This is the second of two guidance articles produced by the British Infection Association (BIA), the Healthcare Infection Society (HIS), the Infection Prevention Society (IPS) and the Royal College of Pathologists (RCPath). Both articles refer to the pandemic of coronavirus disease 2019 (COVID-19) caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Using evidence that emerged during the first wave of the pandemic, the articles summarise aspects of the transmission dynamics of SARS-CoV-2 and provide guidance on how to reduce the risk of transmission. This article focuses on the risks of presymptomatic, asymptomatic and post-symptomatic SARS-CoV-2 transmission, allowing healthcare workers and the public to understand how transmission occurs and to take action to protect themselves and others. The guidance recognises further waves of the pandemic, the possibility of reinfection, the emergence of new variants of the virus and ongoing immunisation programmes.confirmedpresymptomatic transmission  is probable.asymptomatic transmission (meaning that an index case never develops symptoms or signs of infection) is Having considered the evidence, the COVID-19 Rapid Guidance Working Party concluded that:The Working Party was unable to assess the likelihood of post-symptomatic transmission  because of an absence of evidence.The Working Party formulated recommendations for practice taking account of the evidence reviewed. The recommendations were developed for acute healthcare settings , but they might be useful in other health and care settings such as dental practices and care homes. The Working Party also identified areas for future research.people without noticeable symptoms may transmit the SARS-CoV-2 virus to other peopletransmission of SARS-CoV-2 from people without symptoms may occur in all settings in which people are in close proximityhowever, it is likely that the risk of transmission of SARS-CoV-2 is greater from people who have symptoms compared with those who do not.Be aware that:Even in the absence of symptoms, adhere to legislation and guidance regarding measures to reduce the risk of transmission of SARS-CoV-2 .nature of further waves or outbreaks of COVID-19emergence and circulation of SARS-CoV-2 variants of concernpotential for people who have had COVID-19 previously to be reinfectedeffectiveness of available vaccines, including the longevity of immunity they confer.Be aware that the future transmissibility of SARS-CoV-2 from people carrying the virus without symptoms might depend on the:Be aware that it is not yet known to what extent or for how long people recovering from acute infection can transmit the SARS-CoV-2 virus to other people.Covid-19 is a worldwide problem, and we are learning not just how to treat and vaccinate (immunise) people, but also how and when the infection is spread from person to person. Unlike some infections, you cannot necessarily see who is likely to infect another person; this is because sometimes the infection is transmitted before (pre) someone develops symptoms. It is also the case that some people have the infection and can transmit it but never develop symptoms themselves; this we call asymptomatic transmission.This guidance document is one of a pair which have reviewed the scientific evidence on how Covid-19 is spread. This part of the guide provides recommendations on how to help stop the spread of infection before someone becomes obviously ill (presymptomatic) and for those who never become ill themselves (asymptomatic). We did not find evidence for post symptomatic transmission (someone transmitting Covid-19 after they have recovered).The recommendations based on the evidence we have reviewed give confidence that the things we are all doing such as social distancing, hand washing, wearing face coverings and keeping rooms well ventilated by opening windows are the things that we should be doing to prevent people getting infected with Covid-19. We hope that this guide will help everyone try and prevent spreading Covid-19.Coronavirus disease 2019 (COVID-19) was first detected in Wuhan, Hubei province, China; it spread around the world as a pandemic and by November 2021 had affected more than 260 million people [As an emerging and pandemic disease, COVID-19 attracted worldwide attention and interest in understanding the dynamics of SARS-CoV-2 transmission and treatment options for COVID-19 patients. This Working Party Report is the second of two guidance articles developed using evidence published during the first wave of the pandemic to summarise aspects of the transmission dynamics of SARS-CoV-2 and advise on measures to reduce the risk of transmission in health and care settings. The article examines the risks of presymptomatic, asymptomatic and post-symptomatic SARS-CoV-2 transmission. Understanding the risk of transmission according to the index case’s symptom status at the time of exposure of  their close contacts is important to allow healthcare workers and the public to take action to protect themselves and others. The guidance acknowledges the possibility of reinfection, the emergence of new variants of the virus (particularly variants of concern), and ongoing immunisation programmes.Key technical terms used in this guidance article are explained in the accompanying glossary  Guidelines Committee for reviewing this document.The authors received no specific funding for this work. Financial support for time required to identify and synthesise the evidence and to write the manuscript was provided by the authors’ respective employing institutions.No authors reported any conflicts of interest , HIS, the Infection Prevention Society (IPS) and the Royal College of Pathologists (RCPath) commissioned the authors to develop the Working Party Report. The authors are members of the participating organisations and together comprise the COVID-19 Rapid Guidance Working Party convened to develop the guidance. MAM and AB are employed by HIS as guideline developers. Further information is provided in Additional file The views expressed in this publication are those of the authors and have been endorsed by BIA, HIS, IPS and RCPath following rapid consultation.This report is the second in a pair of guidance documents covering key aspects in the prevention of SARS-CoV-2 transmission in health and care settings. The guidance also reviews the evidence for SARS-CoV-2 transmission dynamics in broader settings. The diagnosis and management of COVID-19 in general is outside the remit of this guidance.The Working Party recommendations have been developed systematically through multidisciplinary discussions based on currently available evidence from published, preprint and grey literature sources. They should be used in the development of local protocols for relevant health and care settings such as hospitals, nursing/care homes, primary care and dental practices.The first wave of the COVID-19 pandemic occurred amid uncertainty as to how it could be prevented and controlled. Concern still exists about further waves and new outbreaks occurring. Evidence that emerged during the first wave provides an opportunity to develop evidence-based guidance for preventing and controlling future waves/outbreaks, acknowledging the possibility of reinfection, the context of newly emerging variants of SARS-CoV-2, and ongoing immunisation programmes.The main purpose of the recommendations is to inform clinicians, managers and policy makers about SARS-CoV-2 transmission dynamics and to provide evidence-based guidance to prevent and control the spread of SARS-CoV-2 in health and care settings. The report highlights current gaps in knowledge, which will help to direct future areas of research.The scope of the guidance is to provide advice for the optimal provision of effective and safe health and care services during the period in which COVID-19 remains a health threat. The guidance was developed for acute healthcare settings, but it might be useful in other health and care settings such as dental practices and care homes.Topics for this guidance were derived from initial discussions of the Working Party and specific review questions were developed in accordance with the population–exposure–comparator–outcome (PECO) framework for investigating the likelihood of developing a certain condition after an exposure event. To prepare the recommendations, the Working Party collectively reviewed relevant evidence from published, preprint and grey literature sources. The processes and methods used were in accordance with the National Institute for Health and Care Excellence (NICE) manual for developing guidelines  . The proThe Working Party included infectious diseases, microbiology and virology clinicians, academic infection prevention and control experts, systematic reviewers, and a lay representative.Any healthcare practitioner, manager or policy maker may use this guidance and adapt it for their use. It is anticipated that most users will be clinical staff and infection prevention and control teams. Some aspects of this guidance might also be beneficial to patients, their families/carers, and the public.To provide advice rapidly, the guidance is being produced as two separate articles, each addressing a different review question. Each article will comprise an introduction, a summary of the evidence, and recommendations graded according to the available evidence.The guidance will be considered for update within 1 year of publication to determine whether new evidence exists that would require a change in the recommendations.The aim of the guidance is to evaluate evidence for presymptomatic, asymptomatic and post-symptomatic transmission of SARS-CoV-2 with the intention of preventing transmission in hospitals and other health and care settings.As noted above, the processes and methods used to produce this Working Party Report were aligned with those described in the first Working Party Report . Topics Two electronic databases (MEDLINE and Embase) were searched for articles published between 1 January and 29 May 2020. Search terms were constructed using medical subject headings (MeSH) and free-text terms  or asymptomatic transmission (in which the index case never developed symptoms or signs of infection) was undertaken to aid presentation and interpretation of the evidence.Many of the cluster/outbreak investigations permitted only a categorical  assessment of the credibility of transmission by presymptomatic or asymptomatic people . Other cluster/outbreak investigations allowed calculation of an attack rate  and an associated confidence interval (CI). Stratification of the evidence from cluster/outbreak studies according to the time at which contacts were exposed to SARS-CoV-2 relative to the index case acquiring the virus  was also undertaken.Where cluster/outbreak studies reported the use of personal protective equipment (PPE) this was noted to aid interpretation of the evidence.The possibility of identifying comparative epidemiological studies relevant to the review question had not been anticipated because the pandemic was associated with a novel disease and was still in its early stages when the evidence review was initiated. However, several such studies were identified and included as noted above. For these epidemiological studies  that reported  a measure of transmission risk according to the index case’s symptom status at the time of exposure of their close contacts, the convention of expressing risks based on exposure to people with fewer symptoms compared to risks based on exposure to people with more symptoms was applied where possible.Mathematical modelling studies were included in the review only where they distinguished between transmission risks according to the index case’s symptom status during exposure of their close contacts.Included epidemiological studies were appraised for quality using checklists recommended in the NICE guidelines manual . Criticahttps://www.gradeworkinggroup.org/ for details). The resulting GRADE tables are presented in Additional file .Feedback on the draft guidance was received from the HIS Guidelines Committee and through rapid consultation with relevant stakeholders. The draft report was placed on the HIS website for 10 working days along with the HIS standard response form, including a conflict-of-interest disclosure form. The availability of the draft guidance was communicated via email and social media. Stakeholders were invited to comment on format, content, local applicability, patient acceptability and recommendations. The Working Party reviewed stakeholder comments, and collectively agreed revisions in response to the comments  , 26, 41 The reported cluster/outbreak investigations focused on potential transmission of SARS-CoV-2 in both community and nosocomial settings  is reflected in the evidence tables for the cluster/outbreak studies  , 35, andThree of the mathematical modelling studies included in the review used adaptations of the susceptible–exposed–infected–recovered (SEIR) compartmental modelling framework to model transmission dynamics in hypothetical populations , 29, 45.For each type of study for which it was possible to produce an overall GRADE rating of the quality of the evidence the rating applied was very low  and imprecision associated with no CIs or other measures of precision being reported . Among those cluster/outbreak investigations that evaluated the risk of asymptomatic transmission, several had evidence downgraded for indirectness because the definition of an asymptomatic infection included having mild symptoms (such as a pre-existing cough that might or might not have been associated with or exacerbated by SARS-CoV-2 infection), or signs of infection on a computerised tomography (CT) scan of the chest. See Additional file Another aspect of the evidence from the cluster/outbreak investigations was the use of PPE as recorded in the evidence tables for these studies  relative measures of transmissibility according to the index case’s symptom status during exposure of their close contacts, a frequently occurring reason for downgrading the quality of the evidence was risk of bias associated with potential confounding factors  not being accounted for in the design or analysis of the study. Another common reason for downgrading the quality of evidence from such studies was that CIs for estimated effects crossed default thresholds for defining imprecision according to the GRADE approach. See Additional file The quality of the evidence from the mathematical modelling studies included in the review was downgraded for indirectness in several cases because relative measures of transmissibility according to the index case’s symptom status during exposure of their close contacts were not wholly aligned with the symptom statuses of interest to the Working Party . In one such study, asymptomatic infections and mildly symptomatic infections were grouped together . AnotherThere was strong evidence from 36 cluster/outbreak investigations  , 57, 58 There was moderate evidence from seven cluster/outbreak investigations , 42, 55 There was weak evidence from two further cluster/outbreak investigations , 37 regaThere was moderate evidence from four epidemiological studies reported across six articles , 52, 56 There was inconsistent evidence from four mathematical modelling studies , 29, 45 There was weak evidence from one mathematical modelling study  regardinNo evidence was identified regarding the possibility of SARS-CoV-2 being transmitted by post-symptomatic people.The Working Party’s interest focused on whether transmission occurs as a result of presymptomatic, asymptomatic or post-symptomatic SARS-CoV-2 infection. For the most part, this was evaluated through consideration of absolute risks of transmission. At the start of the evidence review process, it was not anticipated that relative risks of transmission based on the symptom status of an index case would have been examined (because the pandemic was in its early stages and research was just starting to be published). However, it became evident when sifting the results of the systematic literature searches that some studies had investigated relative risks of transmission and this evidence was eligible for inclusion according to the review protocol.The evidence from the cluster/outbreak investigations and epidemiological studies providing estimates of relative risks of transmission based on an index case’s symptom status during exposure of their close contacts was assessed for quality using the GRADE framework. All of the evidence from these studies was classified as being of very low quality. Recurring reasons for downgrading the evidence included: risk of bias  not being accounted for in the case of epidemiological studies providing relative risks of transmission based on the index case’s symptom status during exposure of close contacts); imprecision due to CIs for effect estimates crossing predefined thresholds or being unavailable; and indirectness . The overall assessment of the evidence as being of very low quality did not, however, prevent the Working Party reaching conclusions about characteristics of SARS-CoV-2 transmission and making recommendations for practice (see below).The evidence from the mathematical modelling studies included in the review could not be fully assessed using the GRADE framework, but some GRADE domains were applicable, for example, inconsistency and indirectness. A recurring reason for downgrading the evidence from these studies was indirectness due to relative measures of transmissibility according to an index case’s symptom status during exposure of close contacts not being fully aligned with symptom statuses of interest to the Working Party .confirmedpresymptomatic transmission  is probable.asymptomatic transmission (meaning that an index case never develops symptoms or signs of infection) is Having considered the evidence, the Working Party concluded that:There was uncertainty regarding the evidence related to asymptomatic transmission, with the Working Party noting that a lack of awareness of symptoms or suppressed symptoms  could not be distinguished from a complete absence of symptoms in the reported investigations. The Working Party recognised the potential for subclinical or pauci-symptomatic infection while emphasising that truly asymptomatic infection or carriage of SARS-CoV-2 occurs and transmission is to be expected .The Working Party recognised that the list of symptoms suggesting COVID-19 had expanded during the pandemic, reflecting growing knowledge of the condition. The evidence review and synthesis involved extracting any information about symptoms reported by the study investigators, although it was acknowledged that people’s perceptions of symptoms differ and this could influence the types of symptoms reported. The Working Party emphasised the importance of clarity in defining and reporting symptoms in future research related to COVID-19.The settings in which presymptomatic or asymptomatic transmission was demonstrated mirrored those reported in the first of the pair of guidance articles in which routes of transmission, regardless of the symptom status of the index case, were explored . In partThe Working Party agreed that from the perspective of preventing transmission by people without symptoms, it is immaterial whether they later develop symptoms. The recommendations were therefore phrased in terms of people without symptoms rather than using the terms presymptomatic and asymptomatic. The Working Party anticipated that this phrasing would also make the recommendations more meaningful to the public.The benefits of preventing transmission of SARS-CoV-2 by people without symptoms include the prevention of ill health due to COVID-19 among their close contacts and the prevention of onward transmission to ever greater numbers of people. Possible harms associated with actions intended to prevent transmission of SARS-CoV-2  arise through restriction of personal freedoms and a need to modify behaviours with potential adverse consequences in terms of, for example, mental health and wellbeing. These benefits and harms apply to healthcare workers, patients and their families/carers, and the public. On balance, the Working Party recognised that since anyone might carry the virus without knowing it, or be infected without having noticeable symptoms, the recommendations should reinforce the importance of adhering to existing legislation and guidance intended to reduce the risk of transmission of SARS-CoV-2 in the general population.The Working Party noted that the evidence regarding relative risks of transmission according to symptom status suggested that presymptomatic infections are less transmissible than are symptomatic infections, and that asymptomatic infections are less transmissible than are presymptomatic infections. The Working Party was aware that the viral load associated with asymptomatic and pauci-symptomatic infections is typically lower than that associated with symptomatic infection , lendingAlthough the evidence from the mathematical modelling studies was regarded as indirect, the Working Party noted the reported differences in asymptomatic transmission rates in indoor environments under different ventilation scenarios. This prompted the Working Party to emphasise the importance of ventilation in enclosed spaces in the recommendations.The Working Party was acutely aware that the development of the guidance was occurring during an evolving pandemic. When formulating the recommendations, the Working Party recognised the possibility of reinfection in people who previously had COVID-19 , the emeThe likelihood of post-symptomatic transmission  could not be assessed because of an absence of evidence. The Working Party agreed that post-symptomatic transmission should be prioritised as an area for further research.The Working Party did not undertake a detailed economic analysis because the recommendations focused on raising awareness of the possibility of presymptomatic and asymptomatic transmission of SARS-CoV-2 and reinforcing existing legislation and guidance aimed at preventing transmission. However, the Working Party considered costs and resource use from the perspective of health and care systems and identified that costs associated with transmission that is not prevented include the costs of managing COVID-19 in infected patients and the costs of needing additional resources such as PPE. Considerations related to the value of time as a resource included the time taken to don and doff PPE and time away from work for healthcare workers who are unwell or required to self-isolate. Taken together, these considerations emphasise increased pressure on healthcare systems when COVID-19 is prevalent. The Working Party recognised potential inconvenience and possible adverse consequences  of implementing measures such as social distancing and using PPE. The Working Party also recognised that the cost effectiveness of preventing transmission would be greater in aspects of healthcare focusing on people more vulnerable to COVID-19.when a person who has acquired SARS-CoV-2 becomes infectious andhow long infectivity lasts in the absence of symptoms.As outlined above, the Working Party highlighted several areas for future research. These included consideration of:While the evidence available to the Working Party demonstrated presymptomatic transmission within 7 days of an index case acquiring the virus, later transmission could not be ruled out. Moreover, the available evidence did not permit a detailed analysis of infectivity during the first 7 days since acquiring the virus, which was of interest to the Working Party and could form part of future research. The Working Party also highlighted potential seasonality in transmission rates, and indoor versus outdoor transmission, as areas to explore in future research.The Working Party discussed the relevance and possible consequences of lung damage revealed by CT scans in people who did not report symptoms. The Working Party questioned whether such features might have longer-term consequences for a person who although infected has no noticeable symptoms and recommended this as an area for future research.The Working Party made several observations regarding the quality of the evidence identified in the review. While the importance of rapid evaluation during a pandemic caused by a novel disease such as COVID-19 was appreciated, the value in ensuring robust and efficient research activity was also recognised. The Working Party agreed that this value could be promoted by avoiding duplication and repetition in data collection, analysis, and reporting, and acknowledged the time needed to ensure high quality research outputs. The Working Party highlighted the desirability of concerted global action to coordinate research activity and formalised data gathering and sharing in the event of future pandemics caused by novel diseases. The Working Party acknowledged that some of the areas recommended for future research might already have been addressed in primary studies or systematic reviews published after the searches for the evidence review had been completed. Although the Working Party had considered updating the review to take account of more recently published evidence, the rate at which additional evidence was being published prohibited such an approach. For example, rerunning the MEDLINE and Embase searches in April 2021 indicated that approximately 20,000 further articles would need to be considered; it was, therefore, not feasible to undertake a timely and systematic update of the review using the original search terms. The Working Party emphasised that the research recommendations were intended to build on the evidence review and allow the guidance to be refined or extended, preferably with reference to evidence of higher quality and allowing more focused or nuanced consideration of SARS-CoV-2 transmission dynamics. By November 2021, rerunning the MEDLINE and Embase searches resulted in an additional 30,000 articles, which when filtered to select records containing the phrase ‘systematic review’ in the title, abstract or keywords identified nearly 600 articles. Among these systematic reviews, a handful investigated relative transmissibility of presymptomatic, asymptomatic and symptomatic infections –69; howeThe Working Party noted that evidence included in the review suggested that using PPE (such as face masks or coverings) reduced the risk of transmission of SARS-CoV-2 by people with presymptomatic or asymptomatic infection. The current evidence review was not designed to explore this systematically, whereas the first of the pair of guidance articles  includespeople without noticeable symptoms may transmit the SARS-CoV-2 virus to other peopletransmission of SARS-CoV-2 from people without symptoms may occur in all settings in which people are in close proximityhowever, it is likely that the risk of transmission of SARS-CoV-2 is greater from people who have symptoms compared with those who do not.Even in the absence of symptoms, adhere to legislation and guidance regarding measures to reduce the risk of transmission of SARS-CoV-2 .Be aware that the future transmissibility of SARS-CoV-2 from people carrying the virus without symptoms might depend on the:nature of further waves or outbreaks of COVID-19emergence and circulation of SARS-CoV-2 variants of concernpotential for people who have had COVID-19 previously to be reinfectedeffectiveness of available vaccines, including the longevity of immunity they confer.Be aware that:Be aware that it is not yet known to what extent or for how long people recovering from acute infection can transmit the SARS-CoV-2 virus to other people.Based on the evidence review, which included research published to the end of May 2020, the Working Party considered presymptomatic transmission of SARS-CoV-2 to be confirmed, and asymptomatic transmission to be probable. The evidence for these forms of transmission was sufficient for the Working Party to formulate several strong recommendations with the intention of raising awareness in health and care settings of the potential for transmission in the absence of symptoms. The recommendations were intended to reinforce existing legislation and guidance specifying measures for reducing the risk of transmission from people who have no noticeable symptoms. The Working Party formulated recommendations for future research to address areas of uncertainty, such as the relative transmissibility of presymptomatic, asymptomatic and symptomatic infections, the period of infectivity in people without symptoms, and the possibility of transmission in the post-symptomatic period. The Working Party emphasised the importance of good quality design, analysis and reporting of research studies even in pandemic situations. The Working Party also highlighted the desirability of concerted action to coordinate research activity and share outputs effectively.The rationale for the following research recommendations is presented in "What is the relative transmissibility of SARS-CoV-2 from people with presymptomatic, asymptomatic and symptomatic infection, and how does transmission correlate with quantification of viral shedding?How long after acquiring SARS-CoV-2 do people without symptoms become infectious and how long does infectivity last?To what extent or for how long can people who have acquired SARS-CoV-2 and are post-symptomatic transmit the virus to other people?What are the long-term consequences of lung damage associated with SARS-CoV-2 infection in people who do not report symptoms?What impact do reinfection, variants of concern, and immunisation programmes have on transmission of SARS-CoV-2?Additional file 1. Working Party Report appendices.


Dirofilaria immitis is an important mosquito-borne nematode, being of both veterinary and public health concern. The typical final host is represented by the domestic dog, Canis familiaris, but it is able to infect a large variety of mammalian species. During the present study (March 2016–February 2022), a total of 459 wild carnivore carcasses belonging to 17 species, from Romania, were evaluated for the presence of adult D. immitis by necropsy. Overall, 20 animals (4.36%) were positive: twelve golden jackals, C. aureus (19.05%); four red foxes, Vulpes vulpes (6.67%); one raccoon dog, Nyctereutes procyonoides; two wild cats, Felis silvestris (4.65%); and one European badger, Meles meles (0.87%). This study provides further evidence of the occurrence of the canine heartworm, D. immitis, in Romania, expanding the known host spectrum, reports a new host species for this parasite, the European badger, and a new host for Europe, the raccoon dog. Dirofilaria  is important from a veterinary and public health perspective. The two main species, D. immitis and D. repens, causative agents of canine heartworm disease and subcutaneous dirofilariosis, respectively, have a wide distribution and are able to infect a large variety of domestic and wild carnivorous host species [D. immitis is also of major veterinary concern, as it is associated with a debilitating and eventually fatal disease in infected carnivores [Culex, Aedes, Ochlerotatus, and Anopheles may successfully transmit D. immitis, the main natural vectors are represented by Aedes vexans, A. albopictus, and the Culex pipiens complex [Among mosquito-borne nematodes, the genus  species ,2. Furthrnivores . Althoug complex ,4.Canis familiaris, but the infection was reported in over other 30 mammalian species, including several wild canids, domestic and wild felids, mustelids, ursids, ailurids, pinnipeds, monkeys, rodents, and ungulates [Canis aureus), and grey wolves (C. lupus), seem to have an important epidemiological role in the maintenance and transmission of this parasite [The most frequently infected final host and the most competent reservoir is the domestic dog, ngulates ,6,7. Howngulates ,8. Conveparasite .D. immitis detected in dogs originating from most regions of the country [Dirofilaria spp. infections were detected by necropsy and/or molecular tools also in six species of wild carnivores throughout the country [Romania is endemic for canine cardiac dirofilariosis, with  country ,10,11. F country ,13.D. immitis, in wild carnivores in Romania, to report new host-parasite associations and evaluate their potential reservoir role.The aims of the present study were to provide an update on the distribution of the heartworm, D. immitis in the right ventricle or the pulmonary artery, most of which originated from the southern region of the country  examined animals harboured at least one adult  country .Canis aureus, followed by red foxes, Vulpes vulpes. A single raccoon dog, Nyctereutes procyonoides, was examined and the animal was found to be positive. Within the felids, two wild cats, Felis silvestris , were positive. Of all mustelids, one European badger, Meles meles , was infected.Among canids, the overall prevalence of infection was 12.88% (95% CI 7.68–19.82), with the highest value recorded for golden jackals, The worm burden ranged between one and seven nematodes/animal, with an average value of 2.35, and a median of 1. The individual details of all the infected animals are presented in The present study represents an extension of previous work ,13, and C. aureus (19.05%). This value is similar to the ones reported from our previous studies [V. vulpes, was also high (6.67%), only one out of the four positive individuals harboured nematodes of both sexes, and was positive also for microfilariae , 2 wild cats were positive, harbouring 1 adult male nematode each. Both cases have been published separately [Data regarding heartworm infection in wild felids is still scarce. Out of 48 examined carcasses  were performed using EpiInfo 7 software  and the map was generated using QGIS 3.4 software.Dirofilaria immitis, in Romania, expanding the known host spectrum of this parasite. We report a new host for Europe, the raccoon dog, Nyctereutes procyonoides, and a new host-parasite association for the European badger, Meles meles.The present study provides further evidence of the occurrence of the canine heartworm,


Since conflicting evidence from systematic reviews and meta-analyses (SRs/MAs) on the effectiveness of acupuncture in the treatment of postpartum depression is observed. To systematically collate, appraise and synthesize the evidence from these SRs/MAs, an overview will be performed, and this study is an overview protocol.Eight databases will be searched: Medicine, Web of science, Cochrane Library, Embase, China National Knowledge Infrastructure, SinoMed, VIP, and Wanfang Data. SRs/MAs of acupuncture on postpartum depression will be included. Literature screening, data extraction, and evaluation of the review quality will be performed by 2 independent reviewers. The methodological quality, reporting quality, and evidence quality will be assessed using the assessment of multiple systematic reviews-2 tool, the preferred reporting items for systematic reviews and meta-analyses checklists, and the grading of recommendations, assessment, development, and evaluation system, respectively. The results will be presented in the context of the topic and the objects of the overview. This study will help bridge the implementation gap between clinical evidence and its translation in clinical application, identify flaws in research and guide future high-quality study. Typical PPD occurs within 6 weeks postpartum and recovers on its own within 3 to 6 months, while severe cases can persist for 1 to 2 years. The prevalence of PPD in new mothers is as high as 16%, and the recurrence rate of PPD in the second pregnancy reaches 30%.,4 PPD is characterized by depressed mood, loss of interest, sleep disturbances, psychomotor agitation or retardation, feelings of worthlessness, and even suicidal thoughts and behaviors in severe cases. Given the high prevalence and negative impact of PPD, effective treatment is clearly important.Postpartum depression (PPD) is a type of mood disorder associated with childbirth, the onset of which usually begins between the first day and 4months after delivery. However, these treatments vary in efficacy,–9 are costly, and adverse events are common.,12 Therefore, more effective and safer treatments for PPD are still needed. Alternative treatment strategies for PPD are receiving increasing attention, and acupuncture, in particular, has gained acceptance due to its effectiveness and safety.Treatment of PPD includes pharmacotherapy, psychotherapy, or both, which is consistent with the treatment recommended in guidelines for major depression. There is a gap between the use of evidence and its practical implementation in real-world dynamics. In addition, to ensure the standardization of evidence sources, measurement tools such as assessment of multiple SRs-2, preferred reporting items for systematic reviews and meta-analyses, and grading of recommendations, assessment, development, and evaluation system were released in 2007, 2009, and 2004, respectively. Where multiple SRs/MAs SRs/MAs are published for overlapping topics in a relatively short time frame, an overview is needed to systematically collate, evaluate, and synthesize the evidence from these SRs/MAs. Therefore, we will provide an overview of SRs/MAs on acupuncture for PPD.A number of systematic reviews (SRs)/meta-analyses (MAs) have reported on the efficacy of acupuncture for PPD, but their findings are inconsistent and the credibility of the evidence is unclear. SR/MA is considered the source of the highest level of evidence. However, not all SRs/MAs can provide reliable evidence, and low quality SRs/MAs may instead mislead decision makers.Patients and public will not be involved. This protocol has been registered on the PROSPERO (CRD42021291289).The method used for this protocol will follow the Cochrane Handbook.SRs/MAs enrolled random control trails investigating the therapeutic effects of acupuncture for PPD. No language constraints are placed on this study.Participants are diagnosed with PPD by any recognized guideline, regardless of age, course of the disease, sex, or race.Intervention of interest is the use of acupuncture in PPD, acupuncture group could be with or without conventional medication, with conventional medication as the control.The outcome measures are the following: the Hamilton Rating Scale for Depression, Edinburgh Postnatal Depression Scale, effective rate, estradiol levelsWe will search Medicine, Web of science, Cochrane Library, Embase, China National Knowledge Infrastructure, SinoMed, VIP, and Wanfang. References to the SRs under this topic will also be screened to track relevant studies. The medical subject headings and keywords related to acupuncture, PPD, and meta-analysis comprise the search strategy. Search strategies for each database can be found in Supplementary A.The search results will be entered into NoteExpress to identify and remove duplicates. Literature screening will be performed by 2 independent reviewers Fig. . Any disData of the included studies will be extracted by 2 independent reviewers. The first author, year of publication, country, enrolled trails, tools for quality evaluation, interventions, comparisons, data synthesis methods, outcome measures, and main results will be extracted. Any disagreement will be adjudicated by an experienced third reviewer. will be applied to assess the methodological quality of the included studies by 2 independent reviewers. Any disagreement will be adjudicated by an experienced third reviewer. Furthermore, the reporting quality of the included SRs/MAs will be assessed using preferred reporting items for systematic reviews and meta-analyses checklists. Finally, the evidence quality will be assessed using the grading of recommendations, assessment, development, and evaluation system.The assessment of multiple SRs-2 tool In SRs/MAs included in the overview, data from individual trials may have be pooled multiple times, therefore, meta-analysis will not be conducted in this study, but a narrative synthesis of the findings from the included reviews will be presented. For dichotomous data, findings from included reviews will be shown as risk ratio or odds ratio; for continuous data, as weighted mean difference or standard mean difference with 95% confidence intervals. The results of the quality assessment will be shown in tabular and graphical form.A descriptive analysis will be carried out in an overview.In China, acupuncture has been widely used for the clinical treatment of PPD. However, the researchers emphasize that this therapy is still not fully implemented in a real-world context. There is a gap between evidence-based clinical implementation of acupuncture and its actual implementation in real-world dynamics. High quality SRs/MAs can provide reliable evidence, while low quality SRs/MAs may instead mislead decision makers. Thus, Evidence of uneven quality leads to a gap between its use and its practical implementation in real-world dynamics. In recent years, a growing body of SRs/MAs based on randomized clinical trials has begun to analyze the evidence of acupuncture in the treatment of PPD. However, their results are not completely consistent, and their quality is uneven. These issues all compromise the development of clinical protocols and health decisions. Considering this status quo, an overview of SR/MA of this field is needed. The ultimate goal of this review is to provide a comprehensive evaluation of current evidence on multiple identical topics, to provide more focused high-quality evidence to evidence users, and to identify key flaws in evidence use. This protocol describes the detailed study design for performing an overview of SRs/MAs on acupuncture for PPD. Publication of this protocol will ensure reproducibility of the overview and benefit from feedback from peer expert review.SR/MA are considered the gold standard for assessing the efficacy of clinical interventions, but the evidence derived from them is currently facing challenges due to the various risks of bias generated during the formation of evidence by SRs/MAs.Conceptualization: Fan Bu, Yonghou ZhaoMethodology: Jianbo Chai, Bing BaiSupervision: Wanyu WangValidation: Jianbo Chai, Bing BaiWriting - original draft: Fan BuWriting - review & editing: Yonghou Zhao


This study evaluates practices of infection control in the NICU as compared with the available literature. We aimed to assess providers’ awareness of their institutional policies, how strongly they believed in those policies, the correlation between institution size and policies adopted, years of experience and belief in a policy’s efficacy, and methods employed in the existing literature. An IRB-approved survey was distributed to members of the AAP Neonatal Section. A systematic review of the literature provided the domains of the survey questions. Data was analyzed as appropriate. A total of 364 providers responded. While larger NICUs were more likely to have policies, their providers are less likely to know them. When a policy is in place and it is known, providers believe in the effectiveness of that policy suggesting consensus or, at its worst, groupthink. Ultimately, practice across the US is non-uniform and policies are not always consistent with best available literature. The strength of available literature is adequate enough to provide grade B recommendations in many aspects of infection prevention. A more standardized approach to infection prevention in the NICU would be beneficial and is needed. Neonatal intensive care unit (NICU) admissions have been on the rise in the last two decades . BetweenDespite advances in neonatal medicine, infants requiring neonatal intensive care continue to incur substantial morbidity and mortality ,3. NosocBecause of this burden, multiple interventions are routinely employed to reduce this risk. While scientific evidence is well established in support of the effectiveness of a number of those interventions, no national practice guidelines exist for standard, everyday care and interaction with neonates in the NICU, and compliance rates differ significantly among different facilities . Such vaAn IRB-approved survey was distributed to members of Section of Neonatal Perinatal Medicine of the American Academy of Pediatrics. All subjects gave their informed consent for inclusion before they participated in the study. A systematic review of the literature provided the domains of the survey questions (illustrated in (a)Report on practice patterns and practice changing interventions on hand hygiene compliance within the NICU setting.(b)Association between gloves, gowns, cellphones, accessories, and attire with HAI in the NICU.(c)Type of water utilized for neonatal bathing and infection risk. Sink disinfection practice and infection risk.(d)Siblings care policy and visiting restrictions and its impact on HAI.A literature search was performed on MEDLINE and PUBMED. The search was performed on 18 February 2019 and PRISMA guidelines were followed. Keywords are included in p = 0.0002), their providers were less likely to know them . Across all categories, providers tended to agree more with the effectiveness of each intervention when it is an established policy at their institution (p < 0.001).Of the 3500 surveys that were sent out, 364 were returned, yielding a response rate of 10%. The experience of respondents was 0–10 years (37%), 11–20 years (18%), and 21+ years (45%). The mean number of NICU beds was 47 beds and the most common grouping was 41–80 beds . Twenty-A.Hand Hygiene Compliance:Hand hygiene continues to be one of the most effective and economical methods of infection control and prevention. The large body of evidence that supports its efficacy is well established. Compliance with hand hygiene practice is a major area of focus for all hospitals. Reported data from across practice sites and specialties ranges from 5% to 89%, representing an average compliance of 40% . In the When reviewing the association between type of hygienic compound and compliance, the use of alcohol-based rub was associated with improved compliance . ImproveWhen looking at demographic variances, studies have shown that female providers are more compliant than their male counterparts and nurses are more compliant than physicians ,26,27. FMultiple interventions have been employed and studied to increase hand hygiene either directly by measuring compliance or indirectly by measuring the rates of communicable nosocomial infections . The tabNICUs with low rates of acquired infections have employed immediate and individual feedback to health care workers on their hand hygiene performance and collective feedback to the neonatal ICU on acquired infection rates, while NICUs with high rates of acquired infections only communicated when there was a problem with the acquired infection rate and did not address improper individual hand-washing performance .Unfortunately, studies that report long-term compliance after the individual implementation of the above-described techniques showed the improvement rate often returned to baseline ,44. ThisB.Gloves and Gowns:Using clean, non-sterile gloves in addition to hand hygiene has been shown to decrease neonatal infections in three available studies ,46,47. ITo our knowledge, no study has been performed on the influence of gloves limiting the providers ability to interact with the surrounding environment, specifically cell phones, between hygiene application and patient interaction. This lack of interaction with fomites may account for the reported decrease in rates of infection. Additionally, all three studies used gloves after hand hygiene versus simply donning gloves alone. Any implementation of gloved examination must include hand hygiene prior to donning gloves.While gloves seem to be effective in reducing the risk of neonatal infections, research on gowning in the NICU over the past several decades has not been able to demonstrate a benefit ,50,51,52There are two goals for any intervention that is directed toward hand hygiene: (1) increasing compliance and (2) attaining sustainability. p < 0.01). Additionally, as was the case with hand hygiene practice, if the intervention is an institutional policy, providers are more likely to believe in the efficacy.In our survey, 46% of providers reported that non-sterile gloves are standard practice in their neonatal ICUs and less than 5% reported the mandatory use of gowns during general patient care. The level of support and belief in those interventions mirrors their utilization with 52% of the providers supporting non-sterile glove use and only 7% for gowns. Providers were asked to rate their opinion of each intervention on the Likert scale of 1–5 with 1 being strongly disagree and 5 being strongly agree. Results are summarized in Health care providers have expressed concern that white coats may serve as vectors for infection transmission  with stuA limited number of studies exist on the role of health care providers’ ties and sleeves in hospital acquired infections, and none are specific to the context of the NICU. Weber et al. found that while unsecured ties were associated with an increase in transmission of bacteria, there was no relationship between the length of sleeves and rate of transmission . FollowiA recent systematic review including adult patients from 2016 evaluating 72 studies showed large variation in the rates of contamination of personal clothing and white coats in the hospital setting. The rates of contamination in general were between 0 and 32% for MRSA and Gram-negative rods. Of those studies, four studies evaluated for possible connection between healthcare personnel contaminants and clinical isolates with no clear link identified .Our survey reviewed policies, opinions and general trends in relation to attire and p < 0.001 for both).Providers with less experience (0–5 years) were more likely to believe in those policies than providers with +20 years of experience  to less restrictive measures like pre-entry disinfection (40%), hand disinfection after each use, and placement of mobile devices in plastic bags (40%). Some institutions had overlapping policies and there was a large variation among policies directed towards staff and family members. Seventy percent of respondents had a no jewelry policy in the ICU.We identified only one study that evaluated the difference in infection rates between private rooms and an open layout in the NICU. They found no difference in rates of methicillin-resistant Staphylococcus aureus colonization, late-onset sepsis, or mortality .P. aeruginosa revealed that changing from tap water to sterile water for bathing showed a reduction in the incidence of neonatal bacterial infection [Some NICUs have used sterile water and self-disinfecting sinks as a method to decrease Pseudomonas aeruginosa colonization and infection. A single-center study in a NICU experiencing colonization of nfection . A studynfection . Cost drnfection .Our survey showed about 50% of providers were unfamiliar with the policies in their NICU for water sterilization. Of those who were familiar with the policies for environment and facilities, 10% reported strict use of sterile water for bathing and 4% reported the use of self-disinfecting sinks, with the majority being neutral about their efficacy.While almost every NICU offers full visiting privileges for parents of infant patients, a wide range of visiting policies exist for siblings and other family members . The basEighty percent of the surveyed providers reported restrictions for young relatives’ access during specific seasons. Seventy-three percent reported that parents are required to perform hand hygiene between siblings’ interactions and sharing toys between siblings is reported to be prohibited by 55% of responders. The majority of our respondents believe in the efficacy of those measures.The subject of infection control in NICUs has been the focus of many studies over decades. Those studies have had different outcome parameters, different methodology and have been carried out in different institutional settings. Although the response rate was limited to 10% of the applicable population and thus subject to response bias, our survey confirmed non-uniform practice and found there is disagreement on the effectiveness of each practice among practitioners with different years of experience, hospital setting, and institutional policies. Those two factors limit the ability to draw generalizable conclusions from the reviewed evidence that would be applicable to each and every NICU. Rather, our goal is to provide a framework of organized data that should be utilized to guide policy development at NICU-containing institutions. We outline our recommendations below with the strength of recommendation and level of evidence supporting it.A.High certainty of the evidence: The intervention leads to a large reduction/increase in outcome.B.Moderate certainty of the evidence: The intervention leads to a moderate reduction/increase in outcome.C.Low certainty of the evidence: The intervention leads to a small reduction/increase in outcome.Grading and recommendations were based on the GRADE approach in the Cochrane Handbook for Systematic Reviews of Interventions  and took1.High quality prospective cohort study with adequate power or systematic review of these studies.2.Lesser quality prospective cohort, retrospective cohort study, or systematic review of these studies.3.Case–control study or systematic review of these studies.4.Case series.5.Expert opinion; case report; clinical examples.Levels of Evidence were based on the Levels of Evidence for Prognostic Studies  and are Several studies support a variety of methods to increase compliance and achieve sustainability with hand hygiene practice. Since there is not a single ‘silver bullet’, we recommend that each institution tailors its efforts and policies to its cultural trends and providers’ perception. Our survey did show a statistically significant association between institutional policies and providers’ belief in their efficacy. Specifically, for all above interventions, providers are more likely to believe in the efficacy of a policy if it is instituted at their health care system. That argues that providers are open and receptive to institutional directions and as such would be inclined to adhere to them. It is also possible that the providers surveyed were instrumental in the development of each institution’s policies and, therefore, they believe in their efficacy and, therefore, instituted those policies. Conforming those policies to fit into the dynamics of each institution is also paramount. In an academic facility with high provider turnover (rotating residents and other learners), providers and learners might be better served with videos above sinks and individual on point feedback instead of email reminders and grand round presentations.The only way to achieve sustainability is by periodic interventions and ongoing reassessments. Without those, any intervention aimed at increasing compliance has been shown to lack a long-term effect.−The use of alcohol-based hand hygiene as opposed to soap-based hygiene to improve compliance.−Structured periodic feedback systems to sustain long term hand hygiene compliance.−A multimodal approach to spreading awareness and improving compliance for hand hygiene; we recommend incorporating visual reminders and individual feedback.−The use of non-sterile gloves in addition to hand hygiene during the care of preterm infants and during the RSV season for all infants.We make a grade B recommendation based on level 2 evidence for:Based on level 1 evidence that failed to show improved outcomes with use of non-sterile gowns, we make a grade A recommendation that there is no benefit for their routine use.−We make a grade C recommendation based on level 3 evidence for prohibition of unsecured ties in the NICU to decrease infection transmission.−The current level 2 evidence does not support prohibition of white coats or a bare elbow policy to decrease infection transmission in the NICUs. We recommend resource utilization to reinforce other evidence proven interventions.While the evidence to support attire policies for infection control is lacking, most institutions employ policies related to attire and a large proportion of providers believe in their efficacy. Multiple studies have shown white coats to be colonized with pathogens classically associated with HAI, but the ones investigating an association between attire and HAI failed to establish a link. The literature has also shown that ‘bare elbows’ did not decrease the number of pathogens on providers’ hands and did not improve the practice of hand washing ,59,60. IWhile the evidence supports the potential danger of fomites as vectors for transmission, causality in the NICU setting has not been established. This is reflected in the wide variation of polices, or lack thereof, across different neonatal ICU settings. Given established causality between stethoscopes and infection transmission in the adult based practice, it is reasonable to deploy policies that restrict use of cellphones in the ICU and regulate stethoscope disinfection. A trend that is being seen in both the adult and neonatal setting is the use of disposable stethoscopes to limit infection transmission. It is not clear, however, whether they are beneficial and to what extent lower quality stethoscopes might jeopardize the sensitivity of auscultation.The published evidence on accessories is observational and weak. No study reviews outcomes and the endpoints and none is powered to establish causality—particularly in the NICU setting. It does however suggest that accessories can negatively impact proper hand hygiene and that cellphones are highly colonized with bacteria.−Disinfecting stethoscopes between patient encounters.−Instituting policies that regulate the use of cellphones in the NICU. Not enough data exists to recommend prohibiting or restricting their use. However, hygiene is recommended after each use.We make a grade C recommendation based on level 3 evidence for:We make a grade B recommendation for removal of accessories upon entry to the NICU based on level 3 evidence and their established interference with proper hand hygiene.−We make a grade B recommendation based on level 2 evidence in favor of using sterile water for bathing and self-disinfecting sinks in neonatal ICU settings with high rates of tap water pseudomonal colonization.Not enough evidence exists to recommend a specific ratio of beds to hand-sanitizing stations  for infection prevention, but the evidence shows a trend towards higher rates of HAI with higher ratios. It appears that the majority of providers are either unaware of their hospital policies in regard to water sterilization or indifferent about them. This is consistent with a lack of strong evidence supporting its use.−We make a grade C recommendation based on level 3 evidence for restricting siblings’ access to NICU during RSV seasons.Practice and belief of routine infection prevention in the NICU in the United States is non-uniform and non-conforming to the existing evidence for nosocomial infection prevention. While the literature does not have consistent measures or outcomes, there is enough literature to inform practitioners in development of guidelines. A more standardized approach to infection prevention in the NICU would be beneficial and is needed. We acknowledge the differences in implementation may vary based on state policies, third-party providers, and healthcare structures. This standardized approach will allow for all institutions no matter the size, location, or academic affiliation to adhere to the best practices for all NICU patients. This will effectively promote physician recognition of best care practices and thus limit provider-related infection transmission. We hope that this paper will serve as the first step to creating awareness of the variability of the institutional policies, the lack of awareness to some policies, and the potential dangers of not adhering to such policies. Ultimately, we recommend implementing cost effective national guidelines that will effectively reduce infection risk by increasing awareness and adherence to best policies in all NICUs.


In vitro, cholecystectomy-associated secondary BAs also downregulated monocytes chemokines in the THP-1 derived macrophages through activation of the LXRα-linked signaling pathway. The alterations of mucosal microbiota and fecal BAs profiles were found in the PC patients, characterized as increased species with potential immuno-modulating effects and secondary BAs, which were negatively associated with peripheral monocytes levels. Cholecystectomy-induced secondary bile acids accumulation ameliorated colitis through inhibiting monocyte/macrophage recruitment, which might be mediated by the LXRα-related signaling pathway. Cholecystectomy, after 3 months follow-up, has an immune-regulatory role in murine colitis, preliminarily explaining that no increased risk of IBD had been reported in the PC patients, which still warrants further studies.Although post-cholecystectomy (PC) patients usually have gastrointestinal complications and a higher risk of colorectal cancer, previous studies undetected a heightened risk of inflammatory bowel disease. Thus, we tried to investigate cholecystectomy’s impact and pathophysiological mechanism on murine colitis models and clarify the association among fecal bile acids (BAs), mucosal bacterial microbiota, and immune cells in the PC patients. One month or three months after cholecystectomy, mice have induced colitis and tested BAs and fecal microbiota analysis. Next, mice were treated with various cholecystectomy-accumulated bile acids in drinking water for three months before inducing colitis. All 14 paired PC patients and healthy subjects were enrolled for BAs and mucosal microbiota analysis. Cholecystectomy ameliorated DSS-induced murine colitis, accelerated mucosal repair, and induced a significant shifting of fecal microbiota and BAs profiles under colitis status, which featured a higher relative abundance of species involved in BAs metabolism and increased secondary BAs concentrations. Cholecystectomy-associated secondary BAs  also ameliorated DSS-induced colitis and accelerated mucosal repair in mice. Cholecystectomy and specific secondary BAs treatments inhibited monocytes/macrophages recruitment in colitis mice.  In a population-based cohort study, the risk of IBD did not change significantly between the PC patients and healthy controls (0.062% vs 0.051%).6 The mentioned results are limited to illustrating the role of cholecystectomy in the occurrence of IBD.Cholecystectomy is the most common operation in biliary surgery; however, post-cholecystectomy (PC) patients have higher risks of post cholecystectomy syndrome and even colorectal cancer (CRC) in long-term outcomes.7 Cholecystectomy removes this pacemaker from the enterohepatic circulation and continuously secretes the bile into the intestinal tract. Previous studies reported that the bile acids (BAs) pool had a regular or small size while circulating more quickly after cholecystectomy.9 Consequently, increased secondary BAs concentrations were found in the PC patients.10–13 Cholecystectomy also induced intestinal bacterial dysbiosis, such as an abundant increase of Fusobacterium, Bacteroides, and Escherichia, which was associated with gastrointestinal dysfunction.14–18 These results preliminarily implied cholecystectomy changed BAs and microbiota in the colon, but these alterations’ roles had not been widely investigated in IBD patients.The gallbladder, a reservoir for concentrating and storing bile, allows for 20 ~ 30% emptying during fasting, while 70 ~ 80% emptying after a meal.19 Gut microbiota is involved in BAs metabolism and affects the composition of BAs. Inversely, the altered BAs profiles could further reshape bacterial microbiota.19 Accumulating evidence has shown that gut microbiota dysbiosis is one of the primary triggers in IBD, while its molecular mechanisms and mediators have not been fully understood.20 Recently, BAs and their receptors (BARs), which interact between the host and intestinal microbiota, have been investigated in the IBD patients.21 BAs profile of the IBD patients showed increased chenodeoxycholic acid (CDCA) levels and decreased secondary BAs concentrations, such as lithocholic acid (LCA) and deoxycholic acid (DCA).22 Genomic analysis also implied the depletion of BA biotransformation and production capabilities in the microbiota of IBD patients.23As we all know, the interaction between BAs and intestinal microbiota is bidirectional and highly complex in the IBD pathogenesis.22 A recent study found that DCA and LCA alleviated inflammation in murine colitis models.24 It is worth noting that high-dose and long-term DCA treatment could aggravate intestinal inflammation and accelerate the transition from intestinal adenoma to colonic adenocarcinoma.26 Based above, gut microbiota, BAs, and BARs have complex cooperation to affect the IBD development, which still needs further investigations.Furthermore, several mucosal immune cells  can be activated by BAs and exert immune regulatory effects.Cholecystectomy is one of the crucial factors, which affects BAs-gut microbiota crosstalk, and its effect on IBD pathogenesis has not yet been elucidated. Our data indicated that cholecystectomy-induced secondary BAs accumulation ameliorated colitis through inhibiting monocyte/macrophage recruitment, which the LXRα-related signaling pathway might mediate. The underlying mechanism showed BAs regulated inflammatory homeostasis and preliminarily explained that PC patients had not been reported increased IBD risk.17 We performed cholecystectomy murine models and induced colitis at the first or third month after the operation as described above  colonic epithelial cells (+) cells  cells , and hig+) cells  than the10–13 so we tested fecal BAs in murine fecal samples. Although the PC mice showed similar fecal BA profiles with the NC mice after 3 months, we found increased secondary BAs levels in the PC mice after 3 months compared with pre-operative PC mice . Principal coordinate analysis (PCoA) analysis showed the PCDSS and NCDSS mice had similar clusters at the first month after surgery  was not altered after cholecystectomy under normal and inflammatory status (rd month (but not for the 1st month) between the PC and NC mice, and between the PCDSS and NCDSS mice . As a result, some species belonging to the secondary-BAs-producing genera19  and Helicobacter species were highly colonized at the gut lumen in the PC mice; inversely, lower potential pathogens occupied,27–29 such as Romboutsia ilealis, Romboutsia timonensis, Clostridium disporicum, and Bacteroides intestinalis. Additionally, compared with NCDSS mice, PCDSS mice also have a higher relative abundance of species belonging to secondary-BAs-producing genus19 , immuno-modulating species30–32  and Helicobacter species. In contrast, several potential pathogenic species33–37 including Alistipes ihumii, Campylobacter jejuni, Campylobacter lari, Porphyromonas gingivalis, and Prevotella pleuritidis were reduced in the PCDSS mice .After BAs are secreted in the intestine, an array of bacteria gives rise to secondary bile acids through bio-transformations;DSS mice . The comDSS mice , and theDSS mice . We alsoThese results suggested that cholecystectomy induced a significant undulation of fecal microbiota and BAs profiles under colitis status, which featured a higher relative abundance of species involved in immuno-regulation and BAs metabolism, along with increased secondary BAs levels.+) epithelial cells , CD45+CD3+CD8+ cytotoxic T lymphocytes  and CD45+CD3−B220+ B cells  in the colon.We next investigated which immune cell types were at work under inflammatory status. After isolating immunocytes from colonic tissues, flow cytometry was performed to evaluate immune cell subsets . Per the mild changes in colitis signs, we failed to observe altered immune cells between the PCDSS and NCDSS mice at the first month , but modulation of monocytes, macrophages, and DC cells by BARs has been widely reported.DSS mice ; and thehe colon . Moreove39 we next explored whether cholecystectomy could regulate monocytes’ mobilization into the inflamed colon. Along with the reduced intestinal macrophage population, intestinal monocytes (CD45+CD11b+Ly6C+) were significantly lower in the PCDSS mice than in the NCDSS mice at 3rd month  ligand 2 (Ccl2), Ccl7, Ccl8, and Ccl3 relative to NCDSS mice  reproducibly reduced colonic monocytes and macrophages in colitis mice . The sectis mice . These r19 Thus, we explored whether the BARs were affected in the aforementioned murine models. By comparing colonic mRNA levels of various BARs in colitis animals, most BARs levels were increased in PCDSS mice , such as Gpbar1(TGR5), Vdr, Nr1h3 (LXRα), Nr1h2 (LXRβ), Nr1i2 (PXR), and Nr1i3 (CAR). Similarly, the secondary BAs treatments showed increased levels of some BARs, including Nr1h4(FXR), Gpbar1, Nr1h3, and Nr1h2 . The anti-inflammatory effects of FXR and GPBAR1 in macrophages have been widely reported.40 The LXRs are highly expressed in macrophages.41 To confirm whether the inhibitory action of secondary BAs is also mediated through LXRs, we also tested the LXR target gene  and found the mRNA levels were also increased along with LXRs levels . These results revealed that LXRs signaling might be involved in the process of regulating immune responses.The BAs are modified by microbes metabolically and also serve as signaling molecules, which activate BA receptors (BARs).in vitro. After differentiation into macrophages, THP-1 derived macrophages were cultured with 20, 50, 100, and 200 μM specific secondary BAs  for 24 h and then stimulated with LPS for one hour  levels  were abrogated in the presence of LXR inhibitor  were still suppressed  levels , rather ) levels . The inhnhibitor , but infppressed . The LXRppressed .Figure in vivo findings that low chemokines levels after secondary BAs treatments in colitis models.These results indicated that secondary BAs reduced LPS-induced chemokines expressions through activation of LXRα, following our To confirm whether the alterations of intestinal microbiota and BAs were associated with immune cells in the PC patients, we enrolled 14 PC patients and paired 14 healthy controls (HC) in this study , which were reported to be linked with CRC,47–49 also increased in the PC patients.Our published study showed significant changes of fecal bacteria in PC patients compared with HC subjects;ificance . Then, PWe next conducted fecal BAs analysis, PCoA analysis displayed distinct clusters between the two groups . CompareIn this part, we found that the undulating profiles of gut microbiota and BAs in PC patients, especially increased secondary BAs and related bacterial contents, which were remarkably and negatively associated with the peripheral monocytes level, confirming that cholecystectomy might induce intestinal microenvironment changes to regulating monocytes/macrophages recruitment as mentioned in murine colitis models.5 In our present time-course study, we have shown that cholecystectomy, after a relatively longer duration of post-operation, ameliorated DSS-induced colitis in mice, in keeping with our published research suggested that the follow-up period was a pivotal factor for clinical outcomes in PC patients.17 Based on decades-long duration in the PC patients, further studies are needed a markedly long follow-up year to clarify the role of duration after cholecystectomy in IBD patients.Until now, it is widely shown that PC patients have a higher risk of gastrointestinal complications, but no increased risk of IBD in PC patients has been reported in limited studies, and the mechanism was also not clearly underlined in animals.22 However, we found that cholecystectomy restored normal secondary bile acid levels in DSS-treated mice at the 3rd month. As previously described, PC patients also showed secondary BAs were elevated due to more exposure of BAs to microbiota.10–13 And the depletion of BA biotransformation and production capabilities in the microbiota of IBD patients was also implied.23 In our study, among increased species after cholecystectomy in murine colitis, Bacteroides rodentium, Bacteroides uniformis, Ruminococcus lactaris, Ruminococcus faecis, and Clostridium sphenoidese belong to the Bacteroides, Clostridium, and Ruminococcus, which involved in BAs deconjugation, 7-dehydroxylation, or epimerization.40 Some species with immuno-regulation were also increased. Mucus-penetrating Akkermansia muciniphila is a promising next-generation probiotic, which is negatively associated with metabolic syndromes and auto-immune diseases;30Mucispirillum schaedleri, a mucolytic bacterium, has been reported to been partially modulated by pTreg cells;31Blautia hansenii is classified into genus Blautia, which also has potential probiotic functions.32 As for reduced harmful bacteria, Campylobacter jejuni and Campylobacter lari are the leading pathogens of bacterial diarrheal diseases;37Porphyromonas gingivalis is enriched in CRC patients and accelerates CRC development in mice;34Prevotella pleuritidis has been linked to rheumatoid arthritis and gastric cancer.36 Different from the colitis status, bacterial alteration markedly changed while bile acids slightly altered in PC mice after 3 months under normal status, implying fecal microbiota might be significantly restructured to accommodate more frequent exposures of BAs after cholecystectomy. DSS treatment induced intestinal microbiota dysbiosis, which also includes BA-metabolizing bacteria, resulting in the more apparent alteration of bile acids under colitis status after cholecystectomy, which might relieve murine colitis.Previous studies showed that primary and conjugated BAs were elevated, whereas secondary BAs were decreased substantially in IBD patients.in vitro,19 their effects on colitis have been studied in recent years. As reported, LCA alleviated inflammation in murine colitis models.50 The role of DCA in regulating the immune system is controversial. DCA ameliorated DSS-induced colitis and exerted immuno-modulating effects in vitro;51 however, high-dose and long-term intake of DCA aggravated intestinal inflammation and accelerated the transition from intestinal adenoma to colonic adenocarcinoma.53 DCA has different effects depending on the specific conditions and combination of factors, which still require more studies in the future. HDCA has efficacy in treating animal models of metabolic disorders, such as hyperlipidemia, hypercholesterolemia, and type 2 diabetes mellitus.54–57 Recent studies reported that HDCA suppressed intestinal epithelial cell proliferation, accompanied by alteration of gut bacteria and BAs profiles, and the HDCA analogs protected against TNBS-induced colitis.59 We also confirmed specific secondary BAs  ameliorated DSS-induced colitis and accelerated mucosal repair.Although the anti-inflammatory effects of secondary BAs were widely reported 19 Thus, we tested the intestinal immune responses in colitis models and found the intestinal macrophages response was reduced after cholecystectomy. Cholecystectomy also inhibited the elevation of pro-inflammatory macrophages rather than anti-inflammatory subsets under colitis status, implying that PC might affect the origin of intestinal macrophages. The intestinal macrophages mostly require constant replenishment by circulating monocytes throughout adulthood.60 During intestinal inflammation, inflammatory monocytes are recruited into inflamed tissues, depending on the chemokines , and differentiate to mature intestinal macrophages.60 The PCDSS mice showed dramatic intestinal monocytes and chemokines reductions in this study. These data suggested that cholecystectomy modified the local environment (inhibiting chemokines secretions) to reduce monocyte recruitment, followed by decreased colonic macrophages. Furthermore, Cholecystectomy-accumulated secondary BAs inhibited chemokines expression, accompanied by reduced monocytes and macrophages under intestinal inflammation, revealing the critical role of secondary BAs in the immune-regulatory process.Several mucosal immune cells  can be activated by BAs and exert their anti-inflammatory effects.19 This study also found that LCA, DCA, or HDCA inhibited LPS-induced chemokines and inflammatory cytokine responses and increased LXRα levels in THP-1 derived macrophages. LCA and DCA have been shown to suppress inflammatory cytokines and chemokines from macrophages through activation of BARs  in vitro.40 Whether the inhibitory action of secondary BAs is also mediated through LXRs, which are highly expressed in macrophages,41 has not been elucidated. In addition, the immunoregulation of macrophages through LXRs has also been implicated. LXRs activation inhibits a set of inflammatory genes in activated macrophages.61 Our results revealed that DCA and HDCA (but not LCA) inhibited LPS-induced chemokines expressions through LXRα signaling. The LXRα signaling seems noncontributory in LCA-restrained chemokines secretions.22 Bile acids act as ligands, which affinity is different for bile acid-activated receptors.38 Natural bile acid agonists for FXR are CDCA > CA > LCA > DCA CDCA and GPBAR1 is mainly activated by secondary bile acids (LCA > DCA > CDCA > UDCA > CA).40 HDCA has also been shown as a natural ligand for LXRs and GPBAR1,62 but the affinity of LCA and DCA for LXRs is unknown, which still warrants further studies.BAs also severed as signaling molecules by activating several BARs.10–13 Previous studies and our published results showed significant changes of fecal bacteria in PC patients,14–17 but mucosal microbiota alterations have not yet been reported. In this study, mucosal microbiota varied mildly after cholecystectomy, suggesting that the microbiota, sharped by BAs, could not act directly on the intestinal mucosa. Additionally, most of the secondary BAs-producing bacteria, mainly belonging to the obligate anaerobic Bacteroidetes and Firmicutes phyla, are involved primarily in fecal microbiota,63 which may be the reason for the slight changes in mucosal microbiota. Following the experiments in mice, we also found that the accumulation of secondary BAs was negatively associated with peripheral monocytes levels in the PC patients. These results suggested that cholecystectomy-induced secondary BAs might reduce monocyte recruitment, which still need further investigation in IBD patients with a history of cholecystectomy.Our clinical results showed that PC patients have significantly increased secondary BAs , in consistent with published studies.64 However, these results don’t mean cholecystectomy is harmless. It has been reported that cholecystectomy can increase the risk of CRC.1 Furthermore, the history of cholecystectomy was associated with a higher risk for incident colonic dysplasia in Crohn’s disease patients.65 Cholecystectomy’s potential carcinogenic effect still needs comprehensive consideration in further investigation.It has always been debated whether removing the gallbladder has long-term effects on human bodies. In this study, we found the immune regulatory effect of cholecystectomy on IBD through secondary BAs accumulation. Interestingly, a recent study indicated that cholecystectomy could partially alleviate long-term diabetes-induced gut microbiota dysbiosis.In conclusion, cholecystectomy-induced secondary BAs accumulation ameliorated colitis through inhibiting monocyte/macrophage recruitment, which might be mediated by the LXRα-related signaling pathway . Our finAdult male C57BL/6 J mice (6–8 weeks) were purchased from the Vital River facility . All the mice were housed in specific pathogen-free animal facilities with a 12 h-light/dark cycle. The animal use and humane care were approved by the Institutional Medical Ethics Review Board of Peking University People’s Hospital (No.2021PHE023).The mice were allowed to acclimate for one week and divided into a cholecystectomy group (PC) and a negative control group (NC). After bile emptying of the filled gall bladder, the cystic duct was ligated and the gall bladder was removed in the PC group. The NC mice were received sham operations. One month or three months after the operation, the mice have induced colitis as described below.Some groups of the mice were also treated with various BAs–containing water or regular water (changed every three days) for three months. The BAs tested were hyodeoxycholic acid (HDCA), DCA, LCA . The mice have next induced colitis as below.Murine colitis models were induced with 2.5% DSS  in drinking water for 5 days and then switched to regular water or BAs–containing water for another 5 days. The mice were weighed and evaluated daily with DAI scores throughout experimental colitis. After the mice were sacrificed at specific time points, distal colonic tissue and feces were obtained for further analysis.All subjects were enrolled from Peking University People’s Hospital from January 2018 to October 2018. Post-cholecystectomy patients (PC), who received operations above six months, were recruited. Healthy controls (HC) without biliary diseases, tumors, and traumatic ruptures were enrolled to match with PC patients (age and sex). Before sampling, all subjects were asked to avoid using probiotics and antibiotics for at least two weeks. Subsequently, all subjects were collected the colonic tissues for 16S rRNA sequencing and fecal samples for BAs profiles analysis. This clinical study was approved by the Institutional Medical Ethics Review Board of Peking University People’s Hospital (No. 2018PHB035-01). The written informed consent was obtained from all participants.66 Briefly, colonic tissues were dissected and discarded fatty portions. Then colonic tissues were predigested in Hank’s Balanced Salt Solution (HBSS) containing 5 mM EDTA, 20 mM HEPES, 1 mM DTT, and 2% (vol/vol) FBS for 30 min at 37°C . After discarding epithelium-containing supernatants and washing in PBS, the remaining tissues were minced into small pieces and digested with 0.2 mg/mL collagenase IV (Sigma-Aldrich), 0.2 mg/mL DNase I (Roche), 2% (vol/vol) FBS for 30 min at 37°C. The digested tissues were filtered through a 70-µm strainer, and the solutions were centrifuged at 500 g for 10 min to collect cells. Finally, the pellets were resuspended, and the lymphocytes were isolated by Percoll gradient centrifugation.As described previously, the lamina propria immunocytes were isolated from colonic tissues.Lamina propria lymphocytes were stained with antibodies for 30 min at 4°C. The following anti-mouse antibodies  were used: Alexa Fluor 700-CD45 (Clone 30-F11), APC/Cyanine7-F4/80 (BM8), PerCP-CD11b (M1/70), Brilliant Violet 570-Ly6G (1A8), Brilliant Violet 421-CD86 (GL-1), FITC-CD206 (C068C2), PE-Ly6C (HK1.4), PerCP/Cyanine5.5-CD3 (17A2), BV421-CD4 (GK1.5), PerCP-CD8a (53–6.7) and FITC-B220 (RA3-6B2). All data were acquired on the Gallios instrument (Beckman) and analyzed using FlowJo X (TreeStar) or Beckman analysis software.Colon tissues were fixed by 4% paraformaldehyde, embedded in paraffin, and cut into 4-μm-thick sections. The sections were stained with standard hematoxylin and eosin (H&E) staining and independently scored by two double-blinded investigators. After deparaffinization and rehydration, sections were blocked with 3% hydrogen peroxide. After antigen retrieval, the colon tissue slides were incubated with the primary and secondary antibodies, then 3,3′-diaminobenzidine and hematoxylin were used. The sections were observed under an optical microscope and independently counted by two double-blinded investigators.THP-1 cells, a human monocytic cell line, were cultured in RPMI 1640 medium containing 10% FBS and 1% penicillin–streptomycin . For differentiation into macrophages, cells were incubated with 100 ng/ml Phorbol 12-myristate 13-acetate (PMA) (Biolegend) for 48 h. After exchanging medium, cells were cultured with 20, 50, 100, and 200 μM specific BAs  for 24 h, then stimulated with 500 ng/ml LPS (Sigma-Aldrich) for 1 h. For macrophage BARs signaling assays, cells were pretreated with 100 μM BAs and 10 μM an antagonist of LXR  for 24 h, and then stimulated with 500 ng/ml LPS for 1 h.Total RNA from colon tissues or macrophages was isolated using TRIzol reagent (Thermo Fisher Scientific). Then, a reverse transcriptase kit  was used to generate cDNA. qRT-PCR was conducted on the Applied Biosystems StepOne Plus Real-Time PCR System with SYBR Green Master Mix (Thermo Fisher Scientific). The primer sequences are displayed in Supporting Information Table S2.All of the BA standards were obtained from Steraloids and TRC Chemicals or synthesized in the Metabo-Profile laboratory (China). Ten stable isotope-labeled standards were obtained from C/D/N Isotopes (Canada) and Steraloids (USA). Internal Standard (IS) concentrations were kept constant at all the calibration points. Ultra-Performance liquid chromatography coupled to tandem mass spectrometry (UPLC-MS/MS) system  was used to quantitate BAs in this project. The raw data files generated by UPLC-MS/MS were processed using the QuanMET software (Metabo-Profile) to perform peak integration, calibration, and quantitation for each metabolite. SIMCA 14.1 (32 bit) (Umetrics) was used for data analysis.Mucosal samples from PC patients were collected for Illumina Hiseq 2000 sequencing. The bacterial 16S rRNA V3–V4 region was amplified using paired primers (357 F/806 R). The Illumina reads were sorted into different samples according to their barcoded index sequences. Both Vsearch v2.8.1 and Usearch v10 bit 32 were used in sequencing analysis. Two-side reads were merged, low low-frequency reads were removed, and high-quality reads were used for the subsequent analysis. The amplicon sequence variants (ASVs) method was performed to filter chimeras with Unoise3. ASVs were aligned using the Vsearch and taxonomically classified with the reference sequence rdp_16s_v16_sp.fa. Subsequently, the mice feces were also accumulated for bacterial profiling; while to profile bacterial contents at a resolution of the species, the Oxford Nanopore sequencing was performed with the MinION MK1B device. The raw sequencing data were analyzed with the MinKNOW platform, and reads were annotated with the NCBI 16S database. The sequences generated in the present study are available through the NCBI Sequence Read Archive (accession number).t-test or Wilcoxon matched-pairs signed-rank test. Differences between mice groups were evaluated by one-way analysis of variance (ANOVA) followed by Fisher’s LSD test or Student’s t-test. A P value ≤ .05 was defined as statistically significant.Statistical analysis was performed using GraphPad Prism 9 (GraphPad Software) or SPSS 25.0 (IBM). Continuous variables were displayed as mean and standard error. Paired clinical studies were analyzed by Paired Click here for additional data file.