Background: The German S3- guideline on local therapy of leg ulcers and diabetic foot ulcers is in the process of being updated. Major goals are to improve the guidelines’ applicability and to take steps towards a living guideline according to current methodological standards. The aim of this article is to describe the main measures to achieve these goals. Methods: The context of the guideline in the field of local wound care and the stakeholder requirements are briefly described. Based on a derived framework, the project team adjusted the methods for the guideline. Results: Main adjustments are more specific inclusion criteria, online consensus meetings and the use of an authoring and publication platform to provide information in a multi-layered format. A new set of practice-oriented key questions were defined by the guideline panel to foster the formulation of action-oriented recommendations. Conclusions: The set of new key questions addressing practical problems and patients’ preferences as well as the adjustments made to improve not only the guidelines’ applicability, but also the feasibility of the further dynamic updating processes in the sense of a living guideline, should be steps in the right direction. The German guideline “Local Treatment of Chronic Wounds in Patients with Peripheral Vascular Disease, Chronic Venous Insufficiency, and Diabetes” was first published in 2012. The guideline is currently undergoing a fundamental updating process to improve its applicability and user-friendliness while initiating necessary steps towards a living guideline.www.awmf.org, accessed on 2 August 2021) along with complementary guidelines which focus on the prevention and treatment of the underlying disease as well as concomitant diseases and syndromes whose treatments are a prerequisite for local wound therapy.The guideline concentrates on local therapy of chronic leg ulcers of either vascular or diabetic origin and complements further national and international guidelines aiming at prevention and causal therapy ,2. It aiIn Germany, most guidelines are financed by scientific, medical organizations, rather than national government funds. This means that guideline teams usually have limited resources and most of the work is done by volunteer experts.Due to the complexity of the topic on the one hand, and differing opinions about the guidelines’ scope on the other, it took years for the first version of this guideline to go from initial ideas to final publishing. In addition, the implementation of high methodological standards for guideline development  costs tiIn the first years after publishing the guideline, its applicability had not been systematically evaluated. The reasons for this were limited resources and a simple lack of “voluntary energy”. However, feedback from health professionals involved in patient care indicated that the guideline was difficult to implement. This was mainly attributed to the complexity of the written text. Moreover, the concerted recommendations and statements themselves seemed to be difficult to transfer into practice. A guideline group that was involved in implementation processes in hospitals became particularly aware of this. In the absence of good evidence for the use of most cleansing solutions and dressings, the guideline group formulated a lot of conservative statements indicating that problem. Many of the recommendations on the other hand were formulated vaguely with few concrete recommendations for action. More than half of all randomized controlled trials (RCTs) investigating wound dressings, topical agents, and accompanying physical measures, which have been identified through our systematic searches covering the last 9 years (2011–2020), included less than 100 participants. Overviews of systematic reviews show no improvement in the quality of evidence either ,7. The uThe project team therefore had two major goals: improving the guideline applicability for its users, and establishing measures to improve the feasibility of the development process to initiate the steps towards a living guideline with high validity.The aim of this article is to describe the main measures to achieve these goals. These methods are presented in short, and for the purpose of a better understanding, argumentatively reasoned beforehand. The context of the guidelines in the field of local wound therapy will be briefly explained regarding the reasons of low evidence and its consequences for guideline development. Then, the preparatory work to identify potential areas for improvement is described briefly. The problem areas identified here were the basis of major methodological amendments, in particular a newly defined set of key questions. The guideline development process is reported in the guideline report . Any detThe first version of the guideline was developed in accordance with generally recognized quality criteria as defined in the AGREE instrument .However, implementing high quality standards in a field of low-quality evidence proved to be a challenge. This was mostly due to the fact that it is not easy to formulate trustworthy recommendations in the absence of trustworthy evidence.Noninvasive medical devices, to which most wound treatment products belong, only have to prove their safety and intended purpose before they can be launched on the market. This means that the approval of a wound dressing for example, requires evidence that it absorbs exudate or helps to maintain a moist wound environment. It does not require proof of efficacy on the basis of RCTs in outcomes like “wound healing” or “pain”, which are relevant for patients.By comparison, in the pharmaceutical sector, this proof of efficacy is essential for more than fifty years ,9. If suSince RCTs are not required for the market approval of wound treatment products, these expensive and time-consuming studies are often not carried out at all or are only presented at conferences rather than being published in scientific journals . The majAlthough well-conducted RCTs are possible within the field of wound therapy , they aIn preparation for the update process, we assessed the need for improvement with a critical quality analysis of the guideline documents ,20,21. WTo identify potential for improvement of our methods, the original guideline was critically appraised by two independent experts, according to the AGREE-II-Instrument. The AGREE-II instrument  not onlyExternal assessments of the methodological quality have also been taken into account, like a structured assessment of conflicts of interests from the group “guideline watch”  and peerFor the stakeholder survey , health It was obvious that there was an imbalance between the highly assessed methodological rigor and transparent reporting on the one hand, and the readability for health professionals on the other hand.The quality of a guideline as described in the AGREE II-Instrument and its implementability are strongly correlated. Therefore, it can not only be used to assess a guidelines’ validity and reporting, but with its six domains, it also provides a strategy to develop guidelines . The staConsidering the situation described above, the available resources for reviewing the evidence, and that the guidelines should be constantly updated (in the sense of a “living guideline”) the guideline team decided on the following methodological adjustments for the update:The first version of these guidelines was more than 250 pages long and included detailed summaries, written by more than thirty authors in varying degrees of detail. For the updated version, all chapters will be abridged and prepared by an experienced guideline author in a structured format. An electronic version will be uploaded on an authoring and publication platform called MAGIC (MAGICapp)  to proviThe original guideline version included goal-oriented algorithms. They clearly describe the process from first diagnostic assessment to treatment, while also referring to other guidelines related to prevention and causal therapy. English versions of these are presented in . These aA core component of the guideline update is a new set of key questions, which were agreed on by the panel in a formal moderated session. These questions address practical questions to foster the formulation of action-oriented recommendations. The key questions comprise a range of topics including initial assessment, treatments for different wound symptoms, and organizational issues . The panThe guideline panel came from multiple health disciplines involved in wound therapy. Scientific medical societies within the AWMF appointed representatives with voting rights for final recommendations. Conflicts of interests are handled according to current AWMF standards .Optimal cooperation with the therapeutic team;Good organization of care;High quality of medical-technical care administered by all participating health professions .Patient preferences in wound care were considered in several steps. In preparation of the first version of the guideline in 2012, preferences were identified using a systematic approach. Preferences were synthesized from qualitative studies, surveys, a patient charter and verified with directly affected patients and an official representative appointed from a self-help organization in Germany. The main preferences of patients suffering from wounds within the scope of the guideline referred to:Keeping these requirements in mind, the key questions address several topics related to documentation, organization of care, education of personnel, and interaction with patients .Cochrane Systematic Reviews or other systematic reviews with at least moderate quality (assessed with AMSTAR );Additional RCTs within the scope of the guideline are only considered if they have a sufficient sample size .Whereas the first version even included pilot studies, future releases will use more specific and quality guided criteria to accelerate the synthesis process:In view of the low quality of evidence for interventions in question (as indicated in most Cochrane reviews published in the last years), it is not expected that single studies with small sample sizes would change the confidence enough to have a significant impact on the grading of recommendations. The search was limited to published English and German articles considering patients, interventions, and outcomes within the scope of the guideline. The full search strategy is published with the former guideline report .The validity of systematic reviews was assessed with AMSTAR . CochranFirst, all recommendations from the former guideline version were reviewed by the guideline group . They considered the need for revision in view of new evidence and practice problems, as well as the new set of key questions. All suggestions for newly formulated or adapted recommendations were justified with a short rationale within the team. These informal meetings were initially carried out virtually in winter and spring 2020/21. To respect the limited time resources of the experts and to avoid tedious discussions with the danger of unwanted majority influences, all sessions were limited to one hour. When few experts attended the sessions, or if opinions were quite different, we presented suggestions for revisions of recommendations again in other meetings.In a second step, which should be completed by June 2021, the guideline coordination team will check all recommendations for consistency in the wording, particularly in view of the existing evidence. All recommendations will be summarized with a rationale following the GRADE evidence to decision framework  by consiFormal  consensus meetings are set in summer 2021. They will be moderated by an AMFW-guideline commissioner, with formal consensus methods controlling for conflicts of interests of representatives entitled to vote. This process will follow the standards of the AWMF . Based oIn the last step, the edited guideline and related documents will be sent to all collaborating organizations for final official agreement. All methods will be published in an amendment to the guideline.Local wound therapy is characterized by a high variety of cleansing solutions, dressings, and adjuvant interventions but a low quality of evidence. It is a challenge for a guideline panel in this field to formulate trustworthy recommendations. A stakeholder analysis and critical appraisal of the former version of the guideline indicated that there is a need for action-oriented recommendations addressing questions of the health professionals in practice. Therefore, the guideline underwent a fundamental revision during the update process. Major goals were guideline applicability and user-friendliness. Furthermore, a new set of key questions have been formulated in order to foster action-oriented recommendations to the guideline panel. However, it is a prerequisite that a consensus of the representatives on these recommendations can be achieved, in view of the low quality of evidence.Ensuring a high methodological quality, in view of numerous studies with insufficient sample sizes and high risk of bias on the one hand and limited time resources on the other hand, leads to further adjustments. There are still some limitations. It is possible that even small RCTs provide a high certainty of evidence for an intervention, but it can be assumed that these would be integrated quickly in systematic reviews. Providing high-quality instructional materials will be another challenge. Guidelines in Germany are not supported by national funds and guideline developers are forced to find the best balance between high methodological quality, applicability, and feasibility. National funds to support the evidence synthesis, dissemination into practice, and evaluation of the recommendations’ implementation are rare in Germany  and shouNevertheless, many organizations like the German AWMF with its member societies, AGRRE Enterprise, the GRADE working group, and the MAGIC-team provide support, create quality standards or method handbooks, and develop digital solutions and decision frameworks. The development of guidelines would also be simply impossible without the work of numerous experts and researchers volunteering their support through discussing content, reviewing the extensive evidence, formulating recommendations, or providing high quality evidence syntheses like Cochrane reviews provided by the Cochrane wounds group.This comprehensive preliminary work and ongoing support promotes the development of a trustworthy guideline, which is necessary in a field of high practice variability and great uncertainty in terms of efficacy. However, even the best guideline will not have a significant impact on patients’ care if it is not implemented in a comprehensive way. The set of new key questions addressing practical problems and patients’ preferences as well as the adjustments made to improve not only the guidelines’ applicability, but also the feasibility of further updating processes in the sense of a living guideline should be steps in the right direction.


Clinical Infectious Diseases . https://shionogi-eu-content.com/gb/fetcroja/pi.The prescribing information for cefiderocol is available at: This video is a presentation from Professor Marco Falcone from the University Hospital Pisa, Italy, originally presented at the BSAC conference ‘Into clinical practice: Meeting the challenges of Gram-negative infection management’ on 14 October 2020. Professor Falcone highlights the cefiderocol compassionate use case series that has been published in  This article is part of a promotional Supplement developed and sponsored by Shionogi B.V. The accompanying video formed part of an educational webinar for which the author received a speaking honorarium. The material underwent peer review by the Supplement Editors. Editorial assistance to Shionogi Europe was provided by Page Medical.JAC-AMR Online.The video transcript is available as dlab056_Supplementary_DataClick here for additional data file.


Bile acids play key roles in gut metabolism, cell signaling, and microbiome composition. While the liver is responsible for the production of primary bile acids, microbes in the gut modify these compounds into myriad forms that greatly increase their diversity and biological function. Since the early 1960s, microbes have been known to transform human bile acids in four distinct ways: deconjugation of the amino acids glycine or taurine, and dehydroxylation, dehydrogenation, and epimerization of the cholesterol core. Alterations in the chemistry of these secondary bile acids have been linked to several diseases, such as cirrhosis, inflammatory bowel disease, and cancer. In addition to the previously known transformations, a recent study has shown that members of our gut microbiota are also able to conjugate amino acids to bile acids, representing a new set of “microbially conjugated bile acids.” This new finding greatly influences the diversity of bile acids in the mammalian gut, but the effects on host physiology and microbial dynamics are mostly unknown. This review focuses on recent discoveries investigating microbial mechanisms of human bile acids and explores the chemical diversity that may exist in bile acid structures in light of the new discovery of microbial conjugations.Video AbstractThe online version contains supplementary material available at 10.1186/s40168-021-01101-1. Bile has been implicated in human health for millennia. Hippocrates developed the idea of humourism in the third century BC, which describes the body as being composed of four “humors,” two of which involve bile. When these humors are balanced the body is healthy, but illness occurs when any become unbalanced . Even toa and complete ionization resulting in these compounds being present as anions in vivo [Primary BAs are those synthesized in the liver from cholesterol . The pri in vivo –10. For  in vivo . Relevan in vivo , 13. Due in vivo .Fig. 1DBAs have traditionally been thought to undergo amino acid conjugation solely in the liver. There is a single human enzyme, bile acid-CoA:amino acid N-acyltransferase (hBAAT), that is responsible for acyl-conjugation. These conjugated primary BAs are secreted via the bile canaliculi into the gallbladder where they are stored until consumption of a meal. They are then secreted into the duodenum and travel through the small intestine, only to be subsequently reabsorbed in the terminal ileum and transported to the liver for re-conjugation if necessary, followed by secretion into the gallbladder and recirculation . This enDefa gene family [BAs play an important role in regulating various physiological systems, such as fat digestion, cholesterol metabolism, vitamin absorption, liver function, and enterohepatic circulation through their combined signaling, detergent, and antimicrobial mechanisms . BAs aree family , 24. Taue family . Additioe family . Expresse family . These cStaphylococcus, Balantidium, Pneumococcus, and Enterococcus in addition to members of the phylum Spirochaetes [Staphylococcus aureus, several Lactobacillus species, and several Bifidobacterium species [Bile acids are potent antimicrobials. As such, they play an important role in the innate immune defense within the intestine. Consequently, modifications of BAs are an essential microbial defense mechanism . BAs havochaetes . BAs act species , 29. As  species . ConjugaTraditionally, there have been four distinct pathways related to microbial transformations of BAs: deconjugation, dehydroxylation, oxidation, and epimerization. The latter two methods of BA transformations work hand in hand, as formation of oxo-BAs is a key step prior to epimerization. Research into microbial bile salt hydrolases (BSHs) has been the latest boom in health-related BA research since their discovery in the 1970s with over 260 publications listed on PubMed from within the last 10 years . Additionally, several reviews have been written specifically about the biochemistry, diversity, and implications of microbially transformed BAs on host health , 31, 32.Deconjugation of BAs is considered the “gateway reaction” to further modification . There aBacteroides spp. are among one of the phyla suggested to play a major role in deconjugating primary BAs [Bifidobacterium [Lactobacillus [Clostridium [Enterococcus [Listeria [Stenotrophomonas [Bacteroides [Brucella [Brucella abortus and Listeria monocytogenes, BSH genes are important for virulence and establishing infection within mouse models. A metagenomic study by Jones et al. found BSH-encoding genes are conserved among all major bacterial and archaeal species within the gut [Enzymes capable of catalyzing the deconjugation reaction are found across all major bacterial phyla and within major archaeal species, suggesting that the genes encoding them are horizontally transferable , 35. Bacmary BAs . The divacterium , Lactobabacillus , 39, Clostridium , Enterocrococcus , and LisListeria . Howeverphomonas , Bacteroteroides , and BruBrucella  also con the gut . Bacteri the gut . The merBifidobacterium spp. arose three separate classes of BSH [Bifidobacterium spp., two classes had high activity and differed in substrate specificity. Both classes exhibited a preference for glycine-conjugated BAs but varied in activity for taurine-conjugated BAs. Although BSHs may utilize both taurine and glycine conjugates, encoding many BSHs may allow for slight changes in substrate specificity and more specific manipulation of the bile acid pool. BSH enzymes from Ligilactobacillus salivarius (PDB ID: 5HKE) [Bifidobacterium longum (PDB ID: 2HF0) [Bacteroides thetaiotaomicron (PDB ID: 6UFY) [Clostridium perfringens (PDB ID: 2BJF) [Enterococcus faecalis (PDB ID: 4WL3) [E. faecalis, L. salivarius, B. longum, and B. thetaiotaomicron each maintained the αββα motif indicating that it is essential for activity [B. thetaiotaomicron  , 51, BifD: 2HF0) , 53, BacD: 6UFY) , 55, CloD: 2BJF) , 57, andD: 4WL3)  have beeactivity . The BSHch genus .Fig. 2DClostridium scindens, the bai operon encodes several proteins needed for the sequential oxidation of CA [baiG gene encodes for a bile acid transporter, allowing for CA uptake. BaiG is also capable of transporting CDCA and DCA [4-cholyl-CoA. BaiF is then hypothesized to transfer CoA from 3-oxo-Δ4-cholyl-CoA to CA, yielding 3-oxo-Δ4-CA and cholyl-CoA [cat and lower KM [C. scindens (PDB ID: 4LEH) [Clostridium hylemonae (PDB ID: 4L8O) [, and Peptacetobacter hiranonis  [H) greater than 85% as calculated in Visual Molecular Dynamics (VMD) [C. scindens are encoded by baiN, which is responsible for the sequential reduction of C6-C7 and C4-C5 after dehydroxylation, and by baiA2, which catalyzes the NADH-dependent 3-oxoreduction of both 3-oxodeoxycholic acid and 3-oxolithocholic acid [One of the key transformations by gut microbes is BA dehydroxylation at C7. Within on of CA . The bai and DCA . This isolyl-CoA . BaiF Coolyl-CoA . The ratlower KM . CrystalD: 4LEH) , ClostriD: 4L8O) , and PepD: 4L8P)  Fig. 3)Clostridics (VMD) . The enzlic acid , 66. Bailic acid , 68. 7β-Epimerization of BAs is carried out by gut microbes and further diversifies the chemistry of secondary BAs. This occurs in two distinct steps: oxidation of the hydroxyl group by a position-specific hydroxysteroid dehydrogenase, such as a 7α-HSDH, followed by the reduction of another position-specific hydroxysteroid dehydrogenase, 7β-HSDH. Both reactions do not need to be carried out by the same organism and co-cultures of microbes are known to possess epimerization capabilities . CA can C. scindens, C. hylemonae, C. perfringens, and P. hiranonis have all been observed to produce enzymes capable of hydroxysteroid 3α-dehydrogenation, an important step in the pathway toward 7α-dehydroxylation [C. scindens, C. hylemonae, and P. hiranonis, C. perfringens has not been reported to produce LCA or DCA and its growth is inhibited by both secondary BAs [Clostridia and includes other intestinal organisms such as Blautia producta and Eggerthella lenta (formerly Eubacterium lentum) in addition to environmental species such as Acinetobacter lwoffii [E. lenta 3α-HSDH is capable of utilizing both TaurBAs and GlyBAs as substrates and in the case of CDCA oxidation, 3α-HSDH activity increased when conjugated forms of CDCA were used as substrates [Clostridium baratii among other isolates not yet identified [C. baratii has been shown to epimerize CDCA to UDCA but was not capable of epimerizing glyco- and tauro-BAs and instead deconjugated TaurCDCA prior to epimerization [Ruminococcus gnavus, Clostridium absonum, Stenotrophomonas maltophilia, and Collinsella aerofaciens all contribute to the UDCA pool via conversion of 7-oxo-LCA in an NADH or NADPH-dependent fashion [C. absonum 7β-HSDH functioned optimally at pH 8.5 while R. gnavus and C. aerofaciens functioned optimally at pH 6. 12β-HSDH activity can occur in both acidic and alkaline conditions. R. gnavus, in contrast to C. absonum and C. aerofaciens, displayed a clear preference in catalyzing the conversion of 7-oxo-LCA to UDCA with a specificity constant 55-fold higher than that of the conversion of UDCA to 7-oxo-LCA [R. gnavus as a potential probiotic, and this role should be further investigated.Recently, xylation . Howeverdary BAs . 3α-dehy lwoffii , 72, 73.bstrates . This goentified , 76. C. rization . Epimeri fashion , 77–79. E. lenta demonstrates 12α-HSDH capabilities in addition to 3α-HSDH. E. lenta 12α-HSDH has an estimated molecular weight of 125 kDa and has a broad pH optimum, between pH 8 and 10.5 [+ or NADP+ as a cofactor, though there is a preference for NAD+ [E. lenta 12α-HSDH reaction velocity increased when tested with methylated BAs, suggesting a preference for hydrophobic BAs [E. lenta 12α-HSDH is capable of utilizing both glycine- and taurine-bound BAs [Enterorhabdus mucosicola is also capable of both 3α and 12α oxidation, although 12α-HSDH activity is limited to when C7 position has already been oxidized [C. scindens, P. hiranonis, and C. hylemonae have since been reported to produce 12α-HSDHs and it is hypothesized that Bacteroides species also encode 12α-HSDHs [Clostridium paraputrificum, Clostridium tertium, and Clostridioides difficile [C. paraputrificum 12β-HSDH behaves in a similar manner to established 12α-HSDHs, as shown by its pH optimum and molecular weight. The gene encoding the 12β-HSDH in C. paraputrificum was recently identified, allowing for investigation into the diversity of potential 12β-HSDH producers [C. paraputrificum 12β-HSDH in C. difficile and C. tertium even though both species are capable of 12β-HSDH activity.Several gut bacteria have recently been identified to produce 12α-hydroxysteroid dehydrogenases (12α-HSDH). and 10.5 , 81. Catfor NAD+ , 81. E. obic BAs . Similaround BAs . Enteroroxidized , 83. C. 2α-HSDHs , 65. Acrifficile . These 1ifficile . Based oroducers . PutativCoriobacteriaceae, such as C. aerofaciens, E. lenta, and Lancefieldella parvula (formerly Atopobium parvulum) [C. scindens and P. hiranonis were among the only bacteria capable of completely hydrogenating 3,7,12-trioxolithocholic acid, a fully oxidized derivative of CA, to CA [Members of the gut microbiota are not only capable of reducing BAs with a single position oxidized, but some also reduce BAs oxidized at two or three positions. Similar trends regarding non-target hydroxyl oxidation have been observed by other arvulum) . Not allarvulum) . C. scinA, to CA . OxidatiEnterocloster bolteae, formerly Clostridium bolteae, as a species responsible for their production. In light of their microbial origin and the mechanism mirroring that of host-conjugation, we hereby refer to these compounds as “microbially conjugated bile acids” (MCBAs).A novel set of recently discovered BAs were conjugated at the C24 acyl site similarly to the host conjugation mechanism . InsteadThe exact mechanism of this microbially mediated conjugation has yet to be elucidated, although it may rely on a similar mechanism to hBAAT within the liver involving a Cys-Asp-His triad, with cysteine functioning as the catalytic residue for nucleophilic attack . RegardlThe functions of phenylalanine, leucine, and tyrosine CA conjugates remain mostly unknown, though gavage of mice with these compounds has been shown to result in agonism of FXR. Further investigation into the roles of known and unknown BA conjugates may yield novel drug targets or therapeutic agents for the treatment of numerous enteric diseases. Evidence already points toward BA hydrophilicity playing a major role in activity of several BA modifying enzymes; the three novel conjugates currently reported represent three of the four most lipophilic amino acids based on partition coefficient . Thus, i6, observed in MCA), which can be ɑ- or β-hydroxylated, oxidized to form a ketone, or absent. This backbone can also be present as one of two stereoisomers: 5ɑ-sterol or 5β-sterol, significantly broadening potential BA diversity. Limiting the bile acid backbone to only those known to be conjugated by the host (CA and CDCA) in addition to limiting the amino acid conjugated to those naturally occurring in humans, the potential diversity of the human conjugated BA pool increases over 5-fold from what is currently known , may be involved in severe IBD and Crohn’s disease, as it was identified as one of the most transcriptionally active microbes in the dysbiotic and diseased gut and MCBAs were elevated in these same samples [It is possible that MCBAs may also play a role in disease mechanisms, as PheCA, TyrCA, and LeuCA were more prevalent in patients with inflammatory bowel disease and cystic fibrosis  . However samples , 101. Th samples , 98 and Although BAs have been studied for centuries, recent discoveries show that we still have much to learn. The host BA pool controls microbial diversity, but so too does microbial metabolism of these BAs drive host physiology. In this sense, BAs act as the language of an intricate molecular cross-talk between humans and their gut microbiota. Mechanisms of microbial modification of host BAs continue to be elucidated as do the roles BA metabolism plays in host health. The presence of MCBAs in the human BA pool demonstrates the need for further study of microbial BA modification and further expands the chemical language our gut microbiota uses to communicate with its host.


Introduction: General practitioners (GPs) strive to use a patient centered approach to achieve shared decision making by integrating clinical evidence, clinical judgement, and patient priorities. In order to achieve this standard of care, GPs require relevant, up to date and high quality evidence. Currently there is a gap in the literature regarding the role of GP professional organisations internationally in producing and publishing evidence based guidance and clinical guidelines for GPs. This protocol outlines a scoping review to identify what evidence-based guidance is produced by general practitioner professional organisations internationally in terms of topic content, the structure and methods used to develop guidance and ways of disseminating this guidance, to support general practice clinical decision making.Methods: This scoping review will be conducted using the framework proposed by the Joanna Briggs Institute and the Preferred Reporting Items for Systematic Reviews and Meta-analysis extension for scoping reviews (PRISMA-ScR), will be used to guide the reporting. Two researchers will search electronic databases , grey literature sources and contact international GP professional organisations directly to identify appropriate studies for inclusion. Key information will be categorised and classified to generate a summary of the methods used internationally to develop and implement evidence-based guides for general practitioners and a narrative synthesis will be conducted.Conclusions: This scoping review will examine current practice internationally regarding the role of General Practice professional organisations in producing and publishing clinical guidelines and evidence based guidance to support general practitioner’s clinical decision making to benefit patient care. GPs have a unique role in society, practicing medicine in the context of the family and community. GPs strive to use a patient centred approach to achieve shared decision making by integrating clinical evidence, clinical judgement, and patient priorities.General practitioners (GPs) require evidence-based guidance to support patient careprimarily responsible for the provision of comprehensive and continuing care to every individual seeking medical care irrespective of age, sex and illness GPs in some countries have a gatekeeper role, authorising access to specialty care, hospital care, and some diagnostic tests.There are differences internationally in healthcare systems and the cultural context in which GPs practice, but the role of the GP internationally has similarities in that GPs are ‘. However, a review of 45 UK guidelines reported that many guideline recommendations were based on studies with little or no relevance to primary care. GPs are more likely to use guidelines where the evidence is perceived to be applicable to primary care and where there has been GP input at the guideline development stage.Clinical practice guidelines are systematically developed statements, based on a thorough evaluation of the evidence, to assist practitioner and service users’ decisions about healthcarede novo clinical guidelines for GPs across a range of primary care presentations and commonly managed conditions. Second GP professional organisations may be approached to endorse clinical practice guidelines developed by external organizations and groups. Third, GP professional organisations may disseminate materials to GPs based on national or international guidelines as part of an adopt or adapt approach. While methodological guidance exists to support the process of adoption and adaption of guidelines, there is a need to have standardisation of this process to facilitate reproducibility and reduce duplication of effort.Internationally, GP professional organisations, as key GP opinion leaders, play a varied role in the clinical practice guideline ecosystem. First, in certain countries, GP professional organisations develop ‘de novo’ clinical guidelines. For example, the Dutch College of General Practice (NHG) develop. GP professional organisations are well placed to support GPs to assimilate required evidence through the provision of easily accessible, high level clinical guideline summaries and evidence synopses. However what role, if any, GP organisations take in the dissemination of such evidence is unknown. This gap in the literature limits the ability of GP professional organisations to share both experience and expertise in how best to support GPs in their clinical decision making to support evidence-based patient care. Time pressure and increased workload are established barriers to GP implementation of clinical guidelines and evidence The aim of this scoping review is to identify what evidence-based guidance is published by GP professional organisations internationally to support GPs in their clinical decision making. The objectives are i) to identify the topics covered, both clinical and non-clinical; ii) to review the methods used to develop evidence-based guidance and/or clinical guidelines and how these guidance documents are structured and, iii) to explore how evidence is disseminated to GPs.. While, the overall conduct of the scoping review is informed by the JBI framework, the Preferred Reporting Items for Systematic Reviews and Meta-Analysis extension for scoping reviews (PRISMA-ScR) will be used to guide the reporting of the scoping review.This scoping review will follow the framework proposed by the Joanna Briggs Institute (JBI).As this is a scoping review it will be designed to identify the range of the evidence available and will be represented as a mapping of the identified data, without the act of synthesis or particular reference to methodological quality of relevant studiesOverarching research question: What evidence-based guidance is published by general practice professional organisations to support GPs clinical decision making?Objectives:topics that the organisations are providing?a. What are the contentmethods for developing these guides?b. What are thestructures of the guides and how are they presented?c. What are thedisseminated to GPs?d. How are these guidesGiven the nature of the topic, the fact that many guides may not be published as peer reviewed publications and considerable heterogeneity of the nomenclature , the search strategy will identify both peer reviewed studies and grey literature. This will be achieved through a combination of bibliographic database searching, grey literature searching and contacting key informants. All these processes will be conducted in tandem and are not sequential.. A copy of the search strategy is shown inThe bibliographic database searching will follow a three step strategy, as per JBIfirst step, the limited search, will include searching two appropriate online databases (Medline andEmbase). An analysis of the text words in the titles and abstracts of retrieved papers will be conducted, and of the index terms used to describe the articles.Thesecond step will use all identified key words and index terms to perform a second search of all the following databases: Medline, Embase,Cochrane Library andScopus to identify peer reviewed research papers relating to our aim. This step will be conducted with input from an information specialist.TheThirdly, reference lists of included articles will be searched for additional relevant articles.Grey literature searching will be conducted by searching ‘Guideline Central’ and ‘NHS Evidence Search’ using combinations of the key words and index terms used in the bibliographic database search.Key informants in GP professional organisations, as per the definition of general practice outlined inEndNote X9) and duplicates removed.The final included studies for screening will be downloaded to a reference management software package (Titles and abstracts will be screened for inclusion against the inclusion criteria for the review . For thoThe above steps will be completed by two reviewers (EOB and SD). They will work independently initially and then come together to compare results. Any discrepancies will be resolved by consensus and if consensus is not reached will be referred to a third reviewer (EW).Studies that do not meet the inclusion criteria will be excluded. Reasons for the exclusion will be kept and presented as part of the flow diagram.The final search results will be outlined in a PRISMA flow diagram from the PRISMA-ScR statement, which will be accompanied by a narrative description of the process.. For data extraction the standardised template from the JBI methodology guidance for scoping reviews will be adapted for use.This scoping review is designed to identify the range of the evidence available and represent this as a mapping of the identified data, without the act of synthesis or with particular reference to methodological quality of relevant studiesKey information will be organised in categories based on data from organisational characteristics (evidence source details) e.g., name, country, role, and membership. Details extracted from the source of evidence; characteristics related to the methods , the clinical topics covered and approach to structure and presentation. Modes of dissemination will be recorded as well as implementation strategies.These will be classified and categorised to generate a map of the methods used internationally to develop evidence-based guides for general practitioners and a narrative synthesis conducted.As part of this process one reviewer will independently chart the data from the retrieved articles using the data charting form developed for this review . The sec. Each research question will be reported separately and presented in a tabular form and as a narrative summary. This narrative description will be used to synthesise the study findings based on themes that are generated from the extracted data.Results will be reported using the PRISMA-ScR guidelinesWe intend to disseminate the results through publication in a peer-reviewed journal and conference presentations.Database searches have been completed and title and abstract screening is currently underway.Ethical approval is not required for this scoping review.This scoping review will provide an overview of the evidence-based guidance produced and disseminated by GP professional organisations internationally. This scoping review can contribute to the evidence base for supporting GPs clinical decision making to benefit patient care. Furthermore, the findings of this research can identify potential areas for standardisation internationally to facilitate reproducibility and reduce duplication of effort. The findings of this scoping review will inform future research on the content, presentation dissemination and implementation of evidence-based guidance for GPs.No data are associated with this article. I am happy with this new version.Is the study design appropriate for the research question?YesIs the rationale for, and objectives of, the study clearly described?YesAre sufficient details of the methods provided to allow replication by others?YesAre the datasets clearly presented in a useable and accessible format?YesReviewer Expertise:Clinical practice guidelines, general practice, primary care, person-centered careI confirm that I have read this submission and believe that I have an appropriate level of expertise to confirm that it is of an acceptable scientific standard. This is an interesting study identifying clinical practice guidelines targeting general practice and the role of its professional organisations worldwide. I have one major and a few minor comments. One major comment concerns the search strategy. The authors start with searching online databases and then targeting professional organisation contacts. I would suggest reversing the order. As general practice guidelines are often not included in medical databases, the best way to identify them is to contact professional organisations, for instance, by using the WONCA Network and/or GIN Network. The websites of member organisations could be explored for guidelines published or links to guideline databases. In addition but as a second step, online databases could be searched. In contrast to systematic reviews, a scoping review does not aim to be complete, so efforts could be saved by limiting the search strategy. If the authors still decide to start with searching the online databases, it would be recommended to only follow step 1 as exploration. There is no definition provided of 'guidance'. NICE uses this as an umbrella term, including (evidence-based) guidelines, others use the term to describe all other tools to support decision making in clinical practice that are not evidence based, in contrast to guidelines. I would suggest limiting the review to guidelines, which are evidence-based by definition (according to IOM).The term 'published' also needs clarification. This can be on paper or digital. In the legal context, the term 'endorsed' or 'approved' is more relevant than 'published' as this means that the professional needs to know and to follow the guideline in principle. The term 'published' is more vague. It would be interesting to focus on those guidelines that are part of a programme or set of guidelines used for education, training, and evaluation. As this is a scoping review, the scope can be changed or refined during the study if more focus is desirable to answer the research question.The search strategy identifying general practice could be expanded with terms as 'Primary Health Care' or 'Physicians, Primary Care'. This is broader than 'General Practice' and 'Family Practice' but some countries do not use these terms. In Discussing the potential impact of the findings of this study, the authors could discuss the opportunities to share guidelines and their supporting evidence internationally to reduce duplication of efforts.  Minor comments are:Is the study design appropriate for the research question?YesIs the rationale for, and objectives of, the study clearly described?YesAre sufficient details of the methods provided to allow replication by others?YesAre the datasets clearly presented in a useable and accessible format?YesReviewer Expertise:Clinical practice guidelines, general practice, primary care, person-centered careI confirm that I have read this submission and believe that I have an appropriate level of expertise to confirm that it is of an acceptable scientific standard, however I have significant reservations, as outlined above. Many thanks for reviewing my comments and making minor revisions. I have no further comments to make.Is the study design appropriate for the research question?YesIs the rationale for, and objectives of, the study clearly described?YesAre sufficient details of the methods provided to allow replication by others?YesAre the datasets clearly presented in a useable and accessible format?YesReviewer Expertise:general practice, medical education, dementia care, scoping review, qualitative researchI confirm that I have read this submission and believe that I have an appropriate level of expertise to confirm that it is of an acceptable scientific standard. I have some concerns about this study, and its potential to provide useful evidence. In particular, I do not feel that the rationale for focusing upon the role of GP professional organisations in guideline production is well described. I think that the researchers are suggesting that guidelines produced specifically by GP professional organisations will somehow be better/more applicable than guidelines produced by other organisations, but this case is not made. In addition, I think that current professional focus is generally more upon how you manage patients with more than one condition, and in these circumstances, disease specific guidelines are less applicable. Finally, the authors do not provide any research questions. I would recommend that they do this before embarking on their reviewIs the study design appropriate for the research question?NoIs the rationale for, and objectives of, the study clearly described?PartlyAre sufficient details of the methods provided to allow replication by others?YesAre the datasets clearly presented in a useable and accessible format?Not applicableReviewer Expertise:I am a GP and qualitative researcherI confirm that I have read this submission and believe that I have an appropriate level of expertise to state that I do not consider it to be of an acceptable scientific standard, for reasons outlined above. Many thanks for agreeing to review this Scoping Review protocol. We address your comments on a point by point basis below.Comment I have some concerns about this study, and its potential to provide useful evidence. In particular, I do not feel that the rationale for focusing upon the role of GP professional organisations in guideline production is well described. I think that the researchers are suggesting that guidelines produced specifically by GP professional organisations will somehow be better/more applicable than guidelines produced by other organisations, but this case is not made.Response The rationale for focusing on GP professional organisations is to examine current practice internationally regarding the role of GP organisations in producing and publishing clinical guidelines and evidence based guidance for GPs. There is currently a lack of evidence as to what is being published by GP professional organisations internationally in terms of evidence based guidance and clinical guidelines for GPs to use in their clinical practice. Our initial literature search shows international variation in the role of GP professional organisations in publishing guidelines and evidence based guidance for GPs. For instance in the Netherlands, de novo clinical guidelines are developed and published by the Dutch College of GPs on a range of relevant topics whereas in other countries such as the UK clinical guidelines, aimed at GPs are published by central clinical guideline agencies such as NICE and SIGN while the Royal College of GPs focuses on the publication of other evidence based guidance for GPs.Internationally, GP professional organisations, as key GP opinion leaders, play a varied role in the clinical practice guideline ecosystem. First, in certain countries, GP professional organisations develop ‘de novo’ clinical guidelines. Second GP professional organisations may be approached to endorse clinical practice guidelines developed by external organizations and groups. Third, GP professional organisations may disseminate materials to GPs based on national or international guidelines as part of an adopt or adapt approach. While methodological guidance exists to support the process of adoption and adaption of guidelines, there is a need to have standardisation of this process to facilitate reproducibility and reduce duplication of effort.  Evidence shows that involving clinicians in guideline production relevant to their clinical practice and understanding the context in which the guidelines are to be implemented are very important aspects of ensuring clinical guidelines are implemented in practice. Understanding the role of GP professional organisations internationally in the production and publishing of clinical guidelines can inform best practice in this area and lessons can be shared regarding how different countries approach this issue and how that impacts on implementation.GPs are more likely to use guidelines where the evidence is perceived to be applicable to primary care and where there has been GP input at the guideline development stage To capture the implementation of guidelines published by GP professional organisations internationally a key informant survey of GP professional organisations internationally will be conducted to further inform this scoping review. This will focus on the methods employed by different GP professional organisations in developing guidelines/evidence based guidance, how this is disseminated to GPs and the processes supporting implementation in clinical practice. This approach will give a rounded view of the utility of different approaches to guideline production and publication by GP professional organisations internationally. The introduction has been updated to include a discussion of the barriers to guideline implementation and the possible role GP professional organisation have in facilitating their use.Time pressure and increased workload are established barriers to GP implementation of clinical guidelines and evidence.GP professional organisations are well placed to support GPs to assimilate required evidence through the provision of easily accessible, high level clinical guideline summaries and evidence synopses. However what role, if any, GP organisations take in the dissemination of such evidence is unknown. This gap in the literature limits the ability of GP professional organisations to share both experience and expertise in how best to support GPs in their clinical decision making to support evidence-based patient care.Comment In addition, I think that current professional focus is generally more upon how you manage patients with more than one condition, and in these circumstances, disease specific guidelines are less applicable.Response Multimorbidity is one important area for GPs to have access to up to date information and there are already a number of guidelines developed to support GP decision making in this area e.g. UK NICE guideline for the clinical assessment and management of multimorbidity. This scoping review will provide an overview of the topics that GP professional organisations develop clinical guidelines and evidence based guidance for GPs and will answer the question as to what topics GP professional organisations are prioritising for their GP members.Comment Finally, the authors do not provide any research questions. I would recommend that they do this before embarking on their reviewResponse The overarching research question and objectives for this scoping review are outlined in the Methods section; scoping review framework; stage 1; identifying the research question. For ease of reference we include the research question and review objectives again here;Overarching research question: What evidence-based guidance is published by general practice professional organisations to support GPs clinical decision making?Objectives:topics that the organisations are providing?a. What are the contentmethods for developing these guides?b. What are thestructures of the guides and how are theypresented?c. What are thedisseminated to GPs?d. How are these guidesComment Is the rationale for, and objectives of, the study clearly described? PartlyResponse Given the updates outlined above we would hope that the rationale for the study is now more clearly described.Comment Is the study design appropriate for the research question? NoResponse Given the amendments made to more clearly outline the overall rationale for this study we hope that the reasoning for the choice of study design is also clearer now. A scoping review is defined as a review that is designed to address a broad research question by mapping a body of literature in that area and also to identify gaps in the research knowledge area  Scoping reviews are used not only to determine the extent of the research in a particular area but also the way the research was conducted. As such, we believe it is the appropriate design to answer the research question for this review; ‘What evidence based guidance and guidelines is published by general practice professional organisations to support GPs clinical decision making’. By supplementing the scoping review of the literature with a targeted survey of GP professional organisations internationally the review will be able to provide data on other key aspects of guideline production, dissemination and implementation by GP professional organisations. Thanks for the opportunity to review this interesting scoping review protocol.Abstract: Consider adding a line to briefly explain the importance of this topic - evidence-based guidelines. The aim given here is to 'identify what evidence-based guidance is produced...'. Consider expanding on this identify studies/content topics/methods of development/dissemination.Introduction: While the importance of evidence-based guidance to support patient care is made and also the different approaches used internationally, I feel the authors need to expose and highlight the gap in research. Have any similar reviews been undertaken before or are any scoping/systematic reviews currently ongoing? What literature is out there on the aims/objectives of this study? 'The role of the GP in the healthcare system also has an impact'.....Consider expanding on this sentence to explain to the reader what impact is being discussed. 'GPs who are gatekeepers are more likely to require GP orientated guidelines'...Perhaps this statement could also be further explained?Methods: Search Strategy: Table 1 Eligibility criteria - consider adding rationale why you're excluding papers with <=2 authors. Is that necessary?  Contacting GP professional organisations - Can you further identify which countries/regions will be included in this contact and justify why? Has the protocol been registered? e.g. open science framework/prospero.Is the study design appropriate for the research question?YesIs the rationale for, and objectives of, the study clearly described?YesAre sufficient details of the methods provided to allow replication by others?YesAre the datasets clearly presented in a useable and accessible format?YesReviewer Expertise:general practice, medical education, dementia care, scoping review, qualitative researchI confirm that I have read this submission and believe that I have an appropriate level of expertise to confirm that it is of an acceptable scientific standard. Thanks for the opportunity to review this interesting scoping review protocol.Response Many thanks for agreeing to review this Scoping Review protocol.Abstract: Consider adding a line to briefly explain the importance of this topic - evidence-based guidelines. The aim given here is to 'identify what evidence-based guidance is produced...'. Consider expanding on this identify studies/content topics/methods of development/dissemination.Response Abstract has been amended to include suggestions above. See paragraph 1 Introduction and paragraph 3 conclusion of Abstract‘in terms of topic content, the structure and methods used to develop guidance and ways of disseminating this guidance’Introduction: While the importance of evidence-based guidance to support patient care is made and also the different approaches used internationally, I feel the authors need to expose and highlight the gap in research. Have any similar reviews been undertaken before or are any scoping/systematic reviews currently ongoing? What literature is out there on the aims/objectives of this study?Response There is currently a lack of evidence as to what is being published by GP professional organisations internationally in terms of evidence based guidance and clinical guidelines. There appears to be international variation in the role of GP professional organisations in publishing guidelines and evidence based guidance. For instance in the Netherlands, de novo clinical guidelines are developed and published by the Dutch College of GPs on a range of relevant topics whereas in other countries such as the UK, clinical guidelines aimed at GPs are published by central clinical guideline agencies such as NICE and SIGN while the Royal College of GPs focuses on the publication of other evidence based resources for GPs. See paragraph 3 and 4 of Introduction, research gap highlighted by expanding on the above points and also making clear the rationale for this review.Clinical practice guidelines are systematically developed statements, based on a thorough evaluation of the evidence, to assist practitioner and service users’ decisions about healthcare.However, a review of 45 UK guidelines reported that many guideline recommendations were based on studies with little or no relevance to primary care.GPs are more likely to use guidelines where the evidence is perceived to be applicable to primary care and where there has been GP input at the guideline development stage. 'The role of the GP in the healthcare system also has an impact'.....Consider expanding on this sentence to explain to the reader what impact is being discussed.Response Introduction paragraph 2 has been updated to put both healthcare system and role of GP as gatekeeper into context‘There are differences internationally in healthcare systems and the cultural context in which GPs practice, but the role of the GP internationally has similarities in that GPs are ‘primarily responsible for the provision of comprehensive and continuing care to every individual seeking medical care irrespective of age, sex and illness’ 'GPs who are gatekeepers are more likely to require GP orientated guidelines'...Perhaps this statement could also be further explained?Response See aboveMethods: Search Strategy: Table 1 Eligibility criteria - consider adding rationale why you're excluding papers with <=2 authors. Is that necessary? Response Thank you. The author number restriction has been removed. Contacting GP professional organisations - Can you further identify which countries/regions will be included in this contact and justify why?Response The Key Informant Survey will be circulated to GP professional organisations colleges in Europe, USA, Canada, Australia and New Zealand. We are targeting national GP professional organisations in countries that have a GP system in line with the WONCA definition of General/Family Practice (Table 1 of protocol). See Methods, Search Strategy Stage 2 for updated text.Various avenues will be utilised to contact key informants including WONCA Europe contacts. Has the protocol been registered? e.g. open science framework/prospero.Response Prospero does not accept registration of Scoping Review Protocols. The review has been registered with Open Science Framework.


Severe community-acquired pneumococcal pneumonia is commonly associated with bacteraemia. Although it is assumed that the bacteraemia solely derives from pneumococci entering the blood from the lungs it is unknown if other organs are important in the pathogenesis of bacteraemia. Using three models, we tested the relevance of the spleen in pneumonia-associated bacteraemia.ex vivo to explore permissiveness to bacterial replication, a non-human primate model to check for splenic involvement during pneumonia and a mouse pneumonia-bacteraemia model to demonstrate that splenic involvement correlates with invasive disease.We used human spleens perfused Here we present evidence that the spleen is the reservoir of bacteraemia during pneumonia. We found that in the human spleen infected with pneumococci, clusters with increasing number of bacteria were detectable within macrophages. These clusters also were detected in non-human primates. When intranasally infected mice were treated with a non-therapeutic dose of azithromycin, which had no effect on pneumonia but concentrated inside splenic macrophages, bacteria were absent from the spleen and blood and importantly mice had no signs of disease.We conclude that the bacterial load in the spleen, and not lung, correlates with the occurrence of bacteraemia. This supports the hypothesis that the spleen, and not the lungs, is the major source of bacteria during systemic infection associated with pneumococcal pneumonia; a finding that provides a mechanistic basis for using combination therapies including macrolides in the treatment of severe community-acquired pneumococcal pneumonia.Oxford University, Wolfson Foundation, MRC, NIH, NIHR, and MRC and BBSRC studentships supported the work. There is a consensus that moderate and high severity community-acquired pneumonia (CAP) should be treated with a combination of a macrolide and a beta-lactam, but there is not a clear understanding of the mechanisms underpinning this measure. Recently we showed that intracellular replication of pneumococci in CD169+ tissue macrophages precedes invasive pneumococcal disease (IPD), in murine and porcine models, and that this could be prevented by macrolides, raising the hypothesis that an intracellular phase of bacterial replication could precede bacteraemic disease, but there was no evidence that this occurs during pneumonia or in the human spleen.ex vivo perfusion system, we show that pneumococci are detectable in the splenic macrophages, where they form foci of increasing bacterial numbers over time. In a non-human primate pneumococcal pneumonia model, bacterial foci also formed, and they were still observed after beta-lactam treatment. In a murine model of pneumococcal pneumonia, pneumonia was unaltered histologically and bacteriologically, by treatment with a sub-inhibitory dose of the macrolide, azithromycin. The treatment abolished bacterial counts in spleen and the blood, indicating that it is the bacterial load and functionality of the spleen that determines eventual blood counts and IPD.Using a human spleen The data provide compelling evidence that the spleen, and not the lungs, is the major source of bacteria giving rise to sustained bacteraemia. We suggest that the evidence provides a mechanistic basis for using combination therapy, that includes macrolides, in treating high severity pneumonia, including pneumococcal pneumonia.Alt-text: Unlabelled boxStreptococcus pneumoniae causing more deaths than all other lower respiratory tract aetiologies combined Lower respiratory tract infections are the leading cause of mortality from communicable disease ex vivo perfusion [Recently, we showed that in intravenously infected mice, pneumococcal sepsis is preceded by intracellular replication of the bacteria within splenic macrophages erfusion ,10. Invoex vivo human spleen infections, a non-human primate and murine pneumonia models Direct extrapolation of our previous findings to human disease management is confounded because the architecture of the human spleen displays significant differences to that of other animals ,12. In pSome of the results of these studies have been previously reported in the form of abstracts Human spleen perfusions were conducted at the Leicester General Hospital, Leicester, UK. After written informed consent for utilisation of the organ for research, spleens were obtained from patients during elective partial pancreatectomy in the context of pancreatic cancer. The study was approved by the UK Health Research Authority (IRAS219992), the East Midlands-Leicester South Research Ethics Committee (REC18/EM/0057) and has the ClinicalTrials.gov Identifier NCT04620824.Papio cynocephalus), following intra-bronchial inoculation with S. pneumoniae, had been previously performed at the Texas Biomedical Research Institute (TBRI) in San Antonio, Texas Studies of severe pneumonia in non-human primates  and a more recent clinical isolate TIGR4 (deposited as ATCC BAA-334), respectively serotype 2 and 4, and both not carrying any drug resistance marker [The classical e marker ,18, weree marker ,18. Straex vivo human spleen perfusion was performed as previously described 8 CFU and all other spleens with 2 × 107 CFU. Spleens HSP1 and HPS2 were infected with S. pneumoniae D39, spleen HSP5 and HSP8 with TIGR4, and spleens HSP6, HSP7, HSP9 and HSP12 with a 1:1 mix of both D39 and TIGR4. Two control spleens (HSP3 and HSP4) were not infected and were used as comparator organs for blood gas analysis. Spleens HSP10 and HSP11 where used for a different project. Hemopure samples and tissue biopsies were taken at predetermined time points for quantitative bacterial culture, or for flash freezing in Optical Cutting Matrix  for microscopy analysis. Human tissue samples were excluded from infection experiments if they had been administered antibiotic therapy prior to surgery. Power calculations, statistical analyses, details of immunohistochemistry, the antibodies (Table S2) and microscopy methodologies are detailed in the Supplementary Materials.Normothermic 6 CFU/g in murine lungs with an anticipated standard deviation of 105. We predicted an effect size of a 20% reduction in CFU/g in azithromycin treated animals. The required sample size using these parameters was 4, but 5 animals were subsequently included per group for animal husbandry purposes.For calculation of samples sizes for murine infection experiments, we considered a type-1 error rate of 0.05, and a statistical power of 0.8. Using pilot data derived from previous experiments in our group, we anticipated a mean of 10For human spleen perfusion experiments, we determined samples sizes using pilot data from our published, porcine spleen perfusion model ,10. BriePapio cynocephalus) infected with S. pneumoniae, available from a previous study, were used for this analysis 9 CFU of S. pneumoniae TIGR4, using bronchoscopy Fixed spleen sections from baboons  Act, 1986. This study utilised 5x adult 6-8-week-old female CD1 mice bred and used at the University of Leicester Pre-clinical Research Facility. Animals were randomly allocated into cages in groups of 5 at 8 weeks of age by staff at the pre-clinical research facility who had no involvement in subsequent experiments using an online, random number generator algorithm. Infections, sample processing and image analysis were performed unblinded. All experimental procedures were performed on animals in the same order throughout to minimise confounding factors associated with different treatment times between animals.6 CFU of pneumococci, diluted directly from thawed frozen stocks, intranasally in 50 μl of BHI [6 pneumococci in 100 μl of BHI [Micrococcus luteus ATCC 9341 as a reference microorganism  For intranasal infections, mice  were lightly anaesthetised with isoflurane, before instilling 10l of BHI ,21. For l of BHI . AntibioSlides of paraffin embedded sections of spleens from baboons with pneumococcal pneumonia were used (Table S1) 2O before adding mounting medium containing DAPI  and closing the slides with coverslips. All primary and secondary antibodies used in this study are described in Table S2. Haemotoxylin and eosin stains were performed by the University Hospitals of Leicester histology service using standard protocols and analysed blinded.Fresh frozen samples from murine and human spleens were sectioned to 10 μm thickness using a Leica cryostat . Samples were then fixed in 4% w/v paraformaldehyde for 20 min at room temperature, before being washed three times in PBS. From this stage onwards, formalin-fixed sections subjected to deparaffinisation and antigen retrieval were processed using the same protocol as fresh frozen samples. Samples were permeabilised with 0.1% v/v Triton X-100 in PBS for 10 min and incubated for 30 min in blocking solution . Sections were incubated for 1 h with primary antibodies diluted in blocking solution, washed three times with PBS, and then incubated for 45 min with secondary antibody solution (Table S2). Samples were then washed three times with PBS and once with HHuygens Essential deconvolution software (v 18.04.1p0 64b)  and viewed in Imaris 3D (v 9.3.1) reconstruction software . In order to identify metallophilic CD169+ macrophage distribution in the spleens, sections were scanned using a fully motorised Nikon Eclipse Ti microscope equipped with a Plan Fluor 10X/0.3 objective and an Andor iXonEM+ EMCCD DU 885 camera. Automatic tissue scanning was performed using NIS-Elements software and the images obtained were analysed using ImageJ software.An Olympus FV1000 confocal laser scanning microscope was used to acquire images using 40x (UPlanFLN 40x/NA=1.3) and 60x (UPlanSAPO 60x/NA=1.35) objectives, and the free software Fiji (v 2.0.0-rc 69/1.52p) For scanning microscopy of whole tissue sections, a Vectra Polaris Digital Pathology system  with fluorescence filter cubes was used for imaging of fluorescently labelled tissue sections, and brightfield acquisition for haematoxylin and eosin stained sections. Both types of sample were imaged using a 40x objective (40x/NA=0.75). Image files were exported in .tiff format and quantitatively analysed using Fiji. For quantitative co-localisation analysis, regions of interest were derived using the Image>Adjust>Threshold function in Fiji using fluorescence signal specific for each macrophage population. These regions of interest were then imposed on the bacterial fluorescence channels for particle analysis.For statistical analyses Graphpad Prism (v 9.0.0) was used. A test for the normality of the data was performed prior to the statistical tests. For analysis of categorical 2 × 2 contingency tables, a Fisher's exact test was used. For comparison of multiple groups and conditions/time points, appropriate 2-way analysis of variance (ANOVA) tests were performed.The funders had no role in the study design, data collection, data analyses, interpretation, or writing of report.https://leicester.figshare.com/) with the doi: https://doi.org/10.25392/leicester.data.12957947. Sharing of human tissue is subject to restrictions by the human tissue act and our trial protocol.All original microscopy image files are available through the University of Leicester data repository Figshare  on the margin of splenic follicles cd. ConfoTo investigate whether there is a link between intracellular pneumococcal foci in the spleen and pneumococcal pneumonia, spleen samples from baboons with experimental pneumonia were analysed To characterise the localisation of the splenic macrophages within the overall micro-architecture of the baboon spleen, whole organ sections were analysed by high-resolution fluorescent scanning microscopy. Images showed a diffuse distribution of CD163+ red pulp macrophages b, whereaTo further investigate if intracellular replication of bacteria within the spleen plays a role in the development of pneumococcal bacteraemia, we used a mouse model of pneumonia and bacteraemia, which followed intranasal infection. To selectively inhibit intracellular organisms, mice were treated with a single dose of azithromycin, a drug with a long half-life that is known to concentrate up to 40-fold within macrophages In azithromycin-treated and saline-treated mice, bacterial counts in the lungs, over 48 h after infection, were similar (p>0.05) at each time point a. BacterTo confirm that treatment with azithromycin did not alter the histopathological presentation of pneumonia, which could have influenced translocation of organisms to the blood, sections were prepared of lungs collected from mice 48 h post-infection. Haematoxylin and eosin staining indicated that lung pathology was indistinguishable between saline-treated a and aziMacrolides have been reported to have immunomodulatory effects on macrophages ex vivo perfusion of the human spleen system, together with models of pneumococcal pneumonia in a non-human primate and in the mouse, we have found compelling evidence that the spleen, and not the lungs, is the major source of bacteria giving rise to sustained bacteraemic disease. The evidence also provides a mechanistic explanation for the observed benefit of combination beta-lactam-macrolide antibiotic therapy over beta-lactam monotherapy in the management of moderate and high severity CAP, including pneumococcal pneumonia.Using S. pneumoniae is the most frequent etiological agent [For many years, lower respiratory infections have been the most common cause of mortality from communicable disease and al agent ,2. Thereal agent ,5. This al agent ,34. PossSustained bacteraemia and sepsis following pneumococcal pneumonia is generally assumed to result from continuous seeding of bacteria from the lung into the bloodstream. We proposed a different hypothesis in which bacteria enter the blood from the lung, are filtered by the spleen and only following intracellular replication within splenic CD169+ macrophages do they initiate sustained bacteraemia. This hypothesis does not challenge the primacy of splenic macrophages in clearing capsulated bacteria and that loss of the spleen predisposes to overwhelming sepsis ,38. RathExtrapolation of the observations in mice to humans is constrained by the very different splenic architecture of murine spleens compared to that of humans In spleens of pneumonic baboons, we observed clusters of pneumococci associated with splenic macrophages. The presence of these foci, even following treatment with ampicillin (which does not effectively penetrate macrophages), provides evidence for an extravascular, reservoir for pneumococci where they may be protected from innate immune effectors. The observation of bacterial foci in the spleens of ampicillin-treated baboons indicate that this intracellular niche may allow pneumococci to survive monotherapy with beta-lactams. This is in line with recent data showing persistence of pneumococci within alveolar macrophages of asymptomatic subjects for days after experimental carriage and amoxicillin treatment ex vivo [Next, we sought evidence that in human spleens CD169+ macrophages are permissive for pneumococcal replication. For this, we established a perfusion system in which human organs could be maintained and subsequently infected ex vivo ,42. Micrex vivo . Previouex vivo ,44, but ex vivo , neutropex vivo ,47. Thisex vivo ,48. Macrex vivo ,49.S pneumoniae is the predominant pathogen [Our findings provide a new mechanistic basis for current recommendations for the use of a combination of beta-lactam and macrolide antibiotics in the empirical management of moderate and high severity CAP, particularly in bacteraemic disease where pathogen ,5,6. TheWe used three distinct experimental systems, each of which has inherent technical and physiological limitations. In the human spleen perfusion model, given the limited availability of organ samples, we elected to use a low inoculum of pneumococci. Our rationale was to simulate natural infection and avoid the potential criticism of bacterial overload. This resulted in very few bacteria per biopsy section, especially for the TIGR4 strain. It should be noted that we pooled the data from experiments that used variable conditions for perfusing the artificial blood substitute, a judgement that could be legitimately questioned. The baboon sample analysis was conducted on 5-year-old paraffin embedded spleen samples that were available from a previous experiment whose experimental aims were unrelated to the present study. Potential limitations include: the sample storage medium and unexplained interference of IHC staining of macrophages with our CD169 antibody in the paraffin embedded sections that resulted in poor visualisation of the marker, but the results provide additional support for our hypothesis using a pneumonia model in non-human primates. The murine infection addresses our hypothesis the most directly but is limited by the treatment parameters. Given all these limitations, it is the nevertheless the sum of all three approaches, not any one data set, that provides substantial preclinical evidence to justify further investigation in clinical trials.All authors read and approved the final version of the manuscript. DC, JJW, ZJ and RGH performed experiments and wrote the manuscript, WYC and ARD performed surgery and perfusion, KS supervised microscopy, LMP provided guidance on immunology, MP and WSL provided guidance on clinical relevance, CJO and MIR contributed the baboon experiments, PWA supervised the mouse experiments, and PWA, ERM and MRO did the project planning, contributed funding and verified the underlying data.MRO reports grants from MRC MR/M003078/1, grants from BBSRC BB/S507052/1 , grants from MRC IMPACT Doctoral Training program, during the conduct of the study; grants from GSK co-funding of CASE studentship BBSRC BB/S507052/1, outside the submitted work; and consultancy for Pinsent Masons LLP 2020-21 on work unrelated to this manuscript. MRO had a BactiVac Network grant BVNCP3-05 2019-20 with GSK on topics unrelated to this manuscript. ZJ reports grants from BBSRC CASE GSK studentship BB/S507052/1, outside the submitted work. MP reports grants and personal fees from Gilead Sciences, personal fees from QIAGEN, grants from Sanofi, outside the submitted work. WSL has received core support from the NIHR Nottingham Biomedical Research Centre, and his institution has received unrestricted investigator-initiated research funding from Pfizer for an unrelated multicentre cohort study in which WSL is the Chief Investigator. The other authors have nothing to disclose.


Recently, revisions of two sepsis guidelines, namely, the Japanese Clinical Practice Guidelines for Management of Sepsis and Septic Shock 2020 and the Surviving Sepsis Campaign Guidelines 2021, were published. Although both guidelines were created in accordance with the Grading of Recommendations, Assessment, Development and Evaluation approach, the evidence-to-decision tables differed between them. In addition, certain recommendations may differ between these guidelines for similar clinical questions because of differences in the “PICO” criteria. Other differences in recommendations between the two guidelines are due to unclear evidence, and these ambiguities may provide the basis for further sepsis research. We hope that these two guidelines will contribute to the creation of new clinical evidence in addition to supporting treatment of patients with sepsis. Recently, revisions of two sepsis guidelines, namely, the Japanese Clinical Practice Guidelines for Management of Sepsis and Septic Shock (J-SSCG) 2020 , 2 and tIn the process of revision, many similarities were discovered between the two guidelines. For example, both their aims were to assist in appropriate clinical decision-making to improve the prognosis of patients suffering from sepsis and septic shock. In addition, both guideline working groups included patients for better reflection of their perspectives in the guidelines. However, there are certain differences Tables , 2. The Although both guidelines were created in accordance with the Grading of Recommendations, Assessment, Development and Evaluation (GRADE) approach, J-SSCG 2020 adopted eight domains in the evidence-to-decision (EtD) table, while in SSCG 2021, the resources required, certainty of evidence of required resources, cost-effectiveness, and equity were used for making a recommendation. Guideline recommendations were not based solely on the certainty of evidence, and both guidelines included certain recommendations in areas where no randomized controlled trials (RCTs) had been conducted. In the J-SSCG 2020, expert consensus was made based on EtD table included opinions of panel members and evidence except RCTs. On the other hand, in SSCG 2021, “in our practice statement” were made based on the majority opinion of the guideline panel.In addition, certain recommendations may differ between these guidelines for similar clinical questions because of differences in the “PICO” criteria. The guidelines may have included studies based on different criteria for patients, problem, and population (the population that received an intervention); interventions; comparisons, controls, and comparators (interventions to compare with “I”); and outcomes (events that may occur as a result of the intervention). The resultant differences in included studies might have affected the final recommendations. For example, J-SSCG 2020 recommends enteral nutrition within 24–48 h of initiation of therapy in patients with sepsis, while SSCG 2021 recommends initiation of enteral nutrition within 72 h. As similar RCTs were included in the meta-analyses of the two guidelines, we believe that the differences in recommendations were determined by whether the intervention (the “I” in “PICO”) was set to within 72 or 48 h. Further studies on the optimal timing of the initiation and increase in nutrition and the appropriate dose are required. Similarly, the J-SSCG 2020 recommends the use of lactate as an indicator of tissue hypoperfusion during initial resuscitation, while the SSCG 2021 recommends the use of a decrease in lactate as such an indicator. This difference may also be due to differences in the PICO criteria. The J-SSCG 2020 recommendation is based on the results of systematic reviews in which lactate, or the change in serum lactate concentration was measured in the intervention group, while it seems that SSCG 2021 included studies in which the decrease in lactate was assessed as the intervention.Although J-SSCG2020 did not include any recommendation on capillary refill time (CRT), the SSCG 2021 weakly recommends the use of CRT to guide resuscitation as an adjunct to other measures of perfusion in the absence of advanced hemodynamic monitoring , 4. PrevA number of recommendations that differ between the two guidelines are based on unclear evidence, and such ambiguities may provide a basis for further sepsis research. For example, these guidelines provide different recommendations for initial resuscitation, which is a key point in sepsis treatment. The recommended first- and second-line vasopressors in both guidelines are noradrenaline and vasopressin, respectively. However, J-SSCG 2020 provided no direct comparison between the use of noradrenaline and vasopressin as first-line agents. Although no details were provided, such a comparison was made in SSCG 2021. The SSCG 2021 working group noted that there was evidence to suggest that vasopressin may be superior to noradrenaline in terms of clinical outcomes , 4 but rThe recommendations for vitamin C administration differ between the guidelines despite selecting many of the same sources of evidence. In both guidelines, meta-analyses indicated that the desirable effects of vitamin C outweigh the undesirable effects. However, SSCG 2021 recommended against using vitamin C as the balance of effects did not favor either vitamin C or the placebo. This decision was based on the fact that the point estimate of 90-day mortality favored the control group in the largest RCT . Thus, aNew research questions may be identified when, in addition to reading the recommendations, the evidence for their rationale and the process of decision-making are analyzed. We hope that these two guidelines will contribute to the creation of new clinical evidence in addition to supporting the treatment of patients with sepsis.