaluating new PCVs is the concentration of immunoglobulin G (IgG) that targets the pneumococcal capsule. Because licensure decisions are based on IgG concentrations, this is the primary basis of comparison between new and existing PCVs until vaccine effectiveness (VE) studies are performed . Inevitably, technical advisory groups and decision makers will try to interpret differences in immunogenicity between vaccines and make inferences about effectiveness. For instance, comparing PCV13 and PCV7, there was a weaker IgG response for all of the PCV7 serotypes following the primary doses of PCV13, and weaker response after the booster dose for all of the PCV7 serotypes except 19F7.The first PCV approved for use in children targeted seven serotypes (PCV7), and subsequent PCVs expanded the valency to 10 (PCV10) and 13 (PCV13) serotypesWhile IgG concentrations are the standard basis of comparison, decision-makers are interested in the implications of these differences for VE. Differences in immunogenicity between PCVs can be misinterpreted. There is therefore a need for a framework that contextualizes differences in PCVs and attempts to express any differences in terms of VE.8. This is based on a method by Siber et al. that determines the protective concentration of IgG for the PCVs, based on the vaccine efficacy measured in clinical trials9. The correlate of protection actually varies by serotype and population10, and this variation needs to be taken into account when projecting VE based on differences in immunogenicity.The standard practice in the pneumococcal community has been to interpret an IgG concentration of 0.35 µg/mL as a correlate of protection against IPD, with that same correlate of protection used for all serotypesp) using real-world effectiveness data and then apply Siber’s method “in reverse” to show that a known Cp can be used to predict the effectiveness of higher valency vaccines, and that this can be done in a serotype-specific manner using summary-level data. The objective of this work was to assess the utility of this method for predicting the serotype-specific effectiveness of next-generation PCVs. The assessment is achieved by predicting PCV13’s effectiveness against the PCV7 serotypes and then comparing the predicted effectiveness values with corresponding published values.In the present analysis, we leverage and advance Siber’s method to calculate protective concentrations  along with the Cep Table .Table 2P10), Australia (71% versus 70%11), and Germany (91% versus 90%12).No serotype-specific values of VE for PCV13 were available for comparison, so previously reported aggregate values (for the types in PCV7) were used as a standard. The predicted aggregate VE values are shown in Table Lower bounds of the confidence intervals deviated more widely from the observations in both the United Kingdom and Germany. The deviation of the lower bound is due to the method for VE prediction capturing more variability in VE overall compared to what was observed. The increased variability in effectiveness is reflected at both bounds of the prediction, but because the upper bound of the prediction cannot exceed 100%, the deviation is not as profound at the upper bound. The deviation seen on the lower bound of the aggregate prediction is due to the high variability in the serotype-specific VE input data, in some cases varying from negative values to 100% Table . In casep values, we ran simulations using the method described using the commonly accepted 0.35 µg/mL protective threshold (for every serotype). The results in Table p values ranging from 0.08 to 6.08. The changes in the resulting VE (Table p = 0.35) show that proper, serotype-specific Cp estimation is needed to estimate serotype-specific VE: in cases where the protective thresholds are substantially different than 0.35, the resulting predicted VE values are also substantially different  VE  VE  demonstrates the utility of IgG in pediatric populations, thus mitigating any implied risk. Furthermore, opsonophagocytic killing assay titer values were not used in this work due to the relatively large assay variability (both between laboratories and over time) without a standard comparator assay, like the WHO ELISA for IgG concentrations, which is primarily used for licensure decision and to which concordance can be calculated for titer value normalization. The effectiveness is also the result of both uptake  and efficacy . It can further depend on resulting secondary effects that include the reduced force of infection such as through “herd immunity,” and on changes over time in vaccine uptake or relative prevalence of serotypes (and concomitant changes in cross-protection). Additionally, this method assumes that equivalent antibody concentrations elicited by different PCVs  for a specific serotype yield equivalent levels of protection against disease caused by that serotype. Current data across different manufacturers suggest this is a reasonable assumption  but it needs to be verified with post-licensure VE studies.In addition to these considerations, several underlying assumptions also place limitations on the real-world applicability of our method. The effectiveness prediction does not use a functional assay output, like the pneumococcal opsonophagocytic killing assay, but instead relies on IgG concentration. However, previous work  to PCV7 and PCV13. Last, effectiveness needed to be used rather than efficacy, as randomized controlled trials were not run for the vaccines, regions, and time periods of interest.The method described here can be used to calculate the serotype-specific protective concentrations of antibodies elicited by PCVs, as well their serotype-specific effectiveness. To qualify the method, we applied it to calculate protective concentrations and effectiveness of PCV13 in three different geographic locations  using each country’s respective PCV7 serotype-specific effectiveness as input, as well as immunogenicity data that reflected the dosing regimen used to estimate the PCV7 effectiveness. No serotype-specific effectiveness has been reported for PCV13 against PCV7 serotypes , but aggregate effectiveness was reported against these seven serotypes for PCV13, and this aggregate was compared to the predictions. The serotype-specific predictions were aggregated (weighting by relative incidence rates) and the aggregated results agreed with the previously reported data, qualifying the method.17).Using currently available population-level data, the method can predict serotype-specific effectiveness in next-generation PCVs. As next-generation PCVs are developed in the coming years, it will be important to estimate the shared serotype-specific effectiveness to contextualize differences in immunogenicity between vaccines in terms of expected effectiveness and identify whether next-generation vaccines will maintain  control of serotypes that are currently controlled well. The serotype-specific effectiveness predictions may also be useful in dynamic transmission modeling to assess the potential of breakthrough disease, especially in higher-risk persistent serotypes  is the timepoint used with the current correlate of protection (0.35 µg/mL) timepoint and reflects the immune response elicited within the first year of life when children are at the highest risk of IPD. The immunogenicity data are obtained from publicly available summaries of clinical trials, while the VE data are drawn from real-world evaluations of the vaccines  VE.In addition to the serotype-specific IgG concentrations, Step 1 requires serotype-specific VE Table  as input23. They further assume that the relationship of the immune response and the probability of IPD is a step function , that the antibody concentration measured ∼4 weeks after the primary series immunization of infants predicts up to 5-year effectiveness, and that the reported serotype-specific GMCs and variance from the trials for placebo reflect the true population GMC and variance. It was also assumed that the proportions of subjects who missed vaccine doses were similar between cases and controls in the PCV7 VE publications and that the distribution of IgG concentrations from the study was representative of typical antibody concentration ∼4 weeks after the primary immunization, regardless of adherence. Finally, the reported PCV7 and PCV13 serotype-specific VE values were obtained during an observation period ranging from 2000–2010 (for PCV7) and 2010–2016 (for PCV13). An assumption was made that VE and force of infection remained unchanged across serotypes over those periods. It was further assumed that vaccine efficacy and the value of the Cp did not change .A number of simplifying assumptions were incorporated into step 1. The results assume that once the effects of antibody concentration are accounted for (by the protection model), the resulting efficacy and effectiveness are no longer dependent on the vaccination regimen p previously derived for these serotypes are then used to predict  were used as inputs for Monte Carlo simulation . Each simulation sampled a new GMC and VE from a log-normal distribution; uncertainty in the GSD was accounted for by generating GSD estimates assuming a chi-square distribution.p based on the RCDCs for placebo and PCV7 and the published VE values for PCV7 (step 1). We then solved for the serotype-specific VE values of PCV13 based on the Cp from step 1 and the simulated RCDCs for placebo and PCV13 (step 2). Simulated Cp (µg/mL) and VE (%) were summarized as medians with 95% confidence intervals.Each simulation first solved for a serotype-specific C18. The predicted VE values and confidence intervals were compared with the published values and confidence intervals.Because only aggregate (not serotype-specific) values of VE were available in the literature for qualification of our results, the serotype-specific values calculated by our method were combined with weighting into aggregate medians, 2.5%, and 97.5% bounds. Weighting was based on the relative incidence rate of IPD caused by each serotype in PCV7, obtained from the source publications on VEThese analyses were performed in the R statistical software version 4.1.0.Supplementary Materials