Anti-neutrophil cytoplasmic antibodies (ANCA) play a central role in the diagnosis and pathogenesis of patients with ANCA-associated vasculitis. ANCA-associated vasculitis is a rare disease characterized by necrotizing inflammation of small/medium-sized blood vessels with and without granuloma in different organs. The main syndromes are granulomatosis with polyangiitis, microscopic polyangiitis, and eosinophilic GPA. ANCA in these diseases are almost always directed against proteinase 3 and myeloperoxidase. Most laboratories worldwide use as standard the indirect immunofluorescence technique to screen for ANCA and then confirm positive IFT results with antigen specific immunoassyas for PR3- and MPO-ANCA. New guidelines for ANCA testing have been developed based on a recent European multicentre study, and according to the revised 2017 international consensus recommendations, testing for ANCA in small vessel vasculitis can be done by PR3- and MPO-ANCA immunoassays, without the categorical need for IIF. The new testing strategy for ANCA in vasculitis directly identifies the ANCA target antigen and has a particular value for the AAV sub-classification. Recent studies have shown that AAV can be classified based on ANCA serotype. ANCA presence and the antigen specificity also may have important value as a prognostic factor and may serve as a guide for immunosuppressive therapy. The clinical utility of ANCA depends on the type of assay performed and the appropiate ordering of testing the right clinical setting. Accurate identification of all patients with AAV and the avoidance of misdiagnosis can be achieved using a “gating policy” based on clinical information given to the laboratory at the time of request. However, ANCA has limited specificity as it can be demonstrated in patients with inflammatory bowel disease (IBD), autoimmune liver disease, connective tissue diseases, infections and drug-induced vasculitis, often with multiple antigen specificities and unclear clinical significance.3The detection of anti-neutrophil cytoplasmic antibody (ANCA) as a diagnostic tool and marker of disease in Wegener’s granulomatosis was described in 1985 by Van der Woude and coworkers.Figure 1). Ideally all three tests should be used in each sample.5 Since the establishment of this consensus, many new antigen specific immunoassays have become available, and this has challenged the position of IFT in the testing algorithm for ANCA in vasculitis.7 Furthermore, there is a need for screening algorithm including clinical gating policies to guide the flow of analysis in diagnostic laboratories. In the right clinical context, a positive test for PR3- or MPO-ANCA has high sensitivity and specificity for a diagnosis of AAV.Detection of ANCA in vasculitis is based on primary screening by immunofluorescence test (IFT) on ethanol-fixed neutrophils, and positive indirect immunofluorescence (IIF) test should always be followed by specific PR3- and MPO-ANCA immunoassays  and enzyme-linked immunosorbent assays (ELISAs) that detect ANCA specific for proteinase 3 (PR3) or myeloperoxidase (MPO). For ANCA testing in “new” patients, IIF must be performed on all serum samples. Serum samples containing ANCA […] should be tested in ELISAs for PR3-ANCA and MPO-ANCA”.4 Although this consensus is still widely applied, the position of IIF is being questioned.An international consensus statement for ANCA testing was issued in 1999 and states that “7Over the last 15 years, the performance of ELISA has improved and novel, sensitive and automated technologies, such as fluoro-enzyme immunoassay, chemiluminescence assay and multiplexed flow immunoassay, have been introduced. Besides, there have been advances in assay setup (antigen presentation) with development of second (capture-based) and third (anchor-based) generation assays. Largely, currently available assays for PR3-ANCA and MPO-ANCA are highly sensitive and specific for GPA and MPA.7 In a recent European Vasculitis Study (EUVAS) multicenter study, it has been confirmed that the diagnostic performance of antigen-specific immuno-assays equaled or even exceeded the diagnostic performance of IIF.9 When compared to the study of Hagen and colleagues which was 20 years ago and was a major basis for the consensus statement, there have been marked improvements (mainly in specificity) of the antigen-specific immunoassays.10The availability of reliable antigen-specific immuno-as-says has raised doubts about the two-stage diagnostic strategy currently recommended for ANCA detection.11 Furthermore, it was shown that appropriately designed reference ranges for antibody levels improve interpretation of antigen-specific immuno-assays.12 When new assays are introduced  increase the specificity in case of (low) positive test results. Alternatively, if IIF is used as a screening assay in locally determined best testing algorithms, then the laboratory needs to ensure that the IIF operates at a high sensitivity as there is a large variability in performance of IIF between laboratories.Given these improvements, the revised international consensus demonstrated that high quality antigen-specific immunoassays can be used for the diagnosis of ANCA-associated vasculitis without the need for IIF.11Whereas PR3- and MPO-ANCA have been shown to be highly specific for AAV, their diagnostic value of non-vasculitic conditions is very limited. An overestimation of the diagnostic relevance of a positive ANCA test may erroneously misdirect clinicians and delay adequate treatment. The diagnostic accuracy of ANCA testing in vasculitis should be improved by an increased pretest probability.11 Recently, Arnold and colleagues investigated the impact of a “gating policy” at a single regional center in the year prior to and following the consensus guidelines, and demonstrated that adherence to a “gating policy” for ANCA testing coupled with close liaison between clinician and laboratory does not result in either a missed or delayed diagnosis of AAV.13Accurate identification of all patients with AAV and the avoidance of misdiagnosis can be achieved by use a “gating policy” based on clinical information given to the laboratory at the time of request. This policy limits requests for ANCA testing exclusively to clinical scenarios that may suggest a diagnosis of necrotizing vasculitis.11 The current consensus recommendation applies to ANCA testing for the diagnosis of vasculitis, but does not apply to ANCA testing for the diagnosis of inflammatory bowel disease, autoimmune liver disease or drug-induced autoimmunity.In summary, the new recommendation is that PR3- and MPO-ANCA immunoassays can be used for accurate diagnosis of ANCA-associated vasculitis, without the need for IIF.14Existing classification systems of small vessel vasculitis have relied on combinations of different clinical, radio-graphic and histological findings, but have not included ANCA specificity. Accumulating evidence suggests that ANCA specificity could be better than clinical diagnosis for defining groups of patients, as PR3- and MPO-ANCA are associated with different genetic backgrounds, epidemiology, clinical features, histological findings, and pathogenesis. The new classification approach of AAV is to use ANCA serotypes to classify disease and provide immediate diagnosis based on the presence of PR3- and/or MPO-ANCA. It was demonstrated that serotyping distinguishes distinct classes of ANCA disease: PR3-ANCA associated Vasculitis (PR3-AAV), MPO-ANCA associated Vasculitis (MPO-AAV) and ANCA-negative Vasculitis.15 Moreover, the association with the genes encoding PR3 (PTN3) and its inhibitor α-1-antitrypsin (SERPINA1) with PR3-ANCA disease and/or GPA additionally supports a central pathogenetic role of these autoantigens and their neutralizing counterparts.15The genetic data from genome wide studies (GWAS) point to genetic differences between PR3- and MPO-ANCA patients. These studies have shown that the autoantigen specificities PR3 and MPO correlate better with different HLA risk genes  than with the clinical and pathological phenotypes of GPA and MPA.17 In contrast, MPOAAV is more frequent in kidney-limited disease, displays more severe renal scarring and a worse renal prognosis. Furthermore, patients with MPO-ANCAs are more likely to have renal pathology classified as mixed or sclerotic, and to have a strong association with lung fibrosis compared to patients with PR3-ANCA.19Clinical features differ both in the type and in frequency between PR3-AAV and MPO-AAV. PR3-AAV is associated with granulomatous inflammation, respiratory tract involvement, more extensive extra-renal organ manifestation and a higher relapse rate.21 These data indicate that ANCA specificity is a major determinant of the clinical presentation in AAV.Among patients receiving a clinical diagnosis of GPA, those positive for MPO-ANCAs more frequently have limited disease without severe organ involvement, a higher prevalence of subglottic stenosis, a less frequent need for cyclophosphamide or rituximab therapy and fewer relapses than patients who are positive for PR3ANCA.14In summary, the classification of patients by ANCA specificity (PR3-ANCAs versus MPO-ANCAs) provides practical diagnostic criteria better aligned to patient phenotype, outcomes and treatment responses than does their classification by clinical diagnosis (GPA vs. MPA and EGPA).ANCA serology and the associated clinical manifestation of AAV continue to attract the attention of clinicians and investigators alike. In addition to be providing a useful diagnostic tool for small vessel vasculitis, ANCA testing may be useful in predicting relapses and in guiding therapy. The revised consensus recommendations on ANCA testing state that high quality antigen-specific immunoassays are the preferred screening methodology for the diagnosis of ANCA-associated vasculitis. IIF is no longer deemed suitable as the first screening test, and it adds little to antigen-specific assays in the diagnosis of ANCA-associated vasculitis when the pre-test probability of the disease is high.Notwithstanding, any testing strategy applicable to vasculitis should be able to identify relevant ANCA target antigens (PR3 and MPO), since these correlate best with the clinicopathological aspects, disease activity, propensity for relapse, and response to therapy.


Since the COVID-19 pandemic has occurred, nations showed their unpreparedness to deal with a mass casualty incident of this proportion and severity, which resulted in a tremendous number of deaths even among healthcare workers. The World Society of Emergency Surgery conceived this position paper with the purpose of providing evidence-based recommendations for the management of emergency surgical patients under COVID-19 pandemic for the safety of the patient and healthcare workers.A systematic review was conducted in accordance with the Preferred Reporting Items for Systematic Review and Meta-analysis Protocols (PRISMA-P) through the MEDLINE (PubMed), Embase and SCOPUS databases. Synthesis of evidence, statements and recommendations were developed in accordance with the GRADE methodology.Given the limitation of the evidence, the current document represents an effort to join selected high-quality articles and experts’ opinion.The aim of this position paper is to provide an exhaustive guidelines to perform emergency surgery in a safe and protected environment for surgical patients and for healthcare workers under COVID-19 and to offer the best management of COVID-19 patients needing for an emergency surgical treatment.We recommend screening for COVID-19 infection at the emergency department all acute surgical patients who are waiting for hospital admission and urgent surgery. The screening work-up provides a RT-PCR nasopharyngeal swab test and a baseline (non-contrast) chest CT or a chest X-ray or a lungs US, depending on skills and availability. If the COVID-19 screening is not completed we recommend keeping the patient in isolation until RT-PCR swab test result is not available, and to manage him/she such as an overt COVID patient.The management of COVID-19 surgical patients is multidisciplinary.If an immediate surgical procedure is mandatory, whether laparoscopic or via open approach, we recommend doing every effort to protect the operating room staff for the safety of the patient. An incomparable outbreak of respiratory illness in Wuhan, Hubei Province, China, was detected in December 2019. A novel coronavirus was identified on 12 January 2020 and called the 2019 novel coronavirus (2019-nCoV); on 11 February 2020, the World Health Organization (WHO) officially indicated this viral disease, which affects mostly the lower respiratory tract and manifests as pneumonia in humans, as COVID-19.COVID-19 has rapidly spread and the WHO declared it a Public Health Emergency of International Concern with a risk assessment of very high at a global level.Since the COVID-19 pandemic has occurred, nations showed their unpreparedness to deal with a mass casualty incident of this proportion and severity: hospital leadership and individual providers faced difficult decisions about how to conserve critical resources, such as hospital and intensive care unit (ICU) beds, respirators, transfusion capacity, and personal protective equipment (PPE) that are vital for protecting patients and staff from unnecessary exposure and intra-hospital transmission. It resulted in high mortality rate of infected patients, in particular of fragile patients such as people with multiple chronic comorbidities and polypharmacy, and an unacceptable number of infected healthcare workers and deaths.Since the early phase of the pandemic, surgical theatres were converted into additional ICUs to support critically ill patients and non-urgent, non-cancer surgical procedures were cancelled or postponed until a later date; all nurses and medical staff including residents were reallocated in COVID units.In this apocalyptic scenario, the emergency surgeons accepted their crucial role in the management of infected and non infected patients and the need to work safely to limit the spread of the virus in healthcare facilities and to decrease morbidity and mortality rate, which may result from delay diagnosis and treatment of surgical patients.The World Society of Emergency Surgery educational board (WSES) conceived this position paper with the purpose of providing recommendations for the management of surgical patients in emergency setting under COVID-19 pandemic for the safety of the patient and healthcare workers based on available evidences and experienced surgeons’ opinion.The scientific board of WSES established 11 PICO  questions organized into 4 topics  to build the position paper structure about the management of confirmed, uncertain and negative surgical patients during the COVID-19 pandemic, summarized in Table A systematic review was conducted in accordance with the Preferred Reporting Items for Systematic Review and Meta-analysis Protocols (PRISMA-P)  through A group of experts were designed by a steering committee to review the articles included in the analysis.Synthesis of evidence, statements and recommendations were developed in accordance with the GRADE methodology .The first draft of the paper was submitted to the steering group, consisting of emergency and trauma surgeons, anesthesiologists, emergency physicians, radiologists, infection disease physicians and qualified nursing personnel, for evaluation and approval.Given the limitation of the evidence, the current document represents an effort to provide consensus-based general guidelines for surgical patient management during COVID-19 pandemic in an emergency setting.Symptoms of COVID-19 infection are myriad and may include stroke or myocarditis as the first presentation. COVID-19 infection is suspected in patients presenting with fever, cough, dyspnoea and/or recent direct contact with a confirmed COVID-19 patient (QoE moderate B).d-dimer (QoE moderate B).Characteristic laboratory findings for COVID-19 infection are leucopenia, lymphocytopenia, elevated aspartate aminotransferase, and inflammatory biomarkers such as C-reactive protein, erythrocyte sedimentation rate, elevated lactate dehydrogenase, creatinine, hypersensitive troponin I, fibrinogen and The RT-PCR test in respiratory samples (swab) is the current gold standard method for confirming the diagnosis of COVID-19 (QoE moderate B).The RT-PCR test result heavily relies on the presence of viral genome in sufficient amounts at the site of sample collection that can be amplified. An incorrect sample collection or missing the time-window of viral replication can provide false-negative results and limits the usefulness of qPCR-based assay (QoE moderate B).In the early stage of the disease the detection of SARS-CoV-2, viral RNA is better in nasopharynx samples than the oropharynx (QoE moderate B).For individuals with a high clinical suspicion of SARS-CoV-2 infection with negative RT-PCR test, a combination of repeated nasopharyngeal RT-PCR swab tests and chest imaging may be helpful to confirm early the diagnosis of COVID-19 disease and to evaluate the pneumonia’s severity (QoE moderate B).In the COVID-19 screening, the chest CT scan is the most accurate radiological tool to confirm the diagnosis above all in uncertain cases. The CXR can be helpful in case of unavailability of CT scan (QoE moderate B).The chest CT scan may be useful to complete the COVID-19 screening in patients with a high clinical suspicion of SARS-CoV-2 infection but negative RT-PCR swab test (QoE moderate B).For emergency physicians and emergency surgeons with excellent point-of-care ultrasound (POCUS) skills and limited access to CT, it is reasonable to use lung POCUS in COVID-19 screening, which can help in the diagnosis and at the same time rules out other acute respiratory illnesses (QoE low C).Lungs US can be used as the first COVID-19 screening tool and discriminate low-risk patients  from higher-risk patients , which might require second-level imaging rapidly (QoE very low D).Lungs US may be helpful for patients with a high clinical suspicion of COVID-19 but negative RT-PCR test to confirm the diagnosis if they demonstrate typical lung ultrasound findings for COVID-19 and if skills are available, in the unavailability of CT scan (QoE moderate B).We recommend screening for COVID-19 infection at the emergency department and of all surgical patients with clinical and epidemiological features suspected for COVID-19 disease who are waiting for hospital admission and urgent surgery. The screening provides performing a RT-PCR nasopharyngeal swab test and a baseline (non-contrast) chest CT or chest X-ray or lungs US, depending on skills and availability (strong recommendation based on the moderate level of evidence 1B).Patients presenting with acute abdominal pain are admitted in the emergency department (ED) for surgical evaluation, appropriate laboratory tests and abdominal imaging, if they are required to make diagnosis.There is not yet recommendation about the routinely testing for COVID-19 disease of patients admitted in the ED with acute abdominal pain.Since the COVID-19 infection outbreak, the WHO and CDC , 4 have .In clinical practice, this recommendation turned out rapidly in a large number of infected healthcare workers and a high risk of environmental contamination, worsening the problem of limited resources in terms of PPE availability, hospital beds shortage in ICU and overloaded healthcare personnel.He presents with fever and at least one sign/symptom of respiratory disease and a history of travel to or residence in a country area or territory reporting local transmission of COVID-19 disease during the14 days prior to symptom onset;He presents with any acute respiratory illness, having been in contact with a confirmed COVID-19 case in the last 14 days prior onset of symptoms;He presents with severe respiratory infection, with no other etiology that fully explains the clinical presentation, requiring hospitalization.At ED admission, a patient is defined “suspected” for COVID-19 infection if:Many authors reported that the most common symptoms in patients with confirmed COVID-19 infection are fever > 38.5 °C, breathing difficulties and dry cough, diarrhoea and vomiting are rare , 7.d-dimers , compared with 91% [95% CI: 81–96%] (om onset .The lung point-of-care ultrasound (POCUS) is reported to be useful in confirming clinical suspicion of COVID 19 infection in patients who showed negative RT-qPCR.Vetrugno and Coll  reportedBuonsenso et al.  were theThe early US pulmonary manifestations of COVID-19 pneumonia are patchy distribution of interstitial artefactual signs  that will extend these patterns to multiple areas of the lung surface. The further evolution is represented by the appearance, still patchy, of small subpleural consolidations with associated areas of white lung. The evolution in consolidations, especially in a gravitational position, with or without air bronchograms, and their increasing extension along the lung surface indicate the evolution toward the phase of respiratory insufficiency that requires invasive ventilatory support .The use of a convex or linear transducers. The latter are preferable to study the detail of the pleural and subpleural alterations.The use of a single–focal point modality (no multi-focusing), and set the focal point on the pleural line.Preferably, US scans need to be intercostal  to cover the widest surface possible with a single scan.Soldati et al.  reportedIdeally, 16 areas, if it is possible, should be evaluated: anterior mid-clavicular , right and left; posterior para-spinal , right and left; and lateral axillary , medial right and left, to study the extent of the lungs surface affected.US portability and bed-side evaluation that could decrease the virus exposure of healthcare personnel and environmental contamination derived from moving the patient to the radiology unitEasier sterilization of the device due to smaller surface areasHigher sensitivity (80%) than CXR (no more than 60%) to discriminate a bacterial pneumonia from a non-bacterial infectionUS radiation freeUS instrument costsAdvantages from the use of lung US as first radiological diagnostic investigation in suspected COVID-19 patients are –29:US pThe difficulty to detect a centrally located consolidation from bacterial superinfection;The inability to discern the chronicity of a lesion, limiting its power of early COVID19 diagnosis in the population with preexisting pulmonary conditions.Lung POCUS limitations can be:Given the limits of the currently used nucleic acid detection and CT scan for the diagnosis of COVID-19 used as screening tools, point-of-care test (POCT) of IgM/IgG and ELISA kits for SARS-CoV-2 have been developed to help in the detection of infected patients in the emergency setting.In accordance with other acute viral infections, the antibody profile against SARS-CoV-2 has a typical pattern of IgM and IgG production. The SARS-specific IgM antibodies disappear at the end of week 12, while the IgG antibody can last for a long time –33.Li et al.  studied Moreover, the IgM-IgG combined assay was demonstrated to have better utility and sensitivity compared with a single IgM or IgG test.On the other hand, controversial data are reported about the use of IgM/IgG rapid test in the triage of patients admitted to the ED.Li et al.  reportedFocusing on acute patients, Cassaniti et al.  reportedAll acute surgical patients should complete preoperative COVID-19 screening that includes RT-PCR nasopharyngeal swab test and chest CT scan, when it is available, or a CXR, or lungs US in the ED, whether they are symptomatic or not, to control the in-hospital spreading of SARS-CoV-2 (QoE moderate B).Chest imaging such as a baseline CT scan, a CXR or a lungs US, depending on the availability, are useful diagnostic tools in the unavailability of RT-PCR swab test result to detect potentially infected patients (QoE moderate B).If chest radiological evaluation by CXR, or chest CT scan or lungs US, is inconclusive and the patient needs for immediate surgery, he has to be treated as a COVID-19 patient to limit the risk of contagion and the spreading of the SARS-CoV-2 in the operating theatres (QoE moderate B).After surgery, the uncertain patient has to be isolated as long as the RT-PCR test result is obtained, to be admitted in a COVID (+) or (−) ward. If it is positive, it is recommended repeating the swab test for confirmation. In patients with confirmed COVID-19 diagnosis, the laboratory evaluation should be repeated to evaluate for viral clearance prior to being released from isolation (QoE moderate B).Timing of Acute Care Surgery (TACS) classification system could be a valid tool to evaluate timing of surgery and severity of the surgical disease (QoE low C).We recommend completing the COVID-19 screening  for all acute surgical patients before admission in the surgical ward or operating theatre. If the RT-PCR swab test result is not available to confirm the diagnosis, the patient needs to be isolated and treated such as a COVID-19 (+) patient with all the mandatory precautions. The acute care surgeon is the only responsible for the decision of delaying a surgical procedure in the emergency setting during the pandemic. The TACS classification is a good tool to evaluate timing of surgery. According to this classification, surgery cannot be postponed for class 1 (immediate surgery) and class 2  patients even if diagnosis of COVID-19 is not yet confirmed by the RT-PCR swab test (strong recommendation based on moderate-level evidence 1B).The RT-PCR in respiratory tract samples is the current gold standard method for the diagnosis of COVID-19 infection but it could be time consuming because of the number of test requested and the availability of specialized operators and machines, when the rapid diagnosis is needed for fast intervention decisions. Generally, in very urgent cases, RT-PCR test can be obtained in 4–6 h; in other cases, test can be obtained in 48 h.This is according to the availability and contraindications, if they are present, to performing radiological imaging, chest CT scan, or lung US, if emergency physician or surgeon is skilled to perform it, or CXR can help in confirming the diagnosis of COVID-19 pneumonia in surgical patients requiring immediate surgical procedure, showing pathognomonic features.During this pandemic, the decision of operating or delaying the surgical procedure depends on the emergency surgeon’s evaluation.If an urgent surgical procedure is necessary , the emergency surgeon should check promptly the availability of a dedicated operating room with functional and suitable human and technical resources for COVID-19 and negative patients.Several authors , 36 proplective non-urgent surgery does not always mean optional surgery. Delaying some surgical procedures could become very harmful because the progression of the underlying abdominal disease that can lead to life-threatening complications.Many international societies of surgeons recommended rescheduling non-urgent surgeries but eFocusing on emergency setting, early clinical diagnosis, adequate source control to stop ongoing contamination, appropriate antimicrobial therapy and prompt resuscitation in critically ill patients are the cornerstones in the management of intra-abdominal infections. Timing of surgical intervention is critical for outcomes of patients diagnosed with surgical emergencies. Under this period of limited access to hospital resources, the triage of the patients is fundamental to assess the severity of the intra-abdominal disease underlying the acute abdominal pain. Emergency surgeons have to decide for a non operative (NOM) or operative management of a surgical disease, such as it is recommended in international guidelines.The haemodynamic stability or instability after adequate resuscitative maneuvers remains the main tool to risk-stratify patients for immediate surgery or not.Furthermore, general, or more specific, clinical scores (such as for example the American Society of Anaesthesiologists’ (ASA) score, Alvarado’s score in case of acute appendicitis, SOFA for sepsis), the age of the patient and the presence of comorbidities, such as obesity, diabetes and COPD, can assist the emergency surgeon’s decision making process, associated with clinical  and biological  parameters.The WSES  proposedIn this COVID-19 pandemic, these criteria could guide the acute surgical teams to properly tag each patient to the timing of surgery.Since now, the TACS classification is the only available tool to risk-stratify patients presenting with an emergency disease.The validity of the TACS classification system was evaluated in a tertiary public hospital of a developing country, and data showed that TACS rating improves time for surgeries classified as yellow  .Some authors decided to develop a more functional and robust system six-level classification, the Non-Elective Surgery Triage (NEST), allowing prioritization based on physiologic state and disease process, but no data are available about its application .RT-PCR test remains the reference standard to make a definitive diagnosis of COVID-19 infection and to manage the patient and resources in the correct way (QoE high A).The emergency physician may identify high-risk COVID-19 patients investigating the presence of typical clinical symptoms, laboratory test results and/or epidemiological risk factors as suggested by the WHO, but RT-PCR test confirmation is mandatory to make diagnosis of viral infection (QoE moderate B).Negative chest CT scan is not sufficient to exclude the diagnosis of COVID-19 infection, above all in the early phase of the infection (QoE low C).In case of unavailability of the RT-PCR test, the surgical patient has to be considered potentially infected and managed like a COVID-19 (+) patient (QoE moderate B).If it is not possible to confirm diagnosis of COVID-19 disease in an acute surgical patient by RT-PCR swab test, we recommend managing the patient such as he/she is COVID-19 (+) with all the mandatory precautions against viral infection, which include all the protective measures and a dedicated pathway for the operating room, to decrease the risk of environmental contamination and health personnel exposure. If a dedicated pathway for COVID-19 (+) patients is not available in the hospital, it should be an option to transfer haemodynamic stable suspected patient to the nearest COVID-19 HUB hospital for the appropriate management (strong recommendation based on the moderate level of evidence 1B).COVID-19 RT-PCR test that provides nucleic acid detection in the nasal and throat swab sampling, indicated in early stage of the infection, or other respiratory tract samplingsChest imaging that includes chest radiograph, CT scan or lung ultrasound demonstrating bilateral opacities (lung infiltrates > 50%), lobar or lung collapse. Multiple patchy ground-glass opacities in bilateral multiple lobular with periphery distribution are typical chest CT imaging features of COVID-19 pneumonia.Actual diagnosis of COVID-19 infection is made through:Several authors reported that a patient with RT-PCR-confirmed COVID-19 infection may have a normal chest CT at admission.Fang et al.  reportedBernheim et al.  showed tAi et al. reported  21 of 60In contrast, Pan et al.  reportedThese data confirm that a normal chest CT scan cannot exclude the diagnosis of COVID-19, especially for patients with early onset of symptoms or asymptomatic , 19, 20.Therefore, RT-PCR test remains the reference standard to make a definitive diagnosis of COVID-19 infection despite the false-negative rate.Anyway, emergency physicians may identify high-risk COVID-19 patients by investigating the presence of typical symptoms and/or epidemiological risk factors as suggested by the WHO, but RT-PCR confirmation is mandatory to make diagnosis of viral infection and to manage the patient and resources in the correct way .In case of unavailability of the RT-PCR test, the surgical patient has to be considered potentially infected and to be treated exactly as a COVID-19 patient, requiring all the protective measures to decrease the risk of environmental contamination and health personnel exposure and a dedicated pathway for the operating room.Negative baseline chest CT scan is not sufficient to exclude the diagnosis of COVID-19 infection.If it is not possible to diagnosis COVID-19 infection in a stable potentially surgical patient, it would be better to consider transferring the patient to the nearest COVID-19 HUB hospital for the management.Diagnosis of COVID 19 disease is confirmed through the RT-PCR test (QoE moderate B)Each surgical patient might be considered suspected for COVID-19 disease if clinical signs, imaging features at CXR or/and lungs US or/and chest CT scan and laboratory tests results are compatible with a SARS-CoV-2 infection (QoE moderate B).The COVID-19 screening includes RT-PCR swab test and a chest radiological imaging that could be CXR or lungs US in the unavailability of CT scan (QoE moderate B).If a surgical patient cannot complete the screening for COVID-19 disease and requires immediate surgical procedure, he/she should be managed with all the mandatory precautions against COVID-19 infection (QoE low C).If the RT-PCR swab test is positive, the surgical patient has to be managed as a COVID-19 patient. The chest imaging is useful to assess the severity of the pneumonia (QoE high A).In case of RT-PCR test and chest CT scan unavailability, we suggest screening the patient for COVID-19 pneumonia with CXR or lungs US that can help assessing viral pneumonia radiological signs and severity, exactly such as a chest CT scan, before surgery. In this case, SARS-CoV-2 infection can only be suspected (weak recommendation based on the low level of evidence 2C).We recommend confirming COVID-19 disease with the RT-PCR swab test as soon as possible; if it is not possible, the suspected patient has to be managed as a SARS-CoV-2 infected one (strong recommendation based on the moderate level of evidence 1B).Currently, in the unavailability of RT-PCR test and chest CT scan, we cannot identify COVID-19 patients but only put the suspicion for the infection in the presence of typical symptoms, including fever, dry cough, dyspnoea, myalgia, fatigue or hypolymphaemia, or epidemiological risk factors such as residence in or travel to an area with widespread community transmission or known contact , 7. TherIn case of unavailability of chest CT scan, we can complete COVID-19 screening with a CXR or lungs US.Common CXR findings mirror those described for chest CT scan that are bilateral interstitial pattern/ground-glass opacities, with isolated focal infiltrate.The CXR may be normal early in the disease course but a normal CXR does not rule out the diagnosis, exactly like a chest CT scan. CXR abnormal findings peaked at 10–12 days from the date of symptom onset .In case of positive RT-PCR test and negative CXR, the patient has to be managed as a COVID-19 (+).Chest radiological imaging in COVID-19 (+) patients helps the assessment of the severity of the pneumonia and the necessity to admit the patient in ICU.If a surgical procedure is mandatory, the emergency surgeon should check the availability of a dedicated OR with functional and suitable human and technical resources and of the PPE necessary for protecting both patients and staff from intra-hospital transmission of the virus.Indications for a surgical procedure are not different in confirmed COVID-19 patients (QoE moderate B).Current data about outcome of surgery in COVID-19 patients showed a higher morbidity and mortality rate in comparison with negative patients (QoE moderate B).The risk of environmental contamination and virus exposure in operating room related to the surgical management of a confirmed COVID-19 patient is high in the lack of trained health staff and personal protective equipment (QoE moderate B).During COVID-19 pandemic, it is fundamental to carefully evaluate case by case the necessity for immediate surgical or non operative strategies, as recommended in international guidelines (QoE moderate B).In evaluating the necessity to perform emergency surgery in COVID-19 (+), we recommend complying with international guidelines about immediate surgery or non operative strategies, evaluating case by case and resources. According to TACS classification, class 1 and 2 patients require surgical treatment in a very short delay (strong recommendation based on the moderate level of evidence 1B).Indications for an urgent surgical procedures are not different in confirmed COVID-19 patients; the difference lies in the access to the resources  under the peak of this pandemic and the risk of virus dissemination in the environment and in OR, in the lack of dedicated pathway and staff.The main objective of the ES is not to delay surgery to decrease morbidity and mortality rates in patients in whom a surgical procedure is mandatory.Consequently, triage of the infected patients with TACS classification remains the cornerstone of the management of intra-abdominal surgical diseases. The assessment of the severity of the surgical disease and of the viral pneumonia enables ES to decide on the management of the patient, complying with international guidelines.mild illness: this group of patients does not need ventilatory support or admission in ICU.Patients with moderate viral pneumonia: they could require non-invasive ventilatory support.Patients presenting with severe pneumonia: this is a group of critically ill patients that could present with Acute Respiratory Distress Syndrome (ARDS) and need to be admitted in the ICU to receive ventilatory support or extracorporeal membrane oxygenation (ECMO).Patients with On the assessment of the severity of the COVID-19 pneumonia, patients can be classified in :PatientIn the initial evaluation of a patient presenting with acute abdomen, the haemodynamic status remains the main tool to risk-stratify patients in need of immediate surgery.Then, if the intra-abdominal infection is uncomplicated (i.e. involving the organ and not the peritoneum), NOM could be a valid option, to be considered on a case-by-case basis, according to WSES guidelines .In case of NOM, it is crucial to plan close clinical and radiological surveillance at 12–24-h intervals from the beginning of the intravenous antibiotic therapy until the situation is under control. If the patient presents with persistent abdominal pain, fever or signs of shock, surgical treatment cannot be postponed.Let us critically review the management of the most common surgical diseases, on the basis of the currently evidence.acute appendicitis, the “gold standard” treatment is laparoscopic appendectomy in all patients. In this specific scenario, caution might be taken in the choice of the laparoscopic approach because of concerns about the presence of the virus in the pneumoperitoneum, if it is not possible to decrease the risk of dissemination of the virus in OR. However, NOM with antibiotic therapy has proven to be successful in uncomplicated appendicitis. The Jerusalem WSES guidelines and its 2020 update  and arterial thrombotic events in 3.7% [95% CI: 0–8.2%]. The acute pulmonary embolism was the most frequent thrombotic complication  levels, longer prothrombin time and activated partial thromboplastin time compared to survivors on admission (P < .05); 71.4% of non-survivors and 0.6% survivors met the criteria of disseminated intravascular coagulation during their hospital stay.These rates are very high when compared to a recent RCT conducted on ICU (no COVID) patients that reported the incidence of VTE to be 9.4% . It has platelet . Recentlplatelet  analyzedIt has been proven that the coagulation system can be activated by a variety of different viruses . High pld-dimer; IL-6 levels appeared to increase only 13 days after disease onset, whereas d-dimer levels were already 10-fold increased by that time. This observation suggests that the very high d-dimer levels observed in COVID-19 patients are not only secondary to systemic inflammation, but also reflect true thrombotic disease, possibly induced by cellular activation that is triggered by the virus.While many pro-inflammatory cytokines trigger the coagulation system, Zhou and colleagues  showed tWhether the coagulation cascade is directly activated by the virus or whether this is the result of local or systemic inflammation is not completely understood, anyway, vascular endothelial damage in both small- and mid-sized pulmonary vessels was noted together with disseminated intravascular coagulation, deep vein thrombosis and pulmonary embolism resulting in pulmonary infarction in SARS-CoV and SARS-CoV-2 , 82P = 0.029), but not among the 352 patients with a SIC score, which includes prothrombin time, platelet count and sequential organ failure assessment (SOFA), < 4 .Tang et al.  conducteThe WHO interim guidance statement recommends prophylactic daily LMWHs, or twice daily subcutaneous unfractionated heparin (UFH) .If pharmacological prophylaxis is contraindicated, mechanical VTE prophylaxis (intermittent pneumatic compression) should be considered in immobilized patients . Missed The risk of VTE is increased in critically ill COVID-19 patients. Alterations in pharmacokinetics in this group of patients may necessitate anticoagulation dose adjustment, due to factors relating to absorption, metabolism and renal (or hepatic) elimination of these drugs in the setting of potential organ dysfunction.The anti-thrombotic prophylaxis showed high rate of failure above all in COVID-19 patients admitted in ICU when standard LMWH or UFH doses are used. This is due to a heparin resistance with UFH or sub-optimal anti-Xa peak with LMWH that seems to be common in COVID-19 intensive care unit patients who received therapeutic anticoagulation. Consequently, it is important to measure anti-Xa levels for patients on therapeutic LMWH to ensure adequate dosing and continue with careful monitoring for those on UFH , 87.COVID-19 surgical patient requires a multidisciplinary approach, above all if he/she is admitted in ICU for mechanical ventilation and presents with signs of septic shock (low level of evidence C).Specific pharmacological treatment for COVID-19 disease is not available but when an empirical treatment is administered, it is mandatory to monitor for early detection of complications (moderate level of evidence B).Currently, there are no data about the use of antimicrobial in COVID-19 patients to prevent secondary healthcare infections (low level of evidence C).Initial prompt antibiotic therapy for intra-abdominal infections in surgical patients is typically empirical and depends on the underlying severity of infection, the pathogens presumed to be involved and the risk factors indicative of major resistance patterns. Antimicrobial treatment should be targeted to results from cultures from the site of infections or hemocultures with de-escalation of treatment as early as possible, in accordance with WSES guidelines (moderate level of evidence B).Empirical anti-fungal treatment should only be considered in critically COVID-19 patients, presenting fever of unknown origin, with new pulmonary infiltrate superimposed on a viral pneumonitis pattern, with the aim of confirming the diagnosis by invasive techniques and/or the use of fungal biomarkers (moderate level of evidence B).We recommend carefully administering antibiotics in COVID-19 surgical patients for the high risk of selecting resistant bacteria, especially in patients admitted in ICU for mechanical ventilation. Early empirical antibiotic treatment should be targeted to results from cultures, with de-escalation of treatment as soon as possible (strong recommendation based on the moderate level of evidence 1B).We recommend against empirical anti-fungal treatment in all surgical COVID-19 patients but we suggest considering it in critically ill patients (strong recommendation based on the moderate level of evidence 1B).n = 786; age ≥ 64 years) and that the majority of these patients requires mechanical ventilation and high levels of positive end-expiratory pressure (PEEP). The mortality rate for this group of patients is 26% in this study ), fatigue (25 [73.5%]) and dry cough (18 [52.9%]). Fifteen (44.1%) patients required admission to ICU during disease progression, and 7 patients (20.5%) died after admission to ICU. Compared with patients not requiring ICU admission, ICU patients were older, more likely to have underlying comorbidities, underwent more difficult surgeries, and more severe laboratory abnormalities . The most common complications in non-survivors patients included ARDS, shock, arrhythmia and acute cardiac injury.This retrospective study showed that surgery can have a negative impact on COVID-19 patients even if they are asymptomatic, with a mortality rate of 20.5% and the potentially needing for postoperative ICU care .Another retrospective study carried out in a single thoracic department, enrolling 25 COVID-19 patients reported similar data showing that COVID-19 is associated with poor prognosis for patients undergoing surgery, especially for those with chronic diseases .According to available data, coronavirus infection appears to be a complex systemic disease involving different organs. This lays on that angiotensin-converting enzyme 2 (ACE2) is the functional receptor for SARS-CoV-2.Hammings et al. investigated the immuno-localization of ACE2 protein and reported that it is abundantly present in humans in the epithelia of the lung and small intestine including the duodenum, jejunum and ileum, but not in the colon. Furthermore, ACE2 is present in arterial and venous endothelial cells and arterial smooth muscle cells of various human organs such as oral and nasal mucosa, nasopharynx, lung, stomach, small intestine, colon, skin, lymph nodes, thymus, bone marrow, spleen, liver, kidney and brain. Moreover, ACE2 mRNA was found in many tissues and it is highly expressed in renal, cardiovascular and gastrointestinal tissues.The binding of SARS-CoV-2 on ACE2 causes an elevated expression of ACE2, which can lead to damages primarily on alveolar cells and lately on other organs. Damages to alveolar cells can, in turn, trigger a series of systemic reactions with the secretion of various pro-inflammatory mediators that may play important roles in the pathophysiology of complications .Several studies reported higher in-hospital mortality in patients with COVID-19 infection presenting abnormal kidney function, diabetes and cardiac injury –95.Therefore, COVID-19 patients presenting already with comorbidities such as diabetes, renal abnormalities , chronic obstructive pulmonary disease (COPD) and chronic cardiovascular disorders are at risk for presenting severe coronavirus disease as well as poor outcome after surgery.Moreover, the duration of the surgical procedure and general anaesthesia can increase the risk of postoperative infections.Specific pharmacological treatment for COVID-19 is not available , 97.In this lack, in the early phase of the pandemic, many centres decided to use empirical antiviral therapy with darunavir or lopinavir in combination with ritonavir and oseltamivir and hydroxychloroquine on the basis of early promising results in terms of survival in critical COVID-19 patients reported in several studies.In an open-label clinical study that showed some limitations in the methodology, Gautret et al.  reportedAzithromycin is a broad-spectrum macrolide antibiotic with a long half-life and a large volume of distribution; it is primarily used for the treatment of respiratory, enteric and genitourinary bacterial infections. In addition it has shown to be active in vitro against Zika and Ebola viruses  and to p.In the early phase of this pandemic, lopinavir/ritonavir were administered in severe coronavirus disease cases, in association with other medical treatment. Cao et al.  conducteWhile in healthy, young patients diagnosed with COVID-19 the combination of these drugs are well tolerated with mild side effects, in the elderly patients presenting age-related comorbidities that result in complex polypharmacy, the risk of toxicity with lethal complications is increased.Furthermore, physiological changes related to ageing may affect both pharmacokinetics and pharmacodynamics thereby putting elderly patients at risk of inappropriate prescribing and adverse drug reactions .Both lopinavir/ritonavir and chloroquine and hydroxychloroquine are known to cause QT prolongation and torsades de pointes. In particular, long-term chloroquine and hydroxychloroquine use may increase depolarization length duration and Purkinje fiber refractory period, ultimately leading to atrioventricular nodal and/or His system malfunction and arrhythmia. QT prolongation in individual medical therapy is not always predictable; dose adjustments and/or additional monitoring with electrocardiograms may be appropriate in some cases .COVID-19 patients treated with these drugs need a careful clinical and biological multidisciplinary surveillance.Currently, there are no data about the use of antimicrobial in COVID-19 patients.Streptococcus pneumoniae, Haemophilus influenzae and Staphylococcus aureus being the most frequently reported pathogens. The exact incidence of bacterial superinfection in COVID-19 is unknown. It was reported that the most common co-infections in COVID-19 patients were rhinovirus/enterovirus (6.9%), respiratory syncytial virus (5.2%) and non–SARS-CoV-2 Coronaviridae (4.3%), isolated in the respiratory tract , the patient could be discharged to home if:Appropriate caregivers are available at home, in case of isolated elderly patientsThe patient can be isolated limiting the risk of exposure for the other household members There are no household members who may be at increased risk of complications from COVID-19 infection All the household members are capable of adhering to recommended precautions to avoid the shedding of the virus until the risk of transmission is low.According to the CDC recommendations [The decision to remove precautions is based on two general strategies: a test-based strategy that requires negative respiratory RT-PCR tests obtained after the resolution of symptoms and a symptom-based strategy that recommends keeping patients on contact precautions until a fixed period has elapsed from symptom recovery. The underlying assumption of the symptom-based strategy is that waiting for a fixed period of time is a surrogate for negative RT-PCR testing, which itself is a surrogate for the absence of shedding infectious virus.After hospital discharge, if there is a possibility that the patient is still infectious, clear advice should be given on how to avoid transmitting COVID-19 disease and all the confirmed and suspected COVID-19 patients should be further quarantined in designated COVID (+) hospitals, healthcare facilities or at home, keeping precautions to avoid infection transmission, followed up by RT-PCR tests, to decide to discontinue the isolation, even if the recovered is asymptomatic. COVID-19 patients should be kept in isolation at least until the 14 days have passed since the date of their first positive COVID-19 diagnostic test, as clinical criteria cannot be used to assess where these individuals are in the course of their illness –121.Due to the convenience of operation and the acceptability of patients, the most commonly used specimens at discharge in practice are also oropharyngeal swabs, and sometimes, nasal swabs are collected at the same time. However, in the middle and later stages of the disease, the amount of virus remaining in the pharyngeal cells is small or very low in some patients and they can result  negative. If only the pharyngeal specimens are taken, the viral nucleic acid cannot be detected. Although alveolar lavage fluid is easier to detect viruses, due to its inconvenient operation and high risk of exposure, it is mainly used on critically ill patients who have been intubated This is consistent with prior literature , emphasiIt is important to note that with the recovery of the disease, the positive rate of oropharyngeal swabs in mild patients declines the fastest, and in the later course of the disease, positive results of anal swabs are more than that of pharyngeal swabs .Furthermore, current data showed that several patients meeting criteria for hospital discharge could present positive RT-PCR test during the isolation period. The time from hospital discharge to positive checked RT-PCR after recovery was 5–13 days. All these patients continued to be asymptomatic and chest CT showed no changes from previous images –126.These findings suggested that at least a proportion of recovered COVID-19 patients still may be virus carriers, that intermittent virus shedding might occur in recovered patients and that the number of PCR false-negative test patients at the discharge is not negligible.Serology can help in detecting the infection in suspected/uncertain/asymptomatic cases and in deciding for discontinuation of quarantine. Antibodies IgM and IgG against SARS-CoV-2 infection are usually produced in infected patients, and antibody IgG can persist a very long time. There are no data about the use of serology to follow recovered patients.An observational analysis of 150 patients and HCWs showed that the average time to transition from RT-PCR positive to negative was 24 days after symptom onset, and 10% of patients remained positive even 33 days after symptom onset .Although there is no firm evidence indicating that these recovered patients would transmit the virus to others, after discharge, 4 weeks of further isolation with regular health monitoring  should be considered for COVID-19 patients, such as keeping all necessary precautions  in order to protect family members and the community from infection and further spread of SARS-CoV-2.This position paper represents the result of an extensive analysis of the first consistent evidence about COVID-19 disease. The aim is to provide an evidence-based guidelines for emergency surgeons to perform safe surgery during this pandemic to limit the diffusion of the SARS-CoV-2 infection and to decrease mortality rate related to COVID-19 surgical patients. We recommend screening for COVID-19 infection at the emergency department all acute surgical patients who are waiting for hospital admission and urgent surgery. The screening work-up provides a RT-PCR nasopharyngeal swab test and a baseline (non-contrast) chest CT or a chest X-ray or a lungs US, depending on skills and availability. The management of COVID-19 surgical patients is multidisciplinary. If an immediate surgical procedure is mandatory, whether laparoscopic or via open approach, we recommend doing every effort to protect the operating room staff, for the safety of the patient.


Ubiquitin is a 76 amino acid polypeptide common to all eukaryotic organisms. It functions as a post-translationally modifying mark covalently linked to a large cohort of yet poorly defined protein substrates. The resulting ubiquitylated proteins can rapidly change their activities, cellular localization, or turnover through the 26S proteasome if they are no longer needed or are abnormal. Such a selective modification is essential to many signal transduction pathways particularly in those related to stress responses by rapidly enhancing or quenching output. Hence, this modification system, the so-called ubiquitin-26S proteasome system (UPS), has caught the attention in the plant research community over the last two decades for its roles in plant abiotic and biotic stress responses. Through direct or indirect mediation of plant hormones, the UPS selectively degrades key components in stress signaling to either negatively or positively regulate plant response to a given stimulus. As a result, a tightly regulated signaling network has become of much interest over the years. The ever-increasing changes of the global climate require both the development of new crops to cope with rapid changing environment and new knowledge to survey the dynamics of ecosystem. This review examines how the ubiquitin can switch and tune plant stress response and poses potential avenues to further explore this system.  As global climate changes and the occurrence of more extreme weather patterns becomes more frequent, organisms rely much more than ever on effective mechanisms to buffer the extremes of external environmental fluctuations. This is critical to keep a relatively stable internal environment and maintain active metabolic reactions. To survive from a sudden influx of water resulting from devastating hurricanes, for example, requires the sensing, activation, and expression of water-specific genes such as those encoding aquaporin or vacuolar invertase in plants ,2. On thUb is extremely conserved across the kingdoms of eukaryotic organisms. For example, only three amino acids differ between human and plant Ubs . During Arabidopsis thaliana (Arabidopsis hereafter) seedlings up to fourfold, suggesting that greater than 80% of the total ubiquitylated proteins are degraded by the 26S proteasome (unpublished data). The 26S proteasome is a “cylinder-shaped” enzymatic complex composed of one or two 19S regulatory particles (RP) that cap the two ends of a 20S central core particle (CP) . DR. DR84].  is mild . Additioelements . BPM degrades DREB2A in an NRD-dependent manner. Biochemical data indicate that the NRD domain, by itself, is able to sufficiently interact with BPM. Expressing DREB2A missing the NRD allows for increased accumulation of DREB2A and activation of genes associated with drought response and thermotolerance , ,22, 5,22 to date . AlthougF-box gene family as an example to illustrate future directions in tackling the role of the large group of UPS genes in plant growth and development, particularly in stress response.Functional redundancy due to high rate of gene duplications, low abundance of both E3 and ubiquitylation substrates, short period when a particular UPS pathway is effective, challenges in identifying pairwise relationship between an E3 ligase and its substrate(s), and promiscuous functions of core UPS components could all contribute to the hardship of functional genomic studies in this field [F-box gene family whose members comprise the largest group of CRL E3 ligases. On the basis of the weak correlation of the size of the F-box family with the genome complexity and the large proportion of silenced F-box genes that are under epigenomic suppression, we hypothesized that the plant F-box gene family is under a genomic drift evolution [F-box gene family from previous 18 plant genomes to 111 genomes that allowed us to model the distribution patterns of F-box genes [F-box genes were discovered to be significantly distinct in evolutionary conservation and duplication patterns. While the largest cluster is lineage-specific and on average only present in 3 out of 111 genomes, the second largest cluster is specific to Brassicales. Statistical modeling suggests that the distribution of these two clusters of F-box genes is not a stochastic event. In addition to our unpublished data showing that overexpression of an F-box gene often results in a detrimental phenotype, we proposed a purifying selection model to explain the genomic drift evolution of these two clusters of F-box genes [F-box genes are only limited in a small group of plants. Only when the activity of the F-box genes is suppressed, such as epigenomic suppression in Arabidopsis, would they be able to significantly duplicate in a plant genome [F-box gene family is not determined by the genome complexity but the presence of a mechanism that can suppress the activity of F-box genes. Through genome-wide comparison and phylogenetic analysis, we discovered a novel evolutionary mechanism in the volution ,167. Recox genes . Four clox genes . Becauset genome . TherefoF-box genes may be activated to play adaptive roles through evolutionary innovation [F-box members in Arabidopsis. On the basis of genomic, expression, and evolutionary features of the 83 known Arabidopsis F-box genes, we utilized a neural network machine learning approach to predict a handful number of unknown F-box genes to be active with high confidence [F-box genes characterized, we would be able to effectively identify the functions of active F-box gene members rather than being trapped in a large group of inactive ones. Such an integrative data analysis approach could be also adopted into the functional genomic studies of several other UPS families, in which many members are yet uncharacterized.However, like genetic drifted alleles, some genomic drifted novation . These mnfidence . Thus, t


Altered metabolism is one of the hallmarks of the cancer cells which reciprocally interrelate with epigenetic processes, such as post-translational histone modifications to maintain their desired gene expression profiles. The role of beta-hydroxybutyrate as a ketone body in cancer cell biology and histone modifications are reported. The present study aimed to evaluate the impacts of long-term metabolic reprogramming via glucose restriction and beta-hydroxybutyrate treatment on histone acetylation and butyrylation in MDA-MB231 cells as a model of triple negative stem-like breast cancer. For long-term treatment, cells were set up in three groups receiving DMEM with restricted glucose (250 mg/L), DMEM with restricted glucose but enriched with five millimolar beta-hydroxybutyrate and DMEM with standard glucose (1g\L) and investigated for a month. Histone modifications, including H3 acetylation and butyrylation, were investigated by immunoblotting after an acid extraction of the histone proteins. Neither beta-hydroxybutyrate enrichment nor glucose restriction elicited a significant effect on the butyrylation or acetylation level of histone H3 upon a long-term treatment. Metabolic plasticity of cancer cells, mainly stem-like triple negative breast cancer cells alleviate or neutralize the impact of long-term metabolic reprogramming via restriction of glucose and histone modifications enrichment. These results shed new light upon the mechanism of controversial efficacy of ketogenic diets in clinical trials.  Cancer is still one of the leading causes of death worldwide, and breast cancer is one of the most common types of cancer in the well-developed countries, but over the past 30 years, the trend of breast cancer deaths in developing countries has been increasing . Breast cancer, based on the expression of estrogen and progesterone receptors, and HER-2, are divided into subgroups in which the subgroup lacking those markers are called triple negative breast cancer (TNBC). This type of breast cancer accounts for 10% to 20% of all types of breast cancer, and at the time of diagnosis, they have a high mitotic index, poor prognosis and less response to the conventional treatments . TNBCs are part of basal-like breast cancers which are less differentiated and look a lot like a small population of cancer cells known as the cancer stem cells (CSCs) .+CD24low- phenotype . In contrast to other cancer cell populations, CSCs have specific characteristics that make them more aggressive and more resistant to the current therapies. Such therapies mainly eliminate the dominant sensitive populations of cancer cells which provide better access to the metabolic fuels for remaining resistant CSC and prevail in cancer recurrence and metastases. Among solid tumors, CSCs was firstly identified in breast cancer as the cells with CD44It is well documented that the formation of a cancer cell is accompanied by an increase in the accumulation of genetic and epigenetic changes which gradually confer strong proliferative, and aggressive properties . Epigenetic changes such as CpG island DNA methylation and post-translational changes of histone proteins are related to changes in cell phenotype without alteration in the DNA sequence that alters genes expression . Post-translational changes such as histone acylation , have occurred in histone proteins and cause silencing or activation of the gene expressions . Inappropriate regulation of DNA and post-translational modifications of histones are the frequent occurrences in a large number of diseases, including cancer and neurological abnormalities . Butyrylation has been recently studied in different physiological conditions, such as the differentiation of germ cells and revealed that, like acetylation, it could enhance the expression of certain genes . Recent studies showed a solid reciprocating interrelationship between metabolism and post-translational modification of histone proteins .For years, we know that cancer cells convert glucose to lactate in the presence or absence of oxygen, a phenomenon known as the Warburg effect, which produces ATP via glycolysis. Increasing the conversion of pyruvate to lactate during aerobic glycolysis results in acidosis in the tumor microenvironments that facilitates invasion and metastasis of cancer cells . However, metabolic plasticity is a newfound hallmark of cancer cells which shows that how different population of cancer cells within a tumor are using different metabolic pathways. Elucidation of characteristics of cancer cell metabolism and tailoring the therapeutic strategies are part of current scientific efforts . In addition to glucose, ketone bodies, in the process of intrinsic ketosis due to starvation and prolonged exercise, work as alternative fuels . However, extrinsic ketosis by using ketogenic diet can also increase serum beta-hydroxybutyrate levels . Ketogenic diet by restricting calories from the source of carbohydrates and providing a source of fat leads to induction of ketosis in the recipient . Clinical trials and experimental studies have shown that beta-hydroxybutyrate has anti-cancer and anti-inflammatory effects . Studies have shown that dietary ketones in metastatic cancer mice increase survival rates by 70-60% .On the other hand, it has shown that the use of ketones could lead to tumor growth and metastasis . Contradictory results reveal that the precise mechanism of the function of this type of chemicals in cancer biology is still not well defined. Evidence of the interrelationship between ketone bodies and the regulation of epigenetic modifications has also been reported, which can provide a mechanism for linking energy metabolism in cells and regulating gene expression through histone modifications .The current challenges in the treatment of stem-like triple negative breast cancers and the ambiguity in the impacts of beta-hydroxybutyrate on the post-translational modification of histones, motivated us to investigate the effect of beta-hydroxybutyrate as a ketone body on the occurrence of histone butyrylation of MDA-MB231cells as a model of stem-like triple negative breast cancer.low / - phenotype. The authentication of the cell line was certified by NCBI via STR studies. Initially, cells were cultured in low glucose DMEM medium and were incubated in a CO2 incubator at 37°C, 96% humidity and 5% CO2. Culture vessels were regularly examined for growth and contamination.In this research, the MDA-MB231 cell line was purchased from the Iranian Banking Bank (NCBI) as the TNBC cell line with the CD44highCD24Cytotoxicity study50 was obtained by non-linear fitting using GraphPad Prism 6 software.The MTT assay was used to evaluate cytotoxicity. For an MTT assay, 5000 cells were seeded in each well of a 96-well plate. The plate was placed in an incubator for 24 hours to allow the cells to attach to the wells. Cells were treated with a range of beta-hydroxybutyrate  concentrations between 25 and 1,000 mM in PBS at 37 ° C for 48 hours. MTT solution was added in each well, and the plate was incubated for 4 hours. The formazan crystals were dissolved entirely by adding DMSO. The absorbance was read at 595 nm and 620 nm reference wavelengths using the multimodal reader. The value of ICCell treatmentFor a long-term study, cells were set up in controls (DMEM medium with 1 g/L glucose), ¼ glucose-restricted cells (DMEM with 250 mg/L glucose), and ¼ glucose-restricted cells enriched with 5 mM beta-hydroxybutyrate and all were maintained for 30 days. In all studies, culture media were enriched with 10% FBS, glutamine and bicarbonates. Extraction of histonesCells were Scraped and washed in PBS. Then, they were lysed using RIPA lysis buffer containing appropriate ratio of protease inhibitor cocktail (X100) and were sonicated twice for 5 seconds (power 50% and duty 50%) on ice. The supernatant containing the total protein was extracted by final centrifugation. Sulfuric acid (0.2 M) was added to the total protein and samples were incubated in 4°C overnight on a roller mixer. The next day, the samples were centrifuged  for 15 minutes at and 4°C. The supernatant was transferred to new tubes and the histone proteins were precipitated by TCA (20 %.) after 30 minutes incubation on ice and a 10 minutes centrifugation  at 4°C. The precipitates were washed with 0.1% acetone / HCl and pure acetone. Finally, the precipitates were solved in urea (8 M). The extracted proteins were quantified using Bradford reagent and Bovine serum albumin (BSA) as the standard.Protein electrophoresisFor SDS-PAGE, a 15% separation and a 5% stacking gel were cast and run using Tris-glycine buffers. Five to ten microgram of total protein and four to eight microgram of acid extracted histones were loaded, and the electrophoresis was first performed for 30 minutes at 80 volts and then for 45 minutes at 120 volts. Then, the gel was stained for 1 hour with Coomassie Blue R-250 and stripped off using SDS-PAGE destaining solution (Methanol 30%-Acetic Acid 10%). ImmunoblottingButyrylation and acetylation of histones were assessed by using immunoblotting or western blotting. The proteins were transferred to nitrocellulose membranes using a wet transfer tank at a voltage of 90 V for an hour. Ponceau S staining was performed to ensure that the proteins were transferred to the membrane and stripped off using TBS-T buffer. The membranes were blocked using 5% Skimmed Milk or 5% BSA. Pan anti-butyryllysine , Pan anti-acetyllysine , Histone H3 (PTM-1001) and H4 antibodies  were used as the primary antibodies with 1:1,000 dilution in 5% Skimmed Milk or 5% BSA. Appropriate HRP-conjugated secondary antibodies were utilized to detect the primary antibodies, and the bands were visualized by ECL  solution on X-ray films. +CD24low/- phenotype (96.3%±1.7), the cell line was used without further cell sorting. The impact of long-term treatment (one month) of a triple negative stem-like breast cancer cells with a 5 M concentration of beta-hydroxybutyrate. The toxicity of beta-hydroxybutyrate was measured by MTT assay, and its IC50 value was 103.1 mM . Ketone bodies are the alternative fuels that could be used as a source of energy during starvation or strenuous exercises. However, studies showed that ketogenic diets elicit beneficiary impacts on patients with different diseases including cancers. Beta-hydroxybutyrate is the most abundant and stable intrinsic ketone body, and the number of its salts offered on the over the counter supplements’ market is steadily increasing . Hence, the goal of therapeutic ketogenic diets is to achieve a concentration of 3 to 6 mM beta-hydroxybutyrate in an induction period of 7 days and could be sustained for one to three months. The impact of beta-hydroxybutyrate on epigenetic changes such as post-translational histone modifications and gene expressions have been reported . Histone butyrylation is also a significant histone modification that may regulate the expression of specific genes. Goudarzi (2016) showed that butyrylation, like acetylation, leads to increased gene expression of reproductive cells in male rats. So, in this study the status of CD24-CD44 breast cancer stem cell markers was studied in MDA-MB231 triple negative breast cancers  and due 103.1 mM . No signAfter a long-term treatment, the assessment of K-Ac and K-Bu in Histone H3 in MDA-MB231 cells was not revealed any significant changes in cells with restricted access to glucose or even after enrichment with 5 M bHB . In compIn the other study using MCF-7 cells, it revealed that bHB increases the amount of histone acetylation which in turn increases transcription of genes . The HDAC inhibitory effect of bHB was also could lead to increased histone acetylation . On the other hand, it has also shown that there is no change in the amount of histone acetylation with increasing beta-hydroxybutyrate levels, and only in high concentrations of beta-hydroxybutyrate induces a slight change in the amount of histone acetylation . Our findings with MDA-MB231 cells is consistent with the results of the later study. Such inconsistency in the results could be aroused from the different composition capacity of ketone bodies in different cells and tissue . It is proved that differential histone acylation is regulated by metabolism of the different acyl-CoA forms, which in turn modulate the regulation of gene expression and crotonyl can work as an intracellular source for beta-hydroxybutyrate under normal conditions within the cells by CDYL enzyme . This finding indicates that the histone-forming metabolites within the cells can be converted to each other, beta-hydroxybutyrate could be provided from intrinsic or extrinsic sources. The present study showed that neither K-Ac nor K-Bu of histones were changed after long-term restriction of glucose or even after enrichment with bHB in MDA-MB231 cell line as a model of triple-negative stem-like breast cancer. Such effects of long-term treatments could result of metabolic plasticity of cancer cells  which alleviated the observed induction of metabolic reprogramming by bHB and neutralized the related changes in k-But at histone proteins. Further studies are required to elucidate the exact mechanism of such plasticity and its functional impacts on the expression of different genes,


The existence of a substantial but unclear number of asymptomatic SARS-COV-2 patients worldwide has raised concerns among global public health authorities. In this review, according to the published literature, we provided the evidence that asymptomatic infections can result in person-to-person transmission. Four studies suggested that the virus can be transmitted by asymptomatic patients for at least two consecutive generations, indicating its strong infectivity. Asymptomatic infection tends to be, but is not only, identified among young people (<20 years old). The majority of asymptomatic patients appear to have a milder clinical course during hospitalization, but the severity of the symptoms of the secondary patients infected by SARS-COV-2 from asymptomatic patients varies with their physical constitution. The proportion of asymptomatic individuals among all confirmed cases widely differed (from 1.95% to 87.9%) according to the study setting and the populations studied. The increasing large-scale tests are expected to give more information about the true number of asymptomatic infections in the population. In China and other countries, various guidelines for management of asymptomatic cases have been issued. Importantly, early detection, early reporting, early isolation and early treatment of asymptomatic patients require the joint efforts of policy makers, clinicians, technicians, epidemiologists, virologists and patients. The COVID-19 pandemic caused by the SARS-CoV-2 virus is now straining or overwhelming health care systems worldwide. The clinical manifestations of COVID-19 are protean, including asymptomatic carrier, acute respiratory disease, and pneumonia of varying degrees of severity . Asymptomatic COVID-19 patients are those who carry the virus but do not show any symptoms , and no significant abnormalities on chest radiograph at time of laboratory confirmation In addition to the potent transmissibility of the virus itself, there is increasing evidence that unnoticed, asymptomatic cases in the infected population may greatly accelerate the spread of SARS-COV-2 from person to person 5Table 1). We found that (1) some asymptomatic cases at the time of the earliest test remained asymptomatic throughout the whole duration of laboratory and clinical monitoring, while others, which may account for approximately 80%, experienced clinical symptoms at a later stage of infection (presymptomatic patients) 4 to 1×107 copies per milliliter According to recently published studies, we reviewed the clinical, virological and epidemiological characteristics of asymptomatic infections, which were usually determined by reverse transcription-polymerase chain reaction (RT-PCR) (Asymptomatic transmission could be defined as the transmission of SARS-CoV-2 from an asymptomatic person (source or index patient) to a secondary patient, as ascertained by exposure and symptom onset dates, with no evidence that the secondary patient had been exposed to anyone else with COVID-19 Figure 1). In general, in these clusters, there is a clear history of contact between the source patients and the secondary patients; there is no other explanation for the infection; and according to the epidemiological survey, the source patients were asymptomatic when transmitting the virus to the secondary patients. For example, a study reported in January 2020 identified five patients with COVID-19 pneumonia infected by a 20-year-old asymptomatic woman (the source patient) who was thought to have acquired SARS-COV-2 infection from the epidemic center of Wuhan Figure ). Tong et al. identified 2 COVID-19 patients (patients A and D) after their exposure to an asymptomatic person from Wuhan who was later confirmed to be positive for SARS-CoV-2 Figure ). Li et al. confirmed asymptomatic and human-to-human transmission through close contacts in familial and hospital settings by analyzing the epidemiologic, laboratory, and clinical data of 7 COVID-19 patients in a 2-family cluster Figure ). Qian and colleagues reported a family cluster of asymptomatic COVID-19 transmission in Zhejiang China Figure ). Importantly, the latter three reports provided by Tong et al., Li et al. and Qian et al. together with another report from Luo et al. Figure ), suggesting that the virus has a strong infectivity. In another study in mainland China, the source patient (Case 13), who had a travel history to the city of Huanggang, Hubei province on Jan 19-20, 2020 and was diagnosed as an asymptomatic COVID-19 carrier, transmitted the virus to his cohabiting family members Figure ). In addition, the transmission of SARS-COV-2 infection from asymptomatic contacts has also been reported in other countries outside China, such as Germany As early as January 2020, Chan et al. reported evidence of human-to-human transmission of SARS-CoV-2 in family settings, suggesting that asymptomatic patients might serve as a possible source to propagate the outbreak Figure ), or developed varying degrees of pneumonia . Through comprehensive analysis of 24 asymptomatic COVID-19 patients screened among close contacts, the authors reported that asymptomatic carriers among the close contacts were prone to mild illness during admission. However, the communicable period could last as long as 21 days, and the communicated patients could develop severe illness In all the above reports, the source patients remained without any symptoms of infection during the clinical course  in China, Du et al. identified that presymptomatic transmission might be responsible for 12.6% (59) of confirmed cases Table 1).Now that the transmission of SARS-COV-2 through asymptomatic patients via person-to-person contact has been confirmed, reliable estimations of the proportion of asymptomatic infections among all COVID-19 patients are crucially needed to guide public health policy, especially to clarify the intensity and range of social distancing strategies to be implemented to prevent the spread of COVID-19 According to the National Health Commission (NHC) update, as of April 14, a total of 6,764 asymptomatic infections were reported nationwide in China, close to 8% of all confirmed SARS-COV-2 cases  However, most of the above results were not obtained by random sampling or testing all individuals in the population, but mainly through the analysis of screening data from high-risk groups . Thus, these early results might not accurately reflect the real situation of the entire population. The large-scale population tests launched in increasing countries/regions are expected to reveal the true number of SARS-COV-2 infections in the population and the contribution of asymptomatic individuals. Recently, Iceland reported the results of its population screening based on nucleic acid testing, and found that 43% of the participants who tested positive had no symptoms The current first-line laboratory diagnostic tests for COVID-19 worldwide include nucleic acid testing (mainly RT-PCR) and rapid serological antibody (mainly IgM and IgG) testing. As the transmission of SARS-COV-2 may occur in the early course of infection and a high viral load in respiratory samples could be detected Detecting serum antibodies is an effective supplement to nucleic acid assays. Data show that the positive rate can be increased from 51.9%, for a single RT-PCR test, to 98.6% when combining an IgM antibody assay with an RT-PCR test . Once the individuals complete the 14-day quarantine and test negative twice in a row with one test every 24 hours, they are released from medical observation. Those who show no symptoms and still test positive will be kept under centralized medical observation. If asymptomatic cases display clinical manifestations during the period of centralized medical observation, they shall be immediately transferred to designated medical institutions for standardized treatment in accordance with the confirmed cases Asymptomatic cases are easily neglected in disease screening and epidemic prevention. The asymptomatic COVID-19 patients who have been identified include close contacts of COVID-19 cases during their medical observation period, individuals involved in cluster outbreaks, people who were unknowingly exposed to infected patients and were identified when the infection source was being traced, and individuals who recently traveled to areas/countries/regions with high infection rates  53There are currently no proven pharmaceutical treatments . Rigorous implementation of traditional public health measures such as isolation and quarantine, social distancing and community containment is now the preferred option to reduce transmission of the virus Furthermore, for personal protection, wearing masks is another important public health intervention for limiting the spread of respiratory pathogens. As transmission from asymptomatic infected individuals has been documented for COVID-19, wearing masks in public places would help to intercept the transmission and prevent the spread by these apparently healthy infectious sources (asymptomatic patients) Addressing COVID-19 is now an urgent health and social concern. However, controlling the pandemic only by detecting, isolating and treating symptomatic or critically ill patients is not sufficient because accumulating evidence has confirmed the asymptomatic transmission of SARS-COV-2. Several studies have even suggested that the virus can be transmitted by asymptomatic patients for at least two consecutive generations, indicating its strong infectivity. Therefore, it is of great public health significance to screen and manage asymptomatic patients and their close contacts via multiple strategies to contain potential outbreaks. Several scattered studies have evaluated the contribution of asymptomatic patients in the population. However, there is no comparability between their results due to the differences in the distribution and size of the studied populations, so the true scale of asymptomatic infections remains unclear. Ongoing large-scale population screening is expected to answer this question. Studies have provided a cautionary warning that SARS-COV-2 virus may be transmitted through multiple routes in addition to the most common respiratory droplet spread. It is necessary to fully consider the sample type and sampling time to improve the detection rate. In addition, a combination of molecular and serological tests is needed to identify virus carriers when necessary. Asymptomatic patients themselves should complete the standard isolation and treatment process as required by governments and doctors. Additionally, when considering that the spread of SARS-COV-2 may occur through respiratory droplets, contact with contaminated objects or fecal viral shedding, which is currently being investigated


Thus, some staff are uncomfortable managing a patient using standard precautions, especially if the patient has undergone repeat testing and remains positive for severe acute respiratory coronavirus virus 2 (SARS-CoV-2) as indicated by real-time polymerase chain reaction (RT-PCR) testing. For hospitals that routinely test all admissions, should patients who have recovered from COVID-19 be included? And how should these patients be managed when it comes to preprocedure testing?As the coronavirus disease 2019 (COVID-19) pandemic has evolved, survivors are now returning for care at hospitals and ambulatory sites, creating confusion and uncertainty for the frontline staff regarding proper management of these patients. Although the US Centers for Disease Control and Prevention (CDC) provides clear guidance on both symptom-based and test-based strategies for discontinuation of transmission-based precautions,In the absence of definitive guidance or good-quality published evidence, healthcare epidemiologists must use the best available evidence to make practical and rational decisions. Making these decisions often requires acknowledgement and appropriate balance of 3 important goals: (1) keeping patients and employees safe from harm, which includes avoiding unnecessary delays in care for COVID survivors; (2) ensuring that resources, such as personal protective equipment (PPE) and testing reagents and materials, remain available for the highest-risk situations; and (3) ensuring that staff feel comfortable coming to work and caring for patients. To accomplish these goals, explicitly communicating what is known and not known about the epidemiology, biology, transmission, and mitigation strategies for COVID-19 often becomes necessary while acknowledging that new information will continue to emerge and that guidance may change as a result.2; however, we also have evidence demonstrating that, for uncomplicated, recovering COVID-19 patients, replication-competent virus cannot be cultured >9–11 days after onset of illness.4 The CDC’s unpublished data indicate that the statistically estimated likelihood of recovering replication-competent virus approaches zero by 10 days, which is the basis for their symptom-based strategy for discontinuing isolation precautions.5 In May 2020, the Korean CDC published evidence that patients with COVID-19 who had late positive  RT-PCR testing after interim negative tests did not transmit the infection to close household contacts.6 Of 285 monitored cases with 790 contacts, no case was found that was newly infected solely from contact with a “re-positive” case. They obtained respiratory samples from 108 of these patients and were unable to culture viable virus from any of them. For 23 patients, blood samples were also obtained, and all were positive for neutralizing antibodies. These data support the US CDC’s symptom-based strategy by demonstrating that close contacts of patients with prolonged detection of SARS-CoV-2 by PCR did not become infected and, thus, that these patients were not contagious despite having repeated positive RT-PCR results.The goal of initial SARS-CoV-2 RT-PCR testing is to identify patients who have active COVID-19 for clinical decision making and to identify patients who require transmission-based precautions to prevent the spread of infection. Once a patient has a positive RT-PCR result, 2 options exist for discontinuing transmission-based precautions: (1) test-based strategies, which generally require 2 negative tests at least 24 hours apart to end isolation and (2) symptom-based strategies, which discontinue isolation typically 10 days after symptom onset and resolution of fever for at least 24 hours without antipyretics and improvement in respiratory symptoms. We now have accumulated evidence that SARS-CoV-2 remains detectable by RT-PCR for an average of 3 weeks7 which included primarily ICU patients found infectious viral shedding in only 23 of 129 patients (18%), with a median duration of shedding of 8 days after symptom onset. Interestingly, viable virus was detected in only a small number of patients, with the longest detected at 20 days. The likelihood of detected viable virus was <5% after day 15.Accumulating evidence suggests that symptom-based strategies are sound for uncomplicated cases of COIVD-19, but data evaluating immunocompromised patients, such as solid organ transplant patients and oncology patients, and those with severe illness are scarce. A recent publication from The Netherlands,In light of this evidence, we suggest that most previously positive patients who return for care and who have met the CDC’s symptom-based strategy for discontinuation of transmission-based precautions need not be retested prior to readmission or prior to a procedure. Furthermore, they can be safely managed using standard precautions. For the subgroup of complicated patients who may have the potential to shed live virus for longer, such as those who required intensive care or are severely immunocompromised, a more nuanced approach may be needed. In these cases, the CDC guidance suggests that patients should be isolated for up to 20 days. The above data are reassuring that even in severely ill patients the more conservative time period ensures that no infectious virus is present and even those who persistently test positive could be removed from isolation. Test-based strategies may be considered to help end isolation earlier than 20 days. However, from a practical perspective, it is challenging to simultaneously implement test-based and time-based strategies effectively. One strategy is to discourage repeat testing and base decisions solely on time-based criteria for most patients.Ensuring that frontline staff understand the rationale for institutional recommendations, feel comfortable with the evidence, are willing to follow the guidance, and understand how to manage these patients requires an understanding of the barriers that often hamper the acceptance and roll-out of policies. Frequently modified written policies based on the most up-to-date evidence and that are developed with the input of multiple stakeholders will promote ownership in key areas of the organization. Education of nursing and medical staff through departmental leadership and staff meetings will be required to gain buy-in. Concerns should be addressed regarding areas that may be considered “higher risk,” such as procedural areas, oncology wards, and treatment centers. Especially in situations that are novel and fraught with uncertainty or fear, such as the COVID-19 pandemic, enlisting the trust of hospital staff in hospital leadership is key to success. When resistance is met, a system for the real-time escalation of individual cases to well-informed medical leadership  should be in place (1) to allow hospital leadership to listen carefully to staff concerns, (2) to provide simultaneous education of staff and resolution of the challenging situation, (3) to provide appropriate management and placement of the patient, and (4) to provide clarity to team members, families, and the patient, thereby engendering further trust.