Rhodnius prolixus. Interestingly, the silencing of both genes phenocopied the silencing of ATG6/Beclin1, generating 1) accumulation of yolk proteins in the hemolymph; 2) white, smaller, and yolk-deficient oocytes; 3) abnormal yolk organelles in the oocyte cortex; and 4) unviable F1 embryos. However, we found that the similar phenotypes were the result of a specific cross-silencing effect among the PI3K subunits where the silencing of VPS38/UVRAG and ATG6/Beclin1 resulted in the specific silencing of each other, whereas the silencing of ATG14 triggered the silencing of all three PI3K components. Because the silencing of VPS38/UVRAG and ATG6/Beclin1 reproduced the yolk-deficiency phenotypes without the cross silencing of ATG14, we concluded that the VPS38/UVRAG PI3K complex II was the major contributor to the previously observed phenotypes in silenced insects.In insects the reserve proteins are stored in the oocytes into endocytic-originated vesicles named yolk organelles. VPS38/UVRAG and ATG14 are the variant regulatory subunits of two class-III ATG6/Beclin1 PI3K complexes that regulate the recruitment of the endocytic (complex II) and autophagic (complex I) machineries. In a previous work from our group, we found that the silencing of ATG6/Beclin1 resulted in the formation of yolk-deficient oocytes due to defects in the endocytosis of the yolk proteins. Because ATG6/Beclin1 is present in the two above-described PI3K complexes, we could not identify the contributions of each complex to the yolk defective phenotypes. To address this, here we investigated the role of the variant subunits VPS38/UVRAG  and ATG14  in the biogenesis of the yolk organelles in the insect vector of Chagas Disease Altogether, we found that class-III ATG6/Beclin1 PI3K complex II (VPS38/UVRAG) is essential for the yolk endocytosis and that the subunits of both complexes are under an unknown transcriptional regulatory system. The oocytes of oviparous animals are highly specialized cells committed to the storage of nutrients (yolk) required for the maternally detached embryo development. Most oocytes undergo a massive growth during oogenesis due to their high endocytic activity of yolk macromolecules. In this work, we found that the components of two PI3K complexes are co-transcriptionally regulated, and that one specific PI3K complex (class-III PI3K complex II) is essential for the correct endocytosis and biogenesis of the yolk endocytic organelles. The molecular machinery and regulations that govern the yolk generation are mostly unknown. Thus, these findings are important in the context of general vector’s reproductive biology and for the elaboration of novel strategies for vector’s population control. The oocytes of oviparous animals are highly specialized cells dedicated to the storage of nutrients required for embryo development. Most oocytes undergo a massive growth in volume during oogenesis due to the endocytosis of yolk macromolecules which will be targeted for degradation in a regulated manner throughout embryogenesis –5.Phosphoinositide 3-kinase (PI3K) family members are able to generate phosphatidylinositol-3P (PI3P), an important signal molecule involved in a variety of vesicle trafficking events, in a spatially- and timely-regulated manner. The controlled generation of this important signal inducer regulates diverse cellular processes including cell growth, proliferation, and survival –9. ThreeSaccharomyces cerevisiae but have been found in other species including plants and higher eucaryotes  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,Alessandra Aparecida GuarneriAssociate EditorPLOS Neglected Tropical DiseasesEric DumonteilDeputy EditorPLOS Neglected Tropical Diseases***********************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 are clearly articulated with the hypothesis stated and the design is appropriate to address the stated objectives. However, important things or clarifications must to be address to ensure reproducibility of the results and credibility in the eyes of readers, in other words, a lot of information is missing, mainly in material and methods; that section is the heart of the article because it shows the validity of the work, so please check it and be sure you lead every suggestion (explaining or adding the missing information). 1) Add the corresponding reference to R. prolixus genome and transcriptome databases, for example, Mesquita et al., 2015 or Giraldo-Calderón et al., 2015. 2) Please, consider citing the reference to SIA software and PFAM.3) GenBank/database accession numbers should be provided for all of the genes/transcripts covered in the manuscript . Also, the use of Yeast and Human sequences is missing in Material and Methods.4) Insects section: are the insects also controlled by light and dark cycles? were the females mated? Were females in the first reproductive cycle used? In other words, after the first blood feeding as adult? What was the age of these females? Is the autogeny of these females controlled? All these questions should be answered in the manuscript to cover a rigorous methodology because the reproductive physiology in this specie, including the development of the oocytes, depends of all these parameters. 5) The authors need to give more detail for the extraction methods, cDNA synthesis and qPCR , detail the qPCR reaction , temperature cycle, use of blank without cDNA, detail the characteristics of the primers , how ensure that treatment with DNAse I has degraded all possible contamination with genomic DNA?) The authors might find missing information that should be added using the MIQE.6) For each experiment, did the authors use a pool of tissues or an individual tissue? Please, check it throughout the MS and add the information where necessary. 7) How were the different parts of the ovariole obtained? Perhaps it could be necessary to define the different parts of an ovariole and how they were classified in this work to be dissected. Can the authors ensure that there is no contamination between the different parts? 8) Also, in the Supplementary Fig 1 the author stated “different tissues of the ovary and oocyte stages” but it is actually different parts of an ovariole (not tissues). Please, check it throughout the MS.9) In the supplementary Fig 1, it is stated that all samples were dissected 7 days after the blood meal but in Material and Methods section, it is reported that all organs were dissected 6 days after blood meal. Please, check it. I guess at 7 days PBM the egg laying has started, but again, it depends of all the condition asked in the 4th point. 10) Did the authors separate follicle cells from oocytes? Otherwise, they must to be classified as follicles and not oocyte. Please, check it throughout the MS. Also, chorionated oocytes are actually eggs, right? So, how was “chorionated oocytes sample” defined if the egg laid has already started? I assume that just the eggs that are in the ovary but not in the oviduct. This is not very clear, please explain better. 11) The RNAi experiments were done lack an off-target control. Without this control, off-target effects could not be ruled out. The authors made mention of this point in the discussion, but for future work, it would be interesting to have a second dsRNA with a different sequence to exclude this possibility, specially because mostly the conclusions got by this report are based on RNAi experiments. 12) Why does the Table S1 show two pairs of primers per each transcript to RNAi experiments? Please, explain better the RNAi design. 13) Where is the dsRNA injected? In what part of the body insect? Was the dsRNA diluted in water, PBS or Rhodnius solution? Please, add this information to the text.14) Please, specify in which tissues the knockdown efficiency was confirmed.15) Figure supplementary 2: Mortality curve is not analyzed with parametric statistics since it has a binomial distribution . An analysis using the Log-rank test would be more appropriate.16) PI3P detection by thin-layer chromatography (TLC) and quantification: The TLC plates and the autoradiography of the phospholipids and standards used in this experiment should be show as Supplementary figure.17) The densitometric analysis using Adobe Photoshop CC 2015 software might not be the best option, other applications such as ImageJ are more specific for this analysis. Otherwise, please give references to use Adobe Photoshop CC 2015 software for this analysis. 18) In Material and methods, it is stated that at 10 days post blood meal, the “chorionated oocytes” were collected; however, in the Fig 2 caption, it is stated that the organs were collected at 7 d post feeding. Also, the caption of Fig 2 indicates that PI3P detection by TLC was done using ovaries; however, in Material and methods was pointed out that just “chorionated oocytes” were used for this experiment. 19) Egg homogenates and SDS-PAGE: were the samples centrifugated or just homogenized and used? Please, give details of the method to homogenization and the recipe for PBS. 20) Hemolymph extraction and SDS-PAGE: how was the hemolymph extraction done? Indicate the method used to obtain hemolymph. Was the hemolymph sample hemocyte-free? Please, indicate if the samples were centrifugated.21) Did the authors use around 2 X more proteins for dsVPS38 or dsATG14 than dsMaI to load SDS-gel? It is confusing because an incorrect conclusion could be drawn from this experiment, indeed, the author conclusion is that the overall levels of protein in the hemolymph were increased in knockdown insects respect to controls, but it is complicated to analyze if the protein load for each condition was different. It could be concluded that yolk protein precursors are accumulated in hemolymph if the amount of protein load is the same; otherwise you might see more yolk protein because it loaded more. Please, check it or explain better. 22) In Material and methods, it was written “dsRpATG6 samples”, but I think it was a mistake. 23) “Hemolymphs” should be changed by “hemolymph samples”.24) Light microscopy: were the tissues obtained only from opercula-free oocytes? Because in Material and methods only the preparation of this samples is indicated but then it is stated that transversal sections of the oocytes and eggs were obtained in a cryostat. Please, check it. What was the age of the females used for this experiment? How many days after blood meal? Please, indicate this relevant information in the text. 25) Yolk organelles suspension and Flow Cytometry (FACS): The gating strategy for FACS analysis assessed by Flowing software to discriminate single yolk organelles from aggregate  should be added as Supplementary Figure. Also, “PBS” should be abbreviated because it has already been used. What was the age of the females used for this experiment? How many days after blood meal? Please, indicate this relevant information in the text. 26) Please clarify in Material and methods section the number of independent experiments, and the number of samples  composing each experimental group. This is valid for all the experiments described in the work.Reviewer #2: The objective and hypothesis are well articulated. In general terms, the experimental designs are appropriate. Sample size is adequate to address the hypothesis. Statistical analyses were correctly applied.--------------------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: 1) Results: please, add as Supplementary information the alignment between the different sequences in order to demonstrate the percentage of similarity indicated in “Results”, as well as a picture showing the exons indicated by the authors for each transcript. 2) Figure 1: indicate the statistical significance of the asterisk in the caption. 3) The VPS38 and ATG14 transcript expression showed in Figure 1 is so different to that reported in Figure 2 (control insects). Please, explain this point. 4) In Figure 2 A-B: is the complete ovary used for this experiment? 5) Figure 2 C: please, be careful to discuss about if the complete ovary or just the chorionated oocytes have been used, it should not be used indistinctly. This is valid for all the manuscript.6) Figure 2 C: It is not clear that silenced ovaries are presenting a reduction of 72% in their PI3P levels, it seems closer to 40%.7) Fig 3E is not in Figure 3, please, check the text in results section.8) It can’t be concluded from a simple SDS-gel that the bands noted as Vt/Vg and RHBP are exactly what the authors claim to be. We all know the molecular weights of them, but it would be necessary to indicate it with more care if you don’t use a specific antibody. Please, write in potential (the band is compatible with …) when indicating that these proteins could be Vt/Vg and RHBP, and refer to some other paper where the running profile by SDS-gel has been demonstrated for each protein.9) The model for the transcriptional regulations between ATG6/Beclin1, VPS38/UVRAG and ATG14 is in Figure 6 but in the caption of Figure 5. Please, check it. 10) Please, add the statistical significance missing in the caption of Figure 6.11) Please, clarify if the level of knockdown presented (Knockdown (%) post-treatment) is lowering the expression by presented % or to presented %. Authors could add it in the caption.12) Flow Cytometry (FACS): Is there any reference for this experiment? Otherwise, the authors should explain better what is the aim of measure the complexity of yolk organelles, because it is clear the reason to measure the forward direction (to indicate the relative size of the organelles), but the SSC is use to indicate the internal complexity or granularity of the cell, and here just yolk organelles are analyzed. Could the protein contained in each organelle (after discrimination single organelle from aggregates) give information about the complexity?13) Figure 5: The Fig 5B doesn’t seem a representative density plots of the yolk organelles present in control and silenced chorionated oocytes if it is compared with the frequency showed on the upper quadrants . In the Fig 5B it seems that in the RT, events in dsVPS38 RT are higher than control, and in the LT, events in both dsVPS38 and dsATG14 LT, are higher than control.14) Figure 6: please, add in Material and methods section the use of dsATG6.Reviewer #2: The analysis presented are in agreement with the analysis plan. The results are completely presented. Figures and Tables are of sufficient quality. Minor revision for specific Figures was indicated in "comments to authors".--------------------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: 1) Discussion: it seems that a word is missing in the first sentence. Please, check it. 2) Yolk protein precursors are accumulated in coated vesicles and then, after loosing clathrin coats, in early endosomes. These early endosomes then fuse and form the yolk organelles; in this context, it would be great to discuss what of these potential levels were affected.3) The fact that the expression of ATG14 is not altered by downregulation of VPS38/UVRAG and ATG6/Beclin1 allows to conclude that the impaired yolk uptake phenotype by dsATG6 treated females is the effect of silencing the VPS38 PI3K complex II, which could indicate the VPS38/UVRAG PI3K complex II as the major contributor just for yolk endocytosis, and then, could ATG14 (complex I) be working on further steps, e.g. during the process of protein degradation (autophagy)?Reviewer #2: Most of conclusions are supported by the findings, but discussion needs to be improved. Please, see “comments to authors” for details.--------------------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: Introduction: 1) Since the scope of PLOS Neglected Tropical Diseases is aimed to report studies showing aspects of forgotten diseases affecting the world’s most neglected people, information about the relevance of this work in the current context of Chagas disease should be added, specially because since 2005, the WHO recognized this affection as a neglected tropical disease (NTDs), which is mainly transmitted by the contact of people with faeces/urine of infected blood-sucking triatomine bugs.2) Also, I realized that a good background talking about endocytotic organelles in insect oocytes, or showing the nature of yolk bodies, was missing; it should be added in the introduction to show the relevance of the complexes studied here, in the regulation of autophagy and membrane dynamic of endocytosis.3) Could the authors check the references, and perhaps use more reviews when possible? for example, at the end of a sentence in the introduction there are 14 references added; I understand that it is also part of previous stated, but in that case, it would be better to use after each sentence the corresponding reference.4) ATG9 is named in the 3rd paragraph, I suggest that each time something new is named in the text, it is briefly defined. 5) It may be helpful for the reader to indicate the full name of the abbreviation the first time they are named, for example, UV radiation resistance-associated gene protein (UVRAG) Vacuolar protein sorting 34 (Vps34), AuTophaGy-related (ATG), etc.Reviewer #2: Please, see Summary and General Comments.--------------------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: In this study, the authors performed an analysis on the biogenesis of yolk organelles in the oocytes of the vector Rhodnius prolixus, delving into a study previously published by the same group. Here they report that VPS38/UVRAG and ATG14 subunits of the ATG6/Beclin1-PI3K complexes are essential for the yolk endocytosis. The work is original in the study area on biogenesis of yolk bodies and the conclusions are justified by the evidence presented. Overall, I consider it work contains very interesting and relevant data and deserves to be published. However, it is essential to address a significant number of points to improve the understanding and reproducibility of the experiments. Also, I want to state that the page numbers and line numbers in the manuscript file were missing, so it has been a bit complicated to indicate my observations.Reviewer #2: Comments to the authors:In this work, Veira and co-authors analyzed the role of the variant subunits VPS38/UVRAG and ATG14 in the biogenesis of the yolk bodies in the insect vector of Chagas Disease Rhodnius prolixus. Both genes are members of the ATG6/Beclin1 class-III PI3K complexes, which are involved in the regulation of autophagy and the membrane dynamics of endocytosis. The aims are clear and straightforward and the results are compelling. Even though some conclusions must be revised, the manuscript open horizons to unravel the regulating processes involved in protein uptake and trafficking by developing oocytes, which are largely unknown. Please, find below some points that I suggest revising in order to improve this manuscript. General Comments:- Through the manuscript: Sentences like “control and silenced eggs …” or “chorionated oocytes..” are incorrect. Samples were obtained from control and silenced females. Please, revise. - Material and Methods, page 10- Yolk organelles suspension and Flow Cytometry (FACS): Consider replacing “Suspensions of yolk organelles were obtained by gently disrupting recently dissected chorionated…” by “Suspensions of yolk organelles were obtained by gently disrupting of recently dissected chorionated…”- Results, page 10-Silencing of VPS38/UVRAG and ATG14 results in the accumulation of yolk proteins in the hemolymph and formation of small yolk-deficient oocytes: Consider replacing “Double stranded RNAs were designed to target specific regions of the VPS38/UVRAG and ATG14 mRNAs and injected to the insects hemocoel 2 days before the blood meal” by “Double stranded RNAs designed to target specific regions of the VPS38/UVRAG and ATG14 mRNAs were injected to the insects hemocoel 2 days before the blood meal.- Results, page 11- VPS38/UVRAG and ATG14 silenced eggs are yolk-deficient and do not support embryo development: Consider replacing “… the silenced females laid the same number of eggs as control females”…by “… the silenced females laid similar number of eggs as control females”…Specific Commentsa) Results and Figures:- Silencing of VPS38/UVRAG and ATG14 results in the accumulation of yolk proteins in the hemolymph and formation of small yolk-deficient oocytes (page 11): The authors proposed the measurement of hemolymph yolk protein content in control and silenced females to demonstrate that formation of white/small oocytes was due to a deficiency in the uptake of yolk proteins from the hemolymph. Such an assumption is reasonable but not conclusive if information regarding expression (mRNA and protein levels) of yolk protein precursors such as vitellogenin in the fat body is not provided. In addition, the results showed the amount of total proteins in the hemolymph but not yolk protein content. Authors should perform at least western blots for vitellogenin and RHBP in fat body to better support the conclusion. Please, limit the rationale of the experimental design.-VPS38/UVRAG, ATG14 and ATG6/Beclin1 (page 12) and Figure 6: Authors should provide a hypothesis to explain the downregulation of ULK1/ATG1 in VPS38/UVRAG-silenced samples.- Fig. 3D and Fig. 4F: Did the authors use a loading control for SDS-PAGE? According to Material and Methods section, the amount of proteins loaded were different in samples from control and silenced females.- Legends to Figure 5 and Figure 6 (page 18): The model for the transcriptional regulations between ATG6/Beclin1, VPS38/UVRAG and ATG14 is shown in Figure 6, not in Figure 5. Please, revise. - Supplementary Figure S2: Survival curves are presented in the panel “B”, but the legend stated “E-F”. Please, revise.b) Discussion:- Authors associated the decrease of PI3P in ovaries/chorionated oocytes from VPS38/UVRAG and ATG14 silenced females with “at least some reduction of their protein activity, validating the specificity of the observed phenotypes”. In fact, signaling mediated by PI3P is complex and can be performed even with low levels of PI3P. Please, tone down the conclusion. - Authors scarcely compare their results with others in the literature from different species. Please complete.--------------------what does this mean?). 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Read more information on sharing protocols at  5 Aug 2021AttachmentVieira 2021, authors answers.docxSubmitted filename: Click here for additional data file. 24 Aug 2021Dear Dr Ramos,We are pleased to inform you that your manuscript 'VPS38/UVRAG and ATG14, the variant regulatory subunits of the ATG6/Beclin1-PI3K complexes, are crucial for the biogenesis of the yolk organelles and are transcriptionally regulated in the oocytes of the vector Rhodnius prolixus.' 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. 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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,Alessandra Aparecida GuarneriAssociate EditorPLOS Neglected Tropical DiseasesEric DumonteilDeputy EditorPLOS Neglected Tropical Diseases***********************************************************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: yesReviewer #2: As stated in the first revision, the objective and hypothesis of the MS are well interconnected. The experimental designs are appropriate. Sample size is adequate to address the hypothesis. Statistical analyses were correctly applied.**********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: yesReviewer #2: As stated in the first revision, the analysis presented are in agreement with the analysis plan. The results are completely presented. Figures and Tables meet appropriate quality standards.**********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: yesReviewer #2: Authors made suitable modifications to strengthen the conclusions based on the obtained results. According with the suggestions, the discussion section was largely improved.**********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: -Reviewer #2: (No Response)**********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 have addressed all comments in the revised MS. This is a nice work. Congratulations.Reviewer #2: Comments for the authors:The authors have considered most of the suggestions made by this reviewer. Other aspects related to the methodology and results have also been satisfactorily answered. The authors conducted an exhaustive revision of the entire manuscript, including review of the English language.The revised version of the manuscript has improved substantially.**********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. 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To stablish a consensus on the treatment strategy for advanced non–small-cell lung cancer (aNSCLC) with epidermal growth factor receptor mutation (EGFRm) in Spain.After a systematic literature review, the scientific committee developed 33 statements in 4 fields: molecular diagnosis (10 items); histologic profile and patient clinical characteristics (7 items); first-line (1L) treatment in EGFRm aNSCLC (8 items); and subsequent-line treatment (8 items). A panel of 31 experts completed 2 Delphi online questionnaires rating their degree of agreement/disagreement for each statement through a 1–9 range scale . Consensus was reached if 2/3 of the participants are in the median range.In the first Delphi round consensus was achieved for 24/33 of the statements. One of the assertions was deleted, proceeding to a second round with the eight remaining questions with no consensus or in the range of indeterminacy. Determination of the EGFR status from tissue and analysis of the different biomarkers are two important variables that influenced treatment decision in patients with aNSCLC. 1L treatment should be the best therapeutic option, independently of the subsequent lines of treatment. For patients with the most common activating mutations osimertinib was considered the most efficient and safe 1L option. In case of disease progression, a new biopsy was needed.A consensus document is proposed to optimize the treatment strategy for untreated patients with a NSCLC with EGFR sensitizing mutations. Non-small cell lung cancer (NSCLC) account for 85%–90% of lung cancer, being adenocarcinoma the most common subtype. Epidermal growth factor receptor (EGFR) mutations are found in ∼10%–12% of Caucasians with adenocarcinoma and are more frequent in never smokers, females and in patients of East Asian ethnicity . ResultsAlthough the efficacy of EGFR-TKIs in the treatment of EGFRm aNSCLC patients is well established, the treatment decision-making process is becoming more complex as our knowledge of EGFRm and resistance pathways grows and more treatment options become available. Thus, treating physicians must consider an increasing number of factors, facing real world challenges. Indeed, a consensus developed by experts could help to guide decisions and issues that can occur in clinical practice in case there is not enough evidence.We have establish a consensus about the decision criteria for selecting the best first-line (1L) therapy, the importance of a second biopsy on disease progression to determine the most appropriate subsequent therapies using the Delphi Technique method .The aim of this study was to establish a consensus through Delphi Technique on the treatment strategy options in Spain for aNSCLC harboring EGFRm. An external expert in Delphi methodology guaranteed the quality of the overall process.In September 2019, a scientific committee of six experts in comprehensive lung cancer management met to define and lead this consensus project. The following steps were carried out by the scientific committee: (a) selection of expert panel; (b) generation of clinical statements based on review of the medical literature; (c) definition of the consensus levels and agreement on methodology; (d) interpretation of the results from the two Delphi rounds (DR); (e) develop of the final consensus document.A panel of 31 medical oncologists specialized in lung cancer and outstanding for their clinical and research experience were selected by the scientific committee. In this selection of an expert panel, a balanced territorial representation of Spain was considered.Molecular diagnosis on aNSCLC (10 statements);histologic profile and patient clinical characteristics (7 statements);1L therapy in EGFRm aNSCLC (8 statements);second and subsequent-lines treatment in EGFRm aNSCLC (8 statements).After a narrative literature review, 33 statements organized in 4 major domains were drafted by the scientific committee. These domains aimed to cover from the key diagnostic criteria to different treatment lines according to the patient profile:It was established that these statements would be evaluated in patients with EGFRm aNSCLC with good general condition (ECOG PS 0 and 1) and without medical contraindications to receive treatment. Only reimbursed treatments in Spain are considered.The expert panel completed two DR of the statements through questionnaire in an online platform.Each item was responded using Likert scale with 1–9 range  and the consensus level was calculated by tertiles. Range from 1 to 3 were considered values of disagreement, 4–6 range neither disagree nor agree, and scores from 7 to 9 were considered values of agreement. A statement with consensus existed when more than 2/3 of the answers are in the median range (MR), and not consensus if more than 33.3% are outside the MR. If the MR is in 7–9, there is consensus about agreement between panelists, if MR is between 4–6 exist indeterminate consensus and when the MR is 1–3 there is consensus of panelists disagreement.Consensus/agree: there is consensus between panelist, the statement should not be reviewed.Consensus/disagree: there is consensus that panelists disagree.No consensus/agree: there are more panelists in agreement, but there is no consensus.No consensus/disagree: there are more answers in disagreement, but there is no consensus.Consensus/indeterminate: majority neither agree nor disagree. Statements should be reviewed and to decide whether to delete or keep up once evaluated (no items achieved this score).No consensus/indeterminate: there is no general agreement or disagreement, and, furthermore, there is no consensus on this indeterminacy.The possible consensus levels were the following:All statements reaching consensus in the first round (FDR) did not undergo the second Delphi round (SDR). A SDR was held for the 8 statements without consensus in the FDR.In FDR, started on 30 January 2020, 31 panelists answered the complete questionnaire, which supposes a response rate of 100%. In SDR (23 March 2020) 30 of 31 completed the questionnaire (96.8% response rate). This study consisted of a survey of expert opinions and no patient data were collected, so no specific independent ethical or research review or approval was necessary.α) and intraclass correlation (ri) coefficients. Both statistics were calculated for the complete questionnaire and for each domain.The reliability of the questionnaire was assessed by internal consistency using Cronbach’s alpha coefficient (Cronbach’s Median and interquartile range (p25–p75) were calculated for each item (none of the variables showed Gaussian distribution). As it mentioned above, the score responses were divided in tertiles . The agreement level was calculated according to the tertile in which the MR of the responses was located, and the consensus level was evaluated by the percentage of answers in this median tertile.Spearman's correlation coefficient, following Martinez et al. criteria , was calThe SPSS 21.0. was used for the statistical analyses.This project began in September 2019 and ended in July 2020. During this time and when this article was drafted, newly published literature and congress presentations were reviewed to analyze the clinical implications of any new data in patients with aNSCLC and to provide support for the consensus statements.In the FDR (100% response rate), 33 statements were evaluated, and a consensus was reached for 24 statements (72.7%). One of the non-consensus statements was rejected after reassessed by the scientific committee, and the remaining 8 statements were evaluated in the SDR (96.8% response rate), in which neither achieved consensus.ri Domain 1 and 3)  about the need to know the EGFR mutation status, regardless of PD-L1/ PD-L1 expression, before to establish the treatment (statement 4).The one statement with no consensus asked about the importance of disease stage versus molecular diagnosis (statement 1). On the other hand, the statements with high consensus were focused on EGFR status determination (preferability from tissue samples) and analysis of the different biomarkers, which shows that both variables are key and influence the treatment decision and strategy in patients with aNSCLC.Table Table Osimertinib was considered the best 1L therapy choice in patients with common activating mutations  due the safety profile, PFS and OS of this drug compared to other EGFR-TKI, and for patients with uncommon EGFR mutations.Table In patients treated with osimertinib in front-line, if oligoprogression occured during the therapy, this TKI must be maintained adding local treatment. In case of progression, evaluation of PD-L1 expression was needed in order to assess properly the patient, and the inclusion in a clinical trial would be a priority. To perform a new biopsy was relevant in case of progression, regardless of the type of TKI used on the front-line. Finally, in case of disease progression to osimertinib or in absence of T790M mutation, the best option was to use chemotherapy.In order to assess the correlation between the two rounds as much the total questionnaire, as each domain Table a and of Table (Spearmans’s coefficient range 0.76–1 = strong or very strong correlation) both in total questionnaire and by domains. However, the coefficients are overestimated because the values of the items which reaches consensus at the first round are repeated in the SDR. Kappa index indicates a good qualitative concordance in total questionnaire as well as in each domain.The items between the two phases have moderate and strong correlation values, except for item 1 from Domain 1 with weak correlation Table b.Finally, the coefficient of variation was much less than 10%. Such a low value, which did not even reach 1%, supports the decision not to carry out a new round, since it would not provide variation.The current Delphi study shows that a high degree of consensus exists among experts on the relevance to obtain molecular diagnosis before starting any treatment in aNSCLC, how these patients should be treated in the 1L setting according to the presence of EGFRm, and how the 1L therapy decision in these population may influence subsequent therapeutic approaches. This consensus document complements the information in the European Society of Medical Oncology (ESMO) , SociedaRegarding molecular diagnosis, it is important to highlight the unanimity of agreement among experts about the need to know the definitive EGFRm status, regardless of PD-L1 expression, or patient characteristics like age or smoking habit, before starting aNSCLC treatment. This recommendation follows the most relevant and current guidelines –12. AlthMoving forward the histologic profile and patient clinical characteristics, the experts agreed with the no relevance of smoking habit and age in establishing the 1L therapy and the importance of CNS imaging at diagnosis, given the high incidence of brain metastasis in EGFRm aNSCLC . Regardip < 0.001) and a more favorable toxicity profile due to its lower affinity for wild-type EGFR. Furthermore, osimertinib has also improved efficacy against brain metastases [P = 0.046). At 3 years, 79 of 279 patients (28%) in the osimertinib group and 26 of 277 (9%) in the comparator group were continuing to receive a trial regimen [Regarding the 1L therapy option in EGFRm, osimertinib was considered the best front-line choice in patients with common activating mutations del19, L858R). The FLAURA trial results support this recommendation: osimertinib demonstrated improvement in median PFS  0.46; 95% CI, 0.37 to 0.57; 8R. The F regimen .Focusing on the second and subsequent treatment lines in EGFRm aNSCLC there was a consensus to maintain TKI plus local treatment in case of NSCLC oligoprogression during 1L therapy. This statement has been validated in small trials showing the benefit of this strategy specially with the use of sterotactic body radiation therapy (SBRT) , 22. In Although this consensus document aims to help therapeutic decision-making process, it has several limitations. First, this Delphi project has been developed under the premise of patients with EGFRm aNSCLC with a good general condition (ECOG PS 0 and 1) and without medical contraindications. This may limit its potential applicability to all patients with EGFRm aNSCLC. Also, as the treatment of aNSCLC further evolves, it is important to underline that the SDR took place before the communication of some relevant scientific information such as the results of osimertininb in adjuvant setting in stage IB-IIIA NSCLC .Despite these limitations, the study shows the consistency of the questionnaire created to meet the proposed objectives statements selected: Cronbach's alpha coefficient and intraclass correlation coefficients showed a high internal consistency for the complete questionnaire as well as for each domain with values. The correlation analysis showed strong quantitative correlation and a good qualitative concordance between two DR in total questionnaire as well as in each domain. The Kappa index values corroborate Spearmans’s coefficient estimators in all cases.This consensus document has been generated due to the need to answer key clinical questions such as the best sequencing approach for EGFRm aNSCLC. We would encourage to use this document to help stimulate discussion on future real-world studies that could be carried out to support or question the consensus statements.


Background and purpose — The COVID-19 pandemic has been recognized as an unprecedented global health crisis. This is the first observational study to evaluate its impact on the orthopedic workload in a London level 1 trauma center  before (2019) and during (2020) the “golden month” post-COVID-19 lockdown.Patients and methods — We performed a longitudinal observational prevalence study of both acute orthopedic trauma referrals, operative and anesthetic casemix for the first “golden” month from March 17, 2020. We compared the data with the same period in 2019. Statistical analyses included median (median absolute deviation), risk and odds ratios, as well as Fisher’s exact test to calculate the statistical significance, set at p ≤ 0.05.Results — Acute trauma referrals in the post-COVID period were almost halved compared with 2019, with similar distribution between pediatric and adult patients, requiring a significant 19% more admissions . Hip fractures and polytrauma cases accounted for an additional 11% of the modal number of injuries in 2020, but with 19% reduction in isolated limb injuries that were modal in 2019. Total operative cases fell by a third during the COVID-19 outbreak. There was a decrease of 14%  in aerosol-generating anesthetic techniques used.Interpretation — The impact of the COVID-19 pandemic has led to a decline in the number of acute trauma referrals, admissions (but increased risk and odds ratio), operations, and aerosolizing anesthetic procedures since implementing social distancing and lockdown measures during the “golden month.” The novel coronavirus SARS-COV-2 (COVID-19) was first reported in December 2019 with the first patient hospitalized in the city of Wuhan, China . This was followed a week later by more stringent measures, commonly referred to as a societal “lockdown” , evaluating the trends of acute orthopedic trauma referral caseload and operative casemix before (2019) and during (2020) the COVID-19 lockdown .  All acute referrals, operative notes, inpatient medical records, and discharge summaries were accessed using the electronic medical system.The study period was from the start of social distancing on the morning of March 17, 2020 to April 15, 2020, which also encompasses the morning following more firm “lockdown” measures on March 24, 2020. This was compared with the same 4-week interval in March–April 2019 prior to any COVID-19 related measures to compare its impact 1 year apart.The study criteria comprised all acute orthopedic trauma referrals presenting to the Emergency Department of a busy level 1 trauma center  during the stated intervals 1 year apart, and all orthopedic trauma cases that required an operation, including those from acute orthopedic trauma referrals, within the stated intervals 1 year apart. Those patients listed for an operation prior to the period of data collection were included in the final analysis. We adhered to the STROBE guidelines.Any cases referred internally from other specialties for trauma and orthopedic advice and input, as well as referrals from any external center asking for tertiary advice, were excluded from further analysis. Any patient with a postoperative complication arising in the period prior to the data collection were excluded. For operative trauma cases, those undergoing spinal procedures were excluded as the service is delivered jointly by the neurosurgery service. With respect to infections, all acute and chronic surgical site infections (SSI) and non-SSIs were excluded from the final analysis. All non-urgent semi-elective procedures were excluded from analysis as well, to avoid inaccurate assessment of the impact of any social distancing measures on trauma workloads. Routine elective orthopedic cases were excluded.Demographics including age, sex, and ASA grades were recorded for all patients. Injury characteristics were recorded, including the anatomical location and whether the injury was open or closed. The mechanism of injury was categorized and whether the patient presented as a trauma call. The nature of the operative procedures and the anesthetic techniques were recorded. Patients undergoing multiple procedures were recorded for every episode when they were taken to theatre.All the data were recorded, anonymized, and verified by 2 authors for their accuracy. The median (median absolute deviation) was calculated for both age and ASA grade. Both risk and odds ratios were calculated as well as a Fisher’s exact test for statistical significance, defined as p ≤ 0.05.No formal ethical approval was required as these were audit data. The study was registered and approved with the Trust’s audit department. No identifiable patient data have been kept or reported. This study required no internal or external funding. The authors have no conflict of interests to declare. There were no missing data, as all data points were extracted from electronic patient records .For the pre-COVID period in 2019 there were 193 new referrals. 106 (55%) were male. 31 patients were excluded, which left 162 patients in the pre-COVID period who were acutely referred. 90 (56%) were male. 135 (83%) patients were adults (> 18 years old) .For the post-COVID period in 2020 there were 94 referrals (53% of those in 2019). Sex was split equally.7 patients were excluded, which left 87 new acute trauma referrals in the post-COVID period . 44 (51%) patients were male. 75 (86%) patients were adults.Results have been tabulated as acute referrals, categorized as all referrals, adult referrals, and pediatric referrals between the 2 years . Table 2Although a greater number of the acute referrals required surgery, this was ultimately not statistically significant . Nevertheless, if a patient did require surgery during the COVID outbreak, there was a 131%  increased odds that the operation would be consultant-led  with a 19% increased prevalence of personal involvement compared with 2019. As expected, every attempt was made to minimize reliance on aerosolizing anesthetic procedures wherever possible in order to reduce viral transmission, load, and exposure to the healthcare staff. In 2020, COVID significantly decreased the prevalence of GA  by 15%  as well as decreasing the odds of receiving an aerosolized anesthetic procedure by 80% .There was a notable difference between the number of acute referrals and the operative casemix between the time intervals 1 year apart pre- and post-COVID in a London level 1 trauma center. There was a substantial decrease in the number of relevant acute trauma referrals , number of operations (without significant difference between mechanisms of injury and type of surgery or technique) with a lower number of aerosolizing anesthetic procedures (with significantly less risk and odds ratios). This reduction is likely to have been a direct consequence of the social distancing measures implemented on a national scale.Post-COVID there has also been a reduction in external referrals as compared with 2019, representing the effects of the major service reconfiguration with disbanding of the elective, private, and inpatient practice to cater for the increased space requirement to host acute COVID patients pre- and post-ITU treatment. To reduce the risk of virus transmission and reduce the demands on services, some injuries previously treated at the level 1 trauma center were being treated at level 2 trauma units, a change of practice supported by the British Orthopaedic Association . There aThe comparison of demographics of acute trauma referrals is mostly similar between the 2 periods, as seen in Pre-COVID in 2019, the most common injury pattern for acute referrals was lower limb injuries at 33%. This was followed by upper limb injuries (30%) and together they accounted for nearly two-thirds of all referrals. Post-COVID, both upper and lower limb injuries are still the most common injury but combined they accounted for just 50% of all referrals. Hip fractures and polytrauma (often from road traffic collisions and high-energy injuries) patients, however, accounted for an increased proportion of acute referrals, each accounting for 19% of cases.Usually the result of low-energy falls, HFs often occur indoors, in the garden, and within the property, and the incidence may not be directly impacted by the social distancing measures that were implemented. Therefore, it would be expected that the number of HF referrals would be consistent, as demonstrated by 17 HF referrals in 2020, an increase from the 14 in 2019. HF referrals reflected a higher percentage of all acute referrals during the pandemic study period when compared with 2019 . Yet there was no significant change in risk or odds ratios of HF between the 2 years.Conversely, polytrauma occurs as a consequence of high-energy injuries. This may often be as a result of a road traffic collision (RTC) or fall from a height greater than 1.5 m. RTC rates have not changed significantly following social isolation advice and may account for some of the greater proportion of polytrauma observed. There has been an increase in those falling from a height greater than 1.5 m in 2020 of 10% as compared with 5% in 2019. This could be due to the construction industry being exempt from the lockdown or people spending more time at home committing to home improvements or do-it-yourself tasks.The breakdown of the mechanism of injury has remained largely the same pre- and post-COVID as seen in There has been an 89% reduction in acute referrals due to sporting injuries and a 100% drop in the MOI in the operative casemix 1 year apart . This woDespite the significant reduction in personal vehicle use, there has been a consistent proportion of injuries seen following social isolation attributable to RTCs  at 15% . AlthougAccounting for all exclusions, the total number of operative cases has fallen by a third following COVID. A proportion of this is due to the ceasing of semi-elective operating. However, the reality of practice at our center is that the management of orthopedic injuries during the COVID era has not changed significantly. Non-urgent and elective procedures have been cancelled or postponed following national advice  as the anesthetic methods in 2020 (19%) compared with 2019 (6%) as seen in The limitations of this longitudinal observational study include analyzing 2x4-week snapshots 1 year apart, at a single-center London level 1 trauma center. This may not be representative of the national profile. There has been much literature on the benefits of large-volume centralization of orthopedic trauma to level 1 trauma centers, but this might need to be re-analyzed if smaller peripheral trauma units return to managing grade 1–2 Gustilo Anderson open fractures and simpler closed polytrauma. Further work is required to observe for trends in acute orthopedic referrals and orthopedic trauma surgical casemix as a result of the structural reconfiguration due to COVID. Bias was kept to a minimum and the date range between the two years was dictated by the evolution of the pandemic.The COVID-19 pandemic has had a unique impact on trauma and orthopedic care. Acute trauma referral rates have fallen, with fewer trauma procedures being performed since the implementation of the UK social distancing measures indicating a change in prevalence pre- and post-COVID. Every attempt has been made to substantially reduce the prevalence of aerosol-generating anesthetic procedures with general anesthesia and intubation. We recommend more work to investigate the phenomenon further and whether a similar pattern is seen across the UK.


Average viral load increased 0.559 log10 copies per milliliter. CD4+ T cell count decreased as soon as ART was withdrawn, an average loss of 99.0 cells/mL. Forty-three percent of the mutations associated with antiretroviral resistance in PBMCs disappeared and fifty-seven percent of the mutations in plasma reverted to wild type, which was less than the 100% reversion expected. In PBMC, the PI mutations reverted more slowly than reverse transcriptase mutations. The patients were projected to need an average of 33.7 weeks for PI to revert compared with 20.9 weeks for NRTI and 19.8 weeks for NNRTI. Mutations in the pol gene can cause virological failure and difficulty in re-establishing effective virological suppression.HIV cure studies require patients to enter an analytical treatment interruption (ATI). Here, we describe previously unanalyzed data that sheds light on ATI dynamics in PLHIV (People Living with HIV). We present drug resistance mutation dynamics on the pol gene among individuals with antiretroviral virological failure who underwent ATI. The study involved a 12-week interruption in antiretroviral therapy (ART), monitoring of viral load, CD4+/CD8+ T cell counts, and sequencing of the pol gene from 38 individuals experiencing virological failure and harboring 3-class resistant HIV strains: nucleoside reverse transcriptase inhibitors (NRTI) non-nucleoside inhibitors (NNRTI), and protease inhibitors (PI). Protease and reverse transcriptase regions of the  In the late 1990s and early years of this century, researchers studied the clinical benefit that analytical treatment interruption (ATI) might offer HIV-1 patients experiencing antiretroviral virological failure given the limited options of salvage therapy available at that time. However, these studies led to the conclusion that complete ATI cannot provide sufficient clinical benefit to justify its use as an adjunct to antiretroviral therapy (ART) programs ,3,4,5.At the time the data were gathered for this study, researchers believed that ATI would hasten the reappearance of wild type virus, which in turn would facilitate the subsequent reintroduction of ART ,7,8,9. AFrom June 1999 to June 2000, the authors collected samples from 38 HIV-1 treatment-experienced patients experiencing antiretroviral virological failure who agreed to participate in a study of ATI. This paper analyzes the original data in a more consistent way than conducted previously. Although we no longer consider ATI a viable treatment strategy, there is still much to be learned from these data in terms of the evolution of resistance patterns in the reverse transcriptase and protease portions of the HIV-1 genome. The difference between the virus genotype with and without the selective pressure of ART offers important information about effective treatments for the future. Once infected, the patient’s HIV-1 begins to evolve and continues to do so even as it faces long-term replication suppression ,13. GiveSome anatomical sites such as the central nervous system and genital tract may be inaccessible to some classes of antiretroviral drugs. For example, the brain absorbs protease inhibitor (PI) medications poorly . These iViral fitness, the ability to create more adapted and highly replicating infectious progeny, is inversely related to the number of incorporated antiretroviral resistance related mutations ,26,27. IThe current study describes viral load and CD4+/CD8+ T cell count dynamics. We also characterize the drug-resistant mutation dynamics in the plasma and PBMC. We focus on mutations associated with HIV-1 resistance at the reverse transcriptase and protease regions of the pol gene in DNA and plasma RNA. The expectation of the study team was that, since the use of ART repressed the wild-type virus, it would proliferate on withdrawal of ART and that the resistant strains would disappear due to the greater replicative fitness of the wild type. Finally, we report on the presence of cell activation markers HLA-DR and CD38 on CD8+ T cells. These proteins are associated with activation of latent infected CD4+ T cells, making them available for attack by the patient’s immune system. Prior research has indicated that expression of these activation markers is associated with an increase in viral load and a decrease in CD4+ T cell counts ,34,35,36Previous use of antiretrovirals before the experimental period revealed a variety of antiretrovirals available then. This study included 36 patients with HIV-1 3-class antiretroviral resistance mutations and 2 patients with chronic HIV-1 infection but no detectable HIV-1 resistance related mutations. These latter patients served as controls. There were one male and one female control subjects. The mean age of the controls was 37.0 years (standard deviation of 1.41 years). The patients harboring resistance included 28 males (77.8%) and 8 females (22.2%). The mean age of the resistant group was 38.75 years (standard deviation of 7.77 years). All patients had a virus of subtype B except for patients 3 and 53, who had combination subtype B/F.For the reverse transcriptase and protease regions of the pol gene, the patients were divided based on the degree to which they returned to the wild type. These categories are high reversion, low reversion, and non-reversion. We established the reversion group assignment on the return to wild-type pattern in the reverse transcriptase region of the plasma sequences. The decision rule for assignment to each group was based on a comparison of the number of mutations that the patient experienced between baseline (week 0) and week 12. The “None” group had an equal or greater number of mutations at the end than at the baseline. The “Low” group had fewer but not zero mutations. In other words, they had at least one mutation but less than they had at baseline. The “High” group experienced zero mutations at week 12 after showing one or more at baseline. In this scheme, the controls were those with zero mutations at baseline.The last line of 10 transformation of the raw data) over the twelve weeks of the study for all four groups (three reversion groups and the control group). The data underlying these results, the viral loads, CD4+ counts, and CD8+ counts for all patients at all sample collection times are included in the 10. However, the viral load of the no reversion group rose until week 8 and then declined. It only increased over the study period by 0.136 log10. Overall, the groups, including the controls, increased by an average of 0.568 log10. In addition, the high and low reversion groups’ viral load increase was statistically significant. A paired two-sample t-test of the difference between viral load in all resistant patients  at baseline and at week 12 had a value of t = 6.2919 with 35 degrees of freedom and p < 1.0 × 10−6. The linear increase in viral load in the current cohort contrasts with the rapid rise in viral load reported in a previous study [p-value < 1.0 × 10−6.Focusing on the three groups that showed initial resistance mutations, the decline in CD4 levels between baseline and week 12 was statically significant  did the number of mutations go to zero. In all other cases, there remained a small number of mutations per patient in week 12.That the resistant mutations would revert to the wild-type virus due to ATI among individuals with antiretroviral failure was only partially supported by the data from this study where the proviral DNA profile is concerned. We limited all of the analyses of reversion to wild type to the 36 patients who showed resistant mutations. However, we include in the The plasma sequences collected at baseline and study end (week 12) show an uneven reversion pattern despite the expectation that all mutated codons would revert to wild type after 12 weeks of ATI. Among the patients showing drug resistance, a mean of 14.95 codons evolved between the start and end of the treatment interruption . Of the 434 amino acids in the sequence studied, 111 different codons evolved during the study. Some of these 111 codons had variant amino acids at the end of the survey, so the number of different mutations during the treatment interruption totaled 164. For example, codon 20 showed an evolution of R10K in five patients and I10K in two other patients. We focused our analysis on those codons that evolved in more than one patient. Although our sample was drawn well before IAS began to report drug resistance mutations, we tested whether mutations in patients with drug resistance are consistent with the 2017 IAS list of drug-resistant mutations. In 35 of the 40 codons in the table (87.5%), the patients reverted to wild type as defined in the IAS 2017 interpretations.We conducted a parallel analysis of the PBMC samples from the 36 resistant patients for the evolution of both protease inhibitor and reverse transcriptase inhibitor resistance mutations. We hypothesized that the high reversion level that we would see at the plasma level would not be observed in this compartment. Of the PI codons on the IAS list, we initially had data on 22. These were codons 10, 20, 24, 30, 32, 36, 46, 47, 48, 50, 53, 54, 63, 71, 73, 77, 82, 84, 85, 88, 89, 90, and 93. We eliminated codons 32, 47, and 50 from our analysis as they either had 0 or 1 tests that showed that mutation and therefore did not allow comparison. For codon 89, only patient 3 exhibited a mutation. Codons 77 and 88 showed an increase in the number of patients exhibiting the mutation during the 12 weeks and thus had positive slopes in the regression. Accordingly, it was not possible to calculate a time to 0 patients using regression. However, the Kaplan–Meier survival analysis result showed a probability of the survival of the mutation at codon 77 of 0.318 (95% CI of 0.173–0.587) and at codon 88 of 0.176 (95% CI of 0.063–0.493). Given the overall sample size of 36 and the variable number of patients who showed the mutation , it is not possible to tell if this result is due to a sampling error or to some fundamental observation about these codons. p = 0.00013) using a Pearson’s Product Moment Correlation test. We included a figure with this result in the We evaluated the consistency of results between the time to 0 mutations for each codon and the survival analysis. We found a correlation between these values of 0.69. This correlation was significant  among the PI mutation codons. Graphs for the other codons are found in the data repository for the study .The mean number of weeks for the protease inhibitor mutations is 34.5 weeks with a standard deviation of 27.3 weeks. 2 indicates the degree to which the line explains the points.This figure shows the number of patients with a mutation at each test and a regression of these points. The regression equation shows the parameters determining the line, and the RWe had data on nine of the IAS’s list of 15 NRTI codons . We eliminated codon 65 from further analysis as it had only one patient with a mutation at this location at week 0. Codon 75 just had two data points: one in week 0 and one at week 12. 2 measure indicates the relative variability within the group.Codon 184 (M184V) comes first in the Stanford University database of NRTI mutations because of its 100-fold reduction in patient susceptibility to Lamivudine/Emtricitabine but its enhancement of the susceptibility to Zidovudine and Tenofovir Disoproxil Fumarate . The othp = 1.0). However, the TAMs considered together take a significantly longer time to revert to wild type than codon 184 according to a one-sided Wilcoxon Signed Rank test (p = 0.016). This started with 28 patients exhibiting the Valine mutation, the loss of one carbon atom that distinguishes it from the Isoleucine wild type. By the end of the 12 weeks, the number showing this mutation had dropped to 8, and our linear regression model for this codon projected it would be eliminated by week 16. The TAM groups showed fewer patients with the mutations at the start of the interruption (20.7 for TAM 1 and 17.7 for TAM 2), declining to 8.3 and 7.7, respectively, at the end of the study. The higher rate of reversion for I184V than the TAMS can be seen in greater detail in the panels of Both TAM groups have mean values very close to each other. According to a sample of 3 per group, there is no difference between the two TAM types with a Wilcoxon Rank Sum test (Our data included 7 of the 16 non-nucleoside reverse transcriptase inhibitors (NNRTI) on the IAS list . We eliminated codons 106 and 108 from the analysis as they had insufficient data to permit a regression to be performed. We also removed codons 179 and 188 from the analysis as the counts of patients with the mutation only varied between 1 and 3, which strongly suggested that our sample was not typical of the population. The last line of The average number of weeks for NNRTI mutations to revert to wild type was 19.8 weeks with a standard deviation of 2.2 weeks. Although the graphs and tables we display here show the range of rates of reversion to wild type among the codons studied, the average for each of the three classes of antiretroviral medications indicates that this is a prolonged process that may last as long as 8 months (in the case of protease inhibitor mutations). The study sample showed a greater presence of CD38+ and HLA-DR+ proteins on CD8+ T cells in patients who returned to wild type. The no reversion group, whose mutations remained constant despite the treatment interruption, expressed a significantly lower level of these cell activation biomarkers (measured by the percentage of CD8+ T-cells with both CD38 and HLA-DR present) than did the high reversion group. This result parallels the observation that cell activation markers would be expressed in greater quantity in untreated HIV-1 patients than in patients receiving treatment . 10 viral load by dividing the cell activation markers’ expression by the log10 viral load. As To create an index of cell activity absent viral load, we corrected the cell activation marker levels for the logp-value < 1.0 × 10−6). However, the correlation between cell activation and CD4+ T-cell levels is stronger and statistically significant  showing that the higher the T cell activation, the lower the CD4+ T-cell counts. We compared the T cell activation level with the viral load and CD4+ T-cell levels for all the patients. Overall, the correlation with viral load was low but statistically significant  without changing their HIV genetic profile, which argues in favor of the activity of antiretrovirals in use, although probably without a good adherence to current treatment. As was expected, the viral load increased for patients subject to ATI, and CD4+ T-cell counts decreased. The viral load increase results from an increase in active viral replication after ATI. This is an important factor in replenishing the proviral reservoir even for this short period. This has been shown previously in this same patient population . One facThe picture for reversion to wild type is not clear. This study shows that reversion is not as direct a process as previously thought for resistance-related mutations selected by all three classes of antiretrovirals studied ,30. WhilWhat enables such a result? First, the period chosen for the treatment interruption—twelve weeks—may not be sufficiently long for complete reversion to the wild-type profile at the DNA level, although it was the standard for ATI studies at the time. A 1992 study from Sweden indicates that patients taking zidovudine (AZT) who developed resistance to the medication and subsequently abandoned treatment still carried some of the mutations as long as nine months after stopping treatment . As noteWe also need to consider why each mutation disappears with its dynamics. There appears to be little or no uniformity in the rate of reversion. Theoretically, once ART’s selective pressure that gives an advantage to the resistant mutations is withdrawn, the wild-type strain should reemerge rapidly. Interestingly, once ART is stopped and its selective pressure withdrawn, viruses with distinct resistant-related mutation profiles may reemerge as latent cells resume replication and/or based on the differential replicative capacity of distinct strains in each HIV quasispecies. When single genome amplification of antiretroviral resistant strains is performed, mutations that are detected in the population sequence analyses may not be present in all HIV strains but dispersed among distinct viruses of the HIV quasispecies at a given moment . We mustAfter antiretroviral treatment interruption, HIV recombination at the pol gene may dynamically occur in distinct HIV strains with resistant and wild-type profiles, which may in turn seed new proviral populations. As mentioned before, studies exploring partial treatment interruption show that, once NRTIs are withdrawn and PIs are maintained, wild-type HIV emerges at the reverse transcriptase region of the pol gene while the protease region remains resistant . In contNonetheless, we have previously demonstrated for this same patient population during the ATI period that the disturbance caused by interrupting the selective pressure imposed by antiretrovirals at the pol gene resulted in ancestral viral progeny activation in a very distinct (and distant) HIV genomic region such as the gp120V3 HIV-1 env region, which repopulated the cell reservoir . In contrast with what has been previously demonstrated in vitro, PI-related mutations’ persistence was longer than the persistence of NNRTI-related mutations. As NNRTI mutations do not impair the HIV-1 fitness as with NRTIs and PIs mutations ,45, it wAnother feature that deserves further attention is the fact that M184V waned more rapidly than other studied mutations. Although transmitted drug resistance mutations tend to persist in general, M184V tends to revert to wild type in this specific population . It is aWe recognize that a more detailed analysis of HIV quasispecies in this study would greatly benefit from single genome amplification or next-generation sequencing (NGS) tools. It has been recently demonstrated using NGS that transmitted drug resistance mutations representing small percentages of the viral population do not persist during infection because they are negatively selected in the first year after HIV-1 seroconversion . In thisWe previously described that there are two pathways for NNRTI resistance depending upon the drug used. The use of efavirenz primarily selects for the 103 mutation, which is accompanied by the mutations at positions 100 and 225, whereas nevirapine predominantly selects for the 181 mutation, which is generally accompanied by the 101 and 190 mutations  and 25–3We also recognize that more intensive cell population evaluations, additional immunologic markers, or direct HIV fitness evaluation could provide additional insights into the mechanisms of viral dynamics after ATI among patients experiencing virological antiretroviral failure. However, the number of patients evaluated here is larger than what has been described in other similar studies, and this is the first study analyzing the impact of such a strategy at a DNA level at multiple time points compared with the plasma compartment, where we sequenced only the baseline and study completion (12-week) time points. We, therefore, have been able to show how the reversion of the virus to a wild type after ATI among individuals failing antiretroviral therapy is not a clear and consistent process across the full range of pol codons studied. We have also provided insight into the persistence of distinct resistant-related mutations selected by different antiretroviral classes. We studied specific areas of the pol gene related to their known patterns of resistance to the three classes of ART medications in common use at the experiment time. However, our results suggest that ART and subsequent treatment interruption can induce other viral genome changes in which the impact on resistance profiles is not yet well understood. The region between codons 293 and 560 of the reverse transcriptase region is one area of interest. For example, a small number of studies have indicated that Tyr-318 is subject to a range of mutations that show some history of resistance to NNRTIs . AnotherThirty-six HIV-1 treatment experienced patients who were treated at the Federal University of Sao Paulo, Brazil in 1999, agreed to participate in the study on ATI. Two other patients under the same conditions, who suffered from toxic side effects but had no detected HIV-1 resistance mutations, submitted to ATI during the same period and were included in the evaluation. The samples were collected from June 1999 to June 2000. All patients were followed for 12 weeks. The total sample size of 38 was large for studies of this type at the time it was carried out. A non-systematic review of 13 studies on treatment interruption that parallel this study in intent and method published between 1999 and 2004 had a median number of participants of 24 and an interquartile range of 28 (10–38). Of the studies reviewed, only four had more or the same number of participants as the present study. See Eligible male and female patients met the following criteria: 18 years or older; experienced multiple ART failures while using nucleoside analog reverse transcriptase inhibitors (NRTIs), non-nucleoside analog reverse transcriptase inhibitors (NNRTIs), and protease inhibitors (PIs); had detectable viral loads above 5000 copies/mL; and showed no signs of AIDS. At the time the experiment took place, the design was an interventional study based on the hypothesis that, if the ART drugs were withdrawn, the wild-type virus would replicate and eliminate the presence of resistant strains as the wild type had greater replicative fitness. All patients enrolled on ATI at day zero started a prophylactic regimen against opportunistic agents (data on file). CD4+ T cell counts were taken at 15-day intervals; viral loads were performed monthly; the protease and reverse transcriptase regions of the pol gene were sequenced every four weeks from PBMC; and two plasma genotyping tests were conducted at baseline and week 12 from protease: reverse transcriptase and the p17 region of gag gene. No patient presented AIDS-related symptoms during the study period.HIV-1 RNA in plasma was measured using the NASBA methodology , according to the manufacturer’s instructions. This assay has a limit of detection of 80 copies/mL.At each lymphocyte count, we determined sub-populations of CD4+ and CD8+ T cells by three-color flow cytometry. We used fresh samples for direct staining with appropriate monoclonal antibodies (mAb): CD4-PerCP, CD8-PerCP. Mouse immunoglobulin isotypes conjugated with phycoerythrin (PE), or fluorescein isothiocyanate (FITC) were used as negative controls for non-specific binding. The stained cells were analyzed on a FacScan flow cytometer. For the analysis of genotypic resistance of PBMC, proviral DNA was extracted from cells using QIAamp DNA Blood Mini kit  and plasma RNA was purified using QIAamp RNA kit  and reverse-transcribed using as previously described . The proThe nested-PCR fragments were purified by PCR purification kit  and sequenced using Thermo Sequenase fluorescent-labeled primer cycle sequencing kit—RPN 2436 . The primers were labeled by CY5.5. The products of the sequence reaction were analyzed using a DNA—Long Reader Tower (Visible Genetics Inc.). Base-calling was performed by GeneObjects software , and the bases were aligned and assembled using GeneLibrarian software . The resulting sequences for each sample were interpreted using the 2017 edition of the IAS–USA drug resistance mutations list . TM HIV-1 Genotyping System v2.0  per the manufacturer’s instructions. All codons of the HIV-1 protease region were sequenced. The reverse transcriptase region was sequenced up to codon 320. The HIV-1 gag gene amplification products were sequenced in an ABI 3100 Genetic Analyzer .Nucleic acid isolation, reverse transcription-PCR amplification, and sequencing of the HIV-1 protease and reverse transcriptase regions of the pol gene were performed using ViroSeqThe analysis compared the viral load and CD4+ T cell count dynamics considering patients’ reversion categories. The dynamics of the protease and reverse transcriptase mutations were analyzed by linear regression and Kaplan–Meier survival analysis ,59,60,61The data analysis was conducted using the R Statistical Computing System on a Macintosh (version 3.5.0) . AlgoritWe calculated the number of patients who reverted to wild type for each codon in the protease and reverse transcriptase regions on the IAS resistance mutation list for which we had PBMC data. We used the PBMC data for this analysis rather than the plasma data as the former permitted us to examine the evolution over four tests rather than just looking at the beginning and endpoints of the study. We had data on 9 of the IAS list of 15 nucleoside reverse transcriptase codons (NRTI) and on 7 of the 16 NNRTI codons on the IAS list. Twenty-two of the thirty-five IAS mutations affecting PI appeared in our sample. We aligned the data that had been gathered in 2000 with the codons on the IAS list. We removed those codons with no patients showing the mutation or had missing values for any of the four visits. We then performed a simple linear regression of the number of patients showing the mutation during the study period against the time since ATI initiation and projected from the parameters of the regression line how many weeks would be needed to reach 0 patients, i.e., how many weeks would be required for the mutation to be eliminated from the cohort. In five cases , the regression slope was positive rather than negative, indicating that patients were exhibiting new mutations after the ATI. Additionally, we conducted a Kaplan–Meier survival analysis of how long the mutations persisted in the sample and what was the probability that they would persist at the end of the study. These data indicate many interesting features of the mutational patterns of HIV-1 in response to ART selective pressure and its subsequent withdrawal that can be applied to research on understanding fitness features of the HIV vis-a-vis genetic composition of the virus.