Modern technologies enable the exchange of information about the expansion of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection and the continually increasing number of the coronavirus disease 2019 (COVID-19) cases almost in real time. The gravity of a current epidemiological situation is represented by the mortality rates, which are scrupulously updated daily. Performing autopsies on patients with either suspected or confirmed COVID-19 is of high importance since these might not only improve clinical management but also reduce the risk of SARS-CoV-2 infection expansion. The following paper aimed to present the most crucial aspects of SARS-CoV-2 infection from the point of view of forensic experts and pathologists, recommendations and safety precautions regarding autopsies, autopsy room requirements, possible techniques, examinations used for effective viral detection, recommendations regarding burials, and gross and microscopic pathological findings of the deceased who died due to SARS-CoV-2 infection. Autopsies remain the gold standard for determining the cause of death. Therefore, it would be beneficial to perform autopsies on patients with both suspected and confirmed COVID-19, especially those with coexisting comorbidities. The COVID-19 pandemic, also known as ‘coronavirus disease of 2019′, has become a worldwide concern due to severe acute respiratory syndrome with coronavirus 2 (SARS-CoV-2), going far beyond the actual capabilities of local healthcare systems . A globaDiagnostic procedures depend on the degree of disease severity, and they are different for asymptomatic but suspected patients and for those who present with specific clinical manifestations. In addition, tests for influenza or other pathogens can cause respiratory infections, and specific laboratory tests (complete blood count (CBC), arterial blood gas assessments) and radiological tests (primarily computed tomography (CT), RTG, or ultrasonography (USG)) are performed on a regular basis. The three diagnostic strategies – genetic tests, antigen tests, and antibody tests – currently constitute a routine laboratory diagnosis of SARS-CoV-2 infection. Reverse transcription real-time polymerase chain reaction (rRT-PCR) is currently a basic technique that enables a relatively quick detection of SARS-CoV-2 infection .SARS-CoV-2, recently categorized as an HG3 organism, constitutes a significant risk for employees who face infected patients –10. The Although the clinical features and radiological findings of SARS-CoV-2 infection have been well described so far, the early detection of potential SARS-CoV-2 infection would significantly minimize the epidemiological risk. Invariably, a classical autopsy remains the gold standard for determining the causes and mechanisms of death, as it also allows a gross examination and microscopic examination . It is cSafety requirements and proper techniques for the autopsy would significantly improve the management of a continually increasing number of deceased individuals with COVID-19. In addition, a full autopsy with both macroscopic and microscopic examinations would provide insight into manifestations of SARS-CoV-2 infection that might not be easily detected ante mortem. According to the WHO, bodies are generally not infectious except for patients with hemorrhagic fever (such as Ebola or Marburg) and cholera; however, there is no evidence of whether the deceased with COVID-19 might constitute a possible source of infection . FurtherAccording to Chinese recommendations, there are three types of protection levels whose application depends on the possible risk of infection during the autopsy of patients with either suspected or confirmed COVID-19  announced that the autopsy of people who died due to COVID-19 should follow the same procedures and standards as in patients who died due to any other acute respiratory illness. Since the lungs of patients with pandemic influenza might be infectious when handled improperly, additional respiratory protection is recommended during the autopsy of patients with suspected and confirmed COVID-19 . FurtherAccording to The Royal College of Pathologists, in a deceased individual with SARS-CoV-2 infection, both external and internal examinations are equally important . The uniThe samples collected for further diagnosis should include those from the upper respiratory tract (nose and throat swabs) and the lower respiratory tract , as well as blood for serology , 19. In While conducting an autopsy, standard contact and airborne precautions should be followed . FurtherThe Centers for Disease Control and Prevention (CDC) emphasize that the collection of samples from the deceased differs depending on whether the case is confirmed and whether an autopsy will be performed . When anThe College of American Pathologists (CAP) recommends that the autopsies of patients with either confirmed or suspected COVID-19 be performed only by personnel who are trained in performing autopsies on deceased individuals with emerging infectious diseases using all the necessary PPE . The CAPDuring the autopsy of an individual with suspected COVID-19, the following PPE should be applied: headgear, a double pair of gloves, cut-resistant protective gloves, FFP3, goggles or a protective visor, a long-sleeved gown or a waterproof suit and overshoes. In a confirmed COVID-19 case, with both clinical and imaging confirmation, either postmortem core biopsies or a discretionary autopsy alone should be performed. When there is a need to collect samples from a body of the deceased with either suspected or confirmed COVID-19, it is also recommended that samples from multiple organs  be collected applying core biopsy sampling. Furthermore, a multidisciplinary group including at least one clinician and one radiologist should request sample collection. In patients with suspected or probable COVID-19, both nasopharyngeal and oropharyngeal swabs should be collected; if the tests are negative, an autopsy can be performed . In patiKnowledge about the persistence of SARS-CoV-2 on different surfaces is limited only to laboratory settings, with no precise confirmation in reality. It is yet unknown how long SARS-CoV-2 can persist on the clothes and bodies of the deceased. It was found that the stability of SARS-CoV-2 is similar to that of SARS-CoV-1; however, this was proven only under experimental conditions . AccordiThe results of available studies show that SARS-CoV-2 detection is possible mainly in secretions from the respiratory tract. The highest positive rates of SARS-CoV-2 detection are noted in bronchoalveolar lavage fluid, followed by sputum, nasal swabs, fibrobronchoscope brush biopsy, pharyngeal swabs, feces, and blood  , 39. An Samples for etiological and electron microscopy testing and cryopreservation should be collected first.Samples for etiological gene testing should be stored in Hank’s solution (nucleic acid-free before use) or transfer medium dedicated to virological tests.Tissues that require freezing can be cut into blocks and stored in plastic bottles.Tissues that require an electron microscopy examination can be cut into blocks and stored in 3% glutaraldehyde.Tissues for conventional paraffin embedding can be stored in a 4% paraformaldehyde solution for approximately 48–72 h before further examination .Before an anatomical examination and autopsy, all the samples for further testing should be collected to minimize the risk of potential contamination . GeneralSamples that are recommended to be obtained during an autopsy include 2 samples from the peripheral blood, 1 sample from the blood containing an anticoagulant for genetic examinations, 1 sample from urine, 1 sample from cerebrospinal fluid, and 2 samples from vitreous humor, as well as pleural, pericardial, or peritoneal fluid, if possible , 41. SARThe laboratory diagnosis of SARS-CoV includes sample collection from different sites. Tracheal aspirate and stool samples are believed to have the highest detection yield; throat samples pooled with nasal, rectal, and throat swabs and nasopharyngeal aspirate present a moderate detection yield; and throat washing and urine samples present the lowest detection yield . SARS-CoRegarding MERS-CoV, the specimens include samples from the lower respiratory tract  and upper respiratory tract  .An oscillating or hand-held saw Antiviral disinfectants and specific devices for fumigation decontamination  A separate table for sample preparation for further examinations Medical waste containersVideo transmission of the autopsy for the prosecutors  , 22. The rooms where autopsies are performed should meet specific technical requirements, with a particular emphasis on the ventilation devices. First, two zones should be designated – a 'clear area' and a 'dirty area' – and an additional 'intermediate zone' for the removal of protective clothing after the autopsy . AutopsiThree major examinations are the most important regarding COVID-19 confirmation: real-time polymerase chain reaction, immunochemistry, and electronic microscopy. Serological tests depend on the so-called ‘serological window’, which, in the case of SARS-CoV-2, lasts for 7 to 14 days . Both moThe two most common imaging characteristics of COVID-19 are ground-glass opacities and lung consolidation , 67. LesCurrently, there are no sufficient data on the viability of PMCT as a postmortem diagnostic tool for patients with suspected or confirmed COVID-19. However, according to Italian recommendations, PMCT has been included in the decision tree of the autopsy, and its utility is equal to that of swab collection . The senAn autopsy requires the confirmation of SARS-CoV-2 infection, usually via sample collection and further rRT-PCR tests. The majority of macroscopic features of COVID-19 are observed primarily within the chest cavity, and the most common findings include lung consolidation, pulmonary edema, pleurisy, or pericarditis; additionally, the weight of the lungs might be increased , 43. MacEdler et al. described a cohort of 34 deceased females and 46 males who died because of multiorgan failure related to COVID-19 . The hisSeveral nonspecific manifestations typical of viral infections include edema, hyperplastic pneumocytes, multinucleated giant cells, proteinaceous/fibrinous exudates or patchy inflammation . In someThe lung parenchyma of patients with COVID-19 might contain inflammatory components characterized by numerous CD68-positive macrophages and only a few CD45 lymphocytes that are found primarily in the interstitial space . Lung saPatients who die because of COVID-19 can be embalmed, buried or cremated depending on the preferences . As peopThe Catholic Cemeteries & Funeral Services (CCFS) prepared protocols to handle individuals who died because of COVID-19 that were adjusted for specific situations; mortuary services have continued without interruption in Catholic Church, and the procedures might involve traditional burial, cremation or embalming . The EurGenerally, families and friends may see the body before the burial; however, the restrictions concern physical contact. In addition, under the principles of social distancing, the number of mourners must be limited. Individuals who have certain respiratory diseases should not be allowed to see the body; however, it is possible only if the person wears a protective mask. Furthermore, children, adults over 60 years old, and immunosuppressed individuals should not have contact with the body. During all the activities associated with body preparation for burial, the usual principles of Standard Infection Control Precautions and Transmission-based Precautions should be followed. The risk assessment regarding the funeral premises should include known or suspected infection hazards, the timing of procedures, the number of required staff, and whether additional observers should be excluded . Burial SARS-CoV-2 has been reported to present certain survivability in the bodies of infected patients. There are still speculations as to whether individuals with confirmed COVID-19 should receive an autopsy. However, during times of rapid pandemic expansion and the urgent need for epidemic preservation, the need for an autopsy in both suspected and confirmed cases seems rational since it expands our knowledge of the mechanism of action of SARS-CoV-2 and organs that are targeted by the virus. An autopsy allows the exact macroscopic and microscopic examinations of suspected and confirmed cases. In addition, an autopsy provides an accurate diagnosis and confirms ante mortem laboratory and radiological findings, especially in suspected cases. Furthermore, an autopsy allows the detection of coexisting diseases as well as the assessment of the extent of SARS-CoV-2 infection. Although there have been an increasing number of studies regarding the clinical manifestations and complications of COVID-19, knowledge about the pathologic postmortem manifestations, both macroscopic and microscopic, is still scarce. Since the pandemic is still expanding and the number of infected patients is continually increasing, there is an urgent need to report postmortem findings to expand our knowledge on the pathology of COVID-19. To increase the efficiency of work while minimizing the risk of exposure of medical personnel to the potential source of infection, it is highly recommended to introduce PMCT on a regular basis while simultaneously performing needle biopsy to collect lung parenchyma specimens instead of opening all body cavities. For forensic experts and pathologists, it seems necessary to collect different samples because viral detection might differ depending on the stage of the disease. Minimally invasive biopsy for a postmortem histological examination and the application of a virtual autopsy  might constitute alternatives for a typical autopsy, significantly minimizing the risk of potential infection.1. Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is characterized by survivability in the bodies of the deceased who died because of SARS-CoV-2 infection.2. Autopsies are rational for patients with both suspected and confirmed SARS-CoV-2 infection.3. Postmortem findings of SARS-CoV-2 infection include macroscopic and microscopic changes, as well as altered radiological findings.4. SARS-CoV-2 might be detected in various samples obtained postmortem, such as respiratory tract samples, blood, feces, cerebrospinal fluid, or urine.


Coronavirus disease 2019 (COVID-19) has spread throughout the world since late 2019. Symptoms appear after a two-week incubation period and commonly include fever, cough, myalgia or fatigue, and shortness of breath.A 32-year-old male with a history of opiate abuse presented to the emergency department with altered mental status. The patient was lethargic and hypoxic with improvement from naloxone. Official chest radiograph was read as normal; however, the treating clinicians noted bilateral interstitial opacities, raising concern for underlying infectious etiology. Opiates and cocaine were positive on drug screen, and an arterial blood gas on room air showed hypoxemia with respiratory acidosis. The patient was intubated during the treatment course due to persistent hypoxemia and for airway protection after resuscitation. The COVID-19 test was positive on admission, and later computed tomography showed ground-glass opacities. The patient was extubated and discharged after one week on the ventilator.When screening patients at and during evaluation, physicans should consider a broad differential as patients with atypical presentations may be overlooked as candidates for COVID-19 testing. As screening and evaluation protocols evolve, we emphasize maintaining a high index of suspicion for COVID-19 in patients with atypical symptoms or presenting with other chief complaints in order to avoid spreading the disease. The main objective in this case study was to promote a bottoms-up approach in tackling the coronavirus disease 2019 (COVID-19) pandemic, as it has a wide variety of presentations in different individuals across the globe. Throughout this case, we were able to use multiple points of re-evaluation to uncover COVID-19 induced hypoxemia initially presenting as an opiate overdose.In December 2019, a cluster of patients who had been admitted to various hospitals in the eastern region of China were diagnosed with pneumonia of unknown etiology. After undergoing a series of medical evaluations, it was concluded that these individuals were epidemiologically linked to a seafood and wet-animal wholesale market located in Wuhan, Hubei Province, China. Upon further review, it was determined that these patients had been infected with a novel variation of coronavirus.1Coronavirus is a major pathogen that predominantly targets the human respiratory system. Symptoms of this virus, on average, appear after an incubation period of approximately 5.2 days. Onset of symptoms to death varies between 6–41 days, with a median onset period of 14 days. Mortality is predicated on various risk factors including the patient’s age and comorbidities.At the onset of illness in the setting of the COVID-19 pandemic, most common presentations in patients include fever (77–98%), cough 46–82%), myalgia or fatigue (11–52%), and shortness of breath (3–31%).%, myalgiLab values that help identify COVID-19 have included leukopenia and lymphopenia, aspartate aminotransferase (AST) and alanine aminotransferase (ALT) elevation, increase in acute inflammatory markers, and low procalcitonin.A 32-year-old male presented to the emergency department (ED) with altered mental status secondary to drug overdose as reported by emergency medical services (EMS). EMS gave the patient one round of naloxone with reported improvement of the patient’s respiratory status. Patient chart review was notable for a history of opiate dependence and enrollment in suboxone and methadone centers. History of presenting illness was limited due to the patient’s presenting mental status.Presenting vital signs were notable for a respiratory rate of 25 breaths per minute and oxygen saturation of 75% on room air, with improvement to 95% on a non-rebreather mask. The patient was lethargic but arousable and was able to move all four extremities spontaneously. Initial lab work was notable for a glucose of 87 milligrams per deciliter (mg/dL) (70–130 mg/dL) and a urine drug screen that was positive for opiates and cocaine. The official CXR read was as normal; however, the treating clinicians were concerned about the subtle appearance of bilateral interstitial opacities.After a period of observation, the patient had multiple episodes of emesis. Additionally, he was noted to have no improvement in mental status or ability to oxygenate; therefore, crystalloid fluids, naloxone, ondansetron, and clonidine were given in an attempt to reverse the reported opiate overdose and prevent withdrawal symptoms. On re-assessment, physical exam revealed sonorous respiration and inspiratory stridor; thus, it became more apparent that an underlying pulmonary and metabolic pathology was contributing to the patient’s hypoxemia and lethargy, rather than just opiate use.What do we already know about this clinical entity?Coronavirsus disease 2019 (COVID-19) is a world wide pathogen with a varied symptom course. Major health organizations recommend measures to prevent spread between asymptomatic individuals.What makes this presentation of disease reportable?Early in the COVID-19 pandemic and before routine testing, this patient was identified with COVID-19 infection in addition to opiate overdose with hypoxia.What is the major learning point?As the COVID-19 pandemic wanes, maintaining a high index of suspicion for asymptomatic or occult infection is important in disease control.How might this improve emergency medicine practice?This case highlights the importance of continuous re-evaluation for multiple disease processes underlying a pre-established diagnosis.Once the patient’s family arrived at the hospital, they noted that he had recently traveled to Israel for drug rehabilitation approximately one week prior to his hospital visit. However, they believed that he continued to use intravenous drugs upon arrival home. At that time, Israel already had confirmed COVID-19 positive cases.4The patient was moved into a negative pressure isolation after travel history correlated with the known abnormal CXR and hypoxemia because of a high degree of suspicion of COVID-19. Arterial blood gas on room air showed a pH of 7.26 (7.35–7.45), partial pressure carbon dioxide of 60 millimeters of mercury (mmHg) (35–45 mmHg), partial pressure oxygen of 47mmHg (80–100 mmHg), and bicarbonate of 27 milliequivalents per liter (mEq/L) (22–28 mEq/L), demonstrating hypercarbic hypoxemic respiratory failure, and the patient’s oxygen saturation began to drop to 85% on a non-rebreather.The decision was made to intubate the patient for hypoxemia and airway protection. A post-intubation CXR demonstrated progression of bibasilar opacities from initial CXR , with laDuring the patient’s intensive care unit (ICU) course, the SARS-associated coronavirus ribonucleic acid assay sent from the ED came back positive, indicating COVID-19 pulmonary infection. Repeat lab work also showed characteristic COVID-19 findings of lymphopenia and elevated AST and ALT. After six days on a ventilator, the patient was successfully extubated. He was treated with hydroxychloroquine for COVID-19 and antibiotics for aspiration pneumonia. After a stable course on the general medical floor, he was discharged home with self-quarantining instructions to prevent the spread of COVID-19.Anchoring bias is a common phenomenon in the field of medicine, and especially in emergency medicine. In the acute setting in the ED, a patient’s history and ancillary information may be limited due to various reasons, such as the altered mental status presented in our patient. Because of these limitations, it is critical to maintain a broad and evolving differential diagnosis using thorough physical examinations and continuous re-evaluations.Our 32-year-old patient presented as a drug overdose with agitation and was ultimately admitted to the medical ICU after intubation for profound hypoxemia and airway protection. While we were initially limited to information given by EMS, the lab results and imaging provided us with information that was compatible with patients who had tested positive for COVID-19. Variables such as oxygen saturation and abnormal CXR findings were the initial clues into the presenting viral illness. The fact that the patient’s mental status did not improve, paired with the ancillary history after family arrived in the ED, made it clear that COVID-19 had to be considered underlying the patient’s presentation. After definitive airway control and isolation, a contrast-enhanced CT of the lungs was performed, showing the ground-glass opacities prevalent with COVID-19 positive patients, indicating that the patient was an appropriate candidate for COVID-19 testing. COVID-19 primarily affects the respiratory system, thus directing the clinicians’ focus to respiratory-related symptoms.When screening patients at and during evaluation, clinicians should consider a broad differential because patients with atypical presentations may be overlooked as candidates for COVID-19 testing and treatment. Ramifications of neglecting certain signs and symptoms may include risks such as patients being brought to non-COVID-19 isolation areas and causing further spread of the disease to otherwise healthy individuals. By thoroughly considering all potential risk factors within variable presentations, the capture rate of COVID-19 infected patients should improve. Further discussion and studies should be encouraged as the rapid rise in COVID-19 patients may precipitate mistakes made while they are being triaged. Given the paucity of research on atypical presentations for COVID-19, it would prove beneficial to broaden the knowledge of this pandemic.Hospital protocols are rapidly developing and evolving to account for the wide variety of patient presentations. These include patients with absence of all respiratory symptoms; incidental findings on CT or CXR while investigating other pathology; and exacerbation of chronic disease such as hyperglycemia in diabetic patients. No protocol, however, can effectively capture all patients who may fall out of normal protocol guidelines. In an effort to curb the spread of disease, physicians will need a high index of suspicion for identifying COVID-19 positive patients, especially as the incidence of the disease continues to rise.Keeping a broad differential diagnosis while evaluating patients during the COVID-19 pandemic will aid physicians in controlling the spread of this pathogen. Appropriate use of imaging and labs may expedite diagnosis of COVID-19 in patients and ensure they are properly managed. Atypical manifestations of patients with COVID-19 should not be undermined as research is still limited on the pathophysiology of this virus.


The emergence of cancer immunotherapy has already shown some remarkable results, having changed the treatment strategy in clinical practice for solid tumors. Despite these promising long-term responses, patients seem to lack the ability to respond to immune checkpoint inhibitors, thus demonstrating a primary resistance to immunotherapy. Moreover, a significant number of patients who initially respond to treatment eventually acquire resistance to immunotherapy. Both resistance mechanisms are a result of a complex interaction among different molecules, pathways, and cellular processes. Several resistance mechanisms, such as tumor microenvironment modification, autophagy, genetic and epigenetic alterations, tumor mutational burden, neo-antigens, and modulation of gut microbiota have already been identified, while more continue to be uncovered. In this review, we discuss the latest milestones in the field of immunotherapy, resistance mechanisms against this type of therapy as well as putative therapeutic strategies to overcome resistance in solid tumors. The concept of immune therapy to fight against cancer was first described in 1890 by W. Colley, who observed cancer remissions after inoculating sarcoma patients with erysipelas cultures . An intaThe principal mode of action of the immune system is the recognition by cells of the innate immune system, the release of various cytokines, and complement activation and concomitant phagocytosis. Tumor development is associated with cytotoxic innate and adaptive immune cells, but as the tumor evolves from neoplastic tissue to detectable tumors, tumor cells use different mechanisms in order to create an immunosuppressive recapitulate peripheral immune tolerance .This immune activation is under strict control by inhibitory checkpoints that prevent overstimulation, resulting in autoimmunity and extensive damage to healthy cells . UnfortuResistance to immunotherapy can be categorized as primary and acquired, depending on the presence or absence of initial response and subsequent relapse after the response that has been achieved. The mechanisms involved in the development of resistance include tumor-cell intrinsic and extrinsic factors that can either interfere with the antigen processing and presentation or enable tumors to recruit immune-suppressing cells that antagonize the activity of effectors cells . With thThe significant advance of the checkpoint inhibitors  has indicated as a hopeful therapeutic option in several solid tumors. CTLA-4 antibodies, like ipilimumab and tremelimumab, interrupt CTLA-4 from interacting with B7 and boost T-cell activation. The expression of CTLA-4 on tumors has been linked with poor survival in nasopharyngeal carcinoma  and incrMore specifically, in 2011, ipilimumab was the first approved checkpoint inhibitor, in patients with metastatic melanoma . Pembrolizumab and nivolumab were also among the approved PD-1 inhibitors for advanced melanoma. Synergistic therapy with ipilimumab plus nivolumab led to better PFS compared to each drug being used separately (11.5 vs. 2.9 months only with ipilimumab and 6.9 months only with nivolumab) .The first immunotherapy permitted (2015) was nivolumab for the treatment of patients with metastatic NSCLC. Between patients with advanced non-squamous NSCLC that had progressed during or after platinum-based chemotherapy, OS was better by 41% (nivolumab vs. docetaxel) . MoreoveFurthermore, the combination of nivolumab plus ipilimumab versus sunitinib shows significantly better OS rates among intermediate and poor-risk patients with previously untreated advanced renal-cell carcinoma .Pembrolizumab was also used for second-line treatment of patients with NSCLC that express PD-L1. Additionally, it is authorized for the handling of patients with advanced NSCLC, high PDL1 expression, and metastatic melanoma patients and patients with recurrent/metastatic of the head and neck squamous cell carcinoma .Atezolizumab (PD-L1 inhibitor), is already used for the treatment of patients with locally advanced or metastatic urothelial carcinoma due to its favorable safety profile compared to chemotherapy . MoreovePD-1/PD-L1 blockade appears to be promising immunotherapy with significant clinical benefits and durable responses in multiple tumor types. A percentage of cancer patients never demonstrate a clinical response or stabilized disease after treatment with checkpoint inhibitors. The estimated percentage of US cancer patients who are eligible for ICI  increased from 1.54% in 2011 to 43.63% in 2018. Furthermore, the percentage of cancer patients estimated to respond to ICI was 0.14% in 2011 and increased to 12.46% in 2018 . DiffereThe tumor microenvironment is a complicated structure that includes M2 macrophages, fibroblast, Tregs, myeloid-derived suppressor cells (MDSC), and other stromal cells .Tregs are essential lymphocytes maintaining self-tolerance. This kind of T-cells is characterized by high expression of the FoxP3 transcription factor  and theyA subtype of B-cells, which is known as B regulatory cells or B-regs, has been associated with autoimmunity, inflammation, and tumorigenesis . B-regs MDSCs are characterized by the expression of several molecules, such as CD11b and Gr-1 markers . DiffereAnother essential type of white cells in TME is tumor-associated macrophages (TAMs), including M1 and M2 macrophages ,34. SeveTME is characterized by tumor-associated mast cells or TAMCs. This kind of cells is being recruited by chemotactic molecules that are secreted by cancer, immune and stromal cells such as prostaglandin E₂ (PGE2), vascular endothelial growth factor (VEGF), CXC chemokine ligand (CXCL)12, fibroblast growth factor (FGF)-2, and platelet-derived growth factor (PDGF) . ActivatAutophagy is a well-described catabolic mechanism where molecules and damaged cellular organelles are degraded . The priSeveral studies support the hypothesis that autophagy can alter the pool of antigenic peptides that are presented in the major histocompatibility complexes (MHC) I and II .The fusion of New York esophageal squamous cell carcinoma-1 (NY-ESO-1), a specific antigen frequently over-expressed from melanoma to LC3, leads to the targeted autophagosome increasing response of NY-ESO-1-specific anti-melanoma helper T-cell . MoreoveDendritic cells (DCs) pulsed with such autophagosomes were more efficient in the response of OVA-specific T-cell compared to soluble protein . It appeIt is well known that anti-tumor T-cells are activated via the identification of tumor peptides with immunogenic activity. These peptides are presented on the surface of antigen-presenting cells, such as DCs. Several studies highlight the essential role of the autophagy machinery in the regulation of the major histocompatibility complex (MHC) I and the antigen presentation on the surface of APCs such as macrophages and dendritic cells. In embryo mice, the expression of MHC class I on the cell surface of APCs was increased after treatment with autophagy inhibitors or knockdown of essential genes for autophagy regulation ,56. In aSeveral studies have highlighted the association of autophagy with the cross-presentation (the loading of MHC-I into APCs with immunogenic peptides) of antigens in DCs . IncreasSeveral studies support the hypothesis that epigenetic alteration with DNA methyltransferase (or DNA MTase) and histone deacetylase (or HDAC) inhibitors as a putative target that increases the efficacy of ICI. These types of inhibitors increase the production of cytokines. They also increase antigen presentation by inhibiting Tregs . In a reIn a number of patients, the residual capacity of the immune system can be restored through exogenous tumor-infiltrating lymphocytes (TILs) or T-cells directed to cancer-specific immunogenic peptides. Although cancer cells are evolving in order to trigger antimmunosuppressive environment. This leads to genetic and/or epigenetic alterations that interrupt the formation of neo-antigen . ConsequSeveral molecular mechanisms and oncogenes have been associated with resistance to immunotherapy in solid tumors.+T-cell and tumor cell immunosurveillance. The increasing levels of the PD-L1/PD-1 molecules may be the result of the decreasing production of these miRNAs [The microphthalmia-associated transcription factor (MITF) is a well-known oncogene of melanoma, which is negatively associated with the invasion properties of cancer cells . The MITe miRNAs ,80.The role of signal transduction pathways in cancer is well-described in many studies. In a melanoma animal model, the lack of phosphatase and tensin homolog (PTEN) activation decreased the infiltration of T-cells in tumors and increased the expression of immunosuppressive cytokines. Furthermore, VEGF was increased and led to immune suppression as it was highlighted through clinical samples and B16 murine tumors ,82. MoreLast, beta-2-microglobulin (B2M) is correlated with the heavy chain of MCH- I . MutatioIn the last decade, the association between gut microbiome and response to immune ICI is gaining interest. It is well-established that diet may alter the microbial composition. A study supported the hypothesis that fiber can trigger an immunostimulatory microbial landscape, which may lead to an increased response to ICI . MoreoveFaecalibacterium and Ruminococcaceae) enrichment of specific species and improved response to ICI [Moreover, several studies highlighted the correlation between higher bacterial diversity , most patients with solid tumors do not respond.It is a common belief that PD-L1 expression in tumor cells immunohistochemistry (IHC) with the Tumor Proportional Score (TPS) is the only checkpoint inhibitor that is used as a predictive biomarker approved for NSCLC patients in first- and second-line treatment ,107. UnfThe last years, research efforts revealed the complex and highly heterogeneous structure of TME. As it was mentioned before in the current review, TME is a main resistance mechanism against ICI. The following can be used to reduce the resistance of TME: (a) Upregulation of chemokines (CXCL) 9 and 10. Doxorubicin may induce the activity of CXCL10. The goal of a phase I/II study is to evaluate the effect of doxorubicin hydrochloride when given together with pembrolizumab in patients with sarcoma (NCT02888665); (b) activation of the endosomal toll-like receptors (TLRs) 3, 7, 8 and 9 ; (c) epiAn increasing amount of research data supports the hypothesis that targeting the structure of blood vessels can reduce the function of suppressive cells and promote the anti-tumor activity of immune effector cells within TME . CurrentIn addition, several studies highlight the fact that anti-PD-L1 based immunotherapy appears to be more efficient when combined with chemotherapeutic agents . On the In addition, pembrolizumab as monotherapy could better suit a patient with a low tumor burden and a better performance status, than the combination of pembrolizumab and chemotherapy that would be more beneficial for a cancer patient with a higher tumor burden and a more inferior performance status, for whom a rapid and more probable response to treatment is crucial . FurtherThe crucial role of autophagy as a regulator mechanism for energy and metabolic balance in tumor cells is well described previously. The last decade research efforts have led to the development of agents that modulate autophagy. Chloroquine (CQ) and its derivative, hydroxychloroquine (HCQ), is one of the most studied inhibitors that target the fusion of the autophagosome with a lysosome . SeveralAs it was mentioned, mutation in different genes and the signaling pathways that control have been targeted from many research teams in order to overcome the resistance against ICI. The phase I/II trial studies (NCT04317105) tries to investigate the side effects and best dose of copanlisib  when given together with nivolumab and ipilimumab in treating patients with solid cancers that have spread to other places in the body (advanced) with mutation in PIK3CA and PTEN genes. A novel small inhibitor CGX1321 (inhibitor of WNT pathways) has already entered in human clinical trials as an anti-cancer agent. Keynote 596 uses single agent dose expansion phase in gastrointestinal (GI) tumors and roll-over cohort of CGX1321 and pembrolizumab in subjects who have progressed on single agent CGX1321 and Phase 1b consisting of CGX1321 in combination with pembrolizumab in colorectal tumors. Both phases of this study try to evaluate the safety, pharmacokinetics, and clinical activity of this combinatorial treatment (NCT02675946)In addition, several inhibitor of the histone lysine methyltransferase EZH2 such as CPI-1205 are already developed. In a Phase I/II clinical study (NCT03525795), combine CPI-1205 with ipilimumab in patients with histologically or cytologically confirmed advanced solid tumors. To avoid this resistance caused by gut microbiota, antibiotics, prebiotics , and synbiotics can be used.Moreover, the food industry have been applied recently bacteriophages, to eliminate unfavorable bacteria . FurtherIn In recent years, the vigorous attempt in the field of oncology and immunology has produced an increased amount of knowledge in tumor biology, leading to the development of several therapeutic strategies linked to the immune system. Despite the established successes of immunotherapy in solid tumors, a remarkable amount of patients with different types of cancers do not respond. Several mechanisms such as tumor microenvironment, immune cell infiltration within the tumor, poor antigen presentation, autophagy, molecular mechanisms, and silence of the immune response through cytokine release have already been identified as immunosuppressive factors in solid tumors. However, additional mechanisms continue to be discovered and further reveal the complexity of interactions between the tumor and the immune system. Myriad of research studies and clinical trials try to overcome the resistance mechanism against immunotherapy through the combination of anti-PD-1/PD-L1 or other checkpoint inhibitors with anti-angiogenic factors with traditional chemotherapy. In the future, research efforts should focus on the understanding of tumor microenvironment structure in order to re-active and sensitize tumor cells on immunotherapy. Putative therapeutic strategies will be developed in order to overcome the anti-tumor immune resistance against immunotherapy for different types of cancer.


Polyurethanes are of undoubted interest for the scientific community and the industry. Their outstanding versatility from tailor-made structures turns them into major polymers for use in a wide range of different applications. As with other polymers, new, emerging molecules and monomers with specific attributes can provide new functions and capabilities to polyurethanes. Natural and synthetic coumarin and its derivatives are characterised by interesting biological, photophysical and photochemical properties. Then, the polyurethanes can exploit those features of many coumarins which are present in their composition to achieve new functions and performances. This article reviews the developments in the proper use of the special properties of coumarins in polyurethanes to produce functional and smart materials that can be suitable for new specific applications. In particular, polyurethanes are a very versatile class of polymers, thanks to their tailor-made features from wide-range, controlled molecular structures. From their discovery in 1937 by Otto Bayer, polyurethanes have emerged as a suitable solution for countless applications based on very diverse building blocks and resulting properties. The core chemistry consists of a urethane group as the main repeating unit in polymer backbone . The polBut even nowadays more sustainable and less toxic alternative pathways for PU synthesis are emerging, not only from using biobased isocyanates, but also from new isocyanate-free routes. For example, the synthesis of phosgene-free and isocyanate-free carbamates, and the subsequent condensation reaction with an alcohol can lead to urethanes. Besides, thermoplastic poly(hydroxyurethane)s can be synthesised by reacting different cyclic carbonates with a variety of amines  86]..86].Regarding coumarin into polyurethanes, the first approach in 1992 dealt with creating optically-active polymers by reacting coumarin-derived diamines with diisocyanate to achieve chiral recognition capabilities . Since then, other works focused around combining coumarins, and polyurethanes have increased and led to different results and materials. Therefore, reported polyurethanes have exploited both the photophysical and photochemical properties of different coumarin moieties towards dissimilar objectives. In this sense, the present review intends to present a complete overview of advances in the use of coumarin in polyurethanes, and the wide range of opportunities that can emerge from achieved results.The physical properties of the coumarin ring has always aroused high interest for the scientific community, and proof of this is the wide variety of works devoted to this issue. A large number of the photophysical properties of coumarin motif, such as high thermal stability, high quantum yields and large Stokes shifts, have been studied by emission and EPR measurements , howeverTaking advantage of their high capacity to absorb UV-light, efforts have been devoted to obtaining smart materials that minimise the adverse effects produced by this light on the eye systems. In this sense, Bruin et al. used Coumarin 102 laser dye as an effective UV light absorber in an intraocular lens . Then, aw/w of this dye was characterised by complete absorption of UV radiation below 450 nm.As the UV absorption of artificial intraocular lens must be optimal to avoid the adverse effects of radiation below 400 nm on the retina, chromophores are an important element in polymeric materials ,91. CoumN,N-dimethylacetamide, these were dissolved in acetone, and the coumarin derivatives were introduced as chain extenders  . VelencoN,N-diisopropylethylamine as a nitrogen base and tetrahydrofuran (THF) as the solvent. In this last case, the obtained functional polyol achieved a molecular weight (Mn) and a polydispersity of 1410 g·mol−1 and 1.6, respectively. Furthermore, the spatial conformation seemed to be notably affected by the introduced coumarin moiety into the polymer backbone.The preparation of fluorescent polyol was performed by click chemistry with two different copper catalytic systems to establish the efficiency. Although both routes were successful to obtain fluorescent coumarin-functionalised polyols, the best conditions were obtained with copper(I) bromide as our catalyst, Subsequently, a rigid PU foam was synthesised with a fraction of the synthesised fluorescent polyol (25% parts by weight), a conventional polyether polyol, methylenediphenyl diisocyanate (MDI), and some additives. Then, the coumarin-containing PU foam was compared with the conventional PU. A characteristic UV-visible absorption peak from the incorporated coumarin function was observed, unlike the PU fabricated with the commercial polyol.For a long time, many efforts have been devoted to the preparation of smart functional materials capable of introducing a differential elements leveraging of the photochemical properties induced by the coumarin ring. Although there are works oriented in the biomedical sector, through molecular recognition processes or controlled drug release, much of the effort has focused on the preparation of materials with advanced performance achieved after the UV irradiation process. In the following section, those works focused on various photochemical processes will be highlighted due to the unique photochemical properties of coumarins.To the best of the authors’ knowledge, Chen et al.  preparedIn this scenario, the different synthesised polyurethanes were evaluated as chiral stationary phases for the resolution of enantiomers having aromatic groups and sites for hydrogen-binding by HPLC. Then, three optically active polyurethanes were synthesised by reacting MDI with amine-functional diastereomers and a diester, respectively . Those dThe chiral stationary phases were prepared by absorbing the polyurethanes on silica gels, which were previously surface treated with dimethoxydiphenylsilane as a phenyl functional coupling agent. Then, the expected chromatographic resolution from interaction sites for chiral recognition was evaluated with a series of racemates containing aromatic groups and sites for hydrogen-binding. From some satisfactory results with racemates containing aromatic groups, the authors proposed a discrimination mechanism of more effective diamine-derived polyurethanes based on simultaneous aromatic stacking and a hydrogen-bonding interaction. Besides, the evaluation of the influence of the polarity of the eluents on recognition capabilities demonstrated a negative effect of increased polarity.m,m’-di(hydroxymethyl)azobenzene, IPDI, 1,2-propanediol and poly(ethylene glycol) (PEG) chromen-3-one was higher than that of 2-(2-mthylthiazol-4-yl)-3H-benzo[f]chromen-3-one due to the fluorine substituent, being the 3-(2-methylthiazol-4-yl)-2-H-chromen-2-one derivative the less effective from lower aromaticity than benzo-derivatives. Besides, achieved anti-microbial function was considerable against Gram-positive bacteria, whereas it was just moderate against Gram-negative and mild against fungi.Based again on the reported activity against the bacteria and fungi of coumarin and its derivatives, synthesised molecules were incorporated as functional additives in the same polyurethane coating described above. The antimicrobial activity of the coatings was evaluated with a ratio of coumarin derivatives of 0.5 wt.%, 1.0 wt.% and 1.5 wt.%. Compared to neat control polyurethane formulation, the addition of coumarins led to improved antimicrobial activity from the biologically active coumarin ring and thiazole ring ,136. On The flame retardant properties were evaluated by measuring the limiting oxygen index (LOI), which it increases with lower flammability of the plastic material. The LOI value of the polyurethane coating linearly increased with the content of coumarin derivatives for the same 0.5 wt.%, 1.0 wt.% and 1.5 wt.% concentrations, and it was better than that of the neat reference in all the cases. The thiazole ring provided flame retardancy from a synergy effect between nitrogen and sulphur content, although the other substituents promoted the achieved improvement. Then, 2-(2-(2-(4-fluorobenzylidene)hydrazinyl)thiazol-4-yl)-3H-benzo[f]chromen-3-one became the most efficient molecule from the halogen atom and the three nitrogen atoms in the structure. The formulation with a 1.5 wt.% of this additive, thus, doubled the achieved LOI value if compared to the blank polyurethane coating. Potential improvement in anticorrosive properties from coumarin additives was studied by evaluating blistering, scribe failure and the degree of rusting on painted steel panels after performing a salt spray (fog) test for 500 h of exposure. Organic heterocyclic compounds are considered organic structures with some effective corrosion inhibition properties . TherefoPolyurethanes are a very versatile tool to create unique materials with a wide range of properties from tailor made structures and compositions. The incorporation of coumarins into polyurethanes as functional molecules opens a window on new functions and opportunities. The distinctive photophysical and photochemical properties of natural and synthesised coumarin species lead to diverse polymeric materials with extensive performance.This review summarises the potential applications of coumarins and emphasises the great increment of their use into polyurethane materials. A comprehensive description of the diverse achieved properties and functionalities in polyurethanes allows us to update this field with all relevant related activity. Besides, it encourages the polymer scientist to expand future possible combinations. The possibilities to new functional polyurethanes could be endless.Different new coumarin molecules with different reactive groups arise as potential chemicals to provide polyurethanes and any other organic material with different and useful responsible characteristics for the future.


Clustered regularly interspaced short palindromic repeats (CRISPR)-Cas9 technology allows the modification of DNA sequences in vivo at the location of interest. Although CRISPR-Cas9 can produce genomic changes that do not require DNA vector carriers, the use of transgenesis for the stable integration of DNA coding for gene-editing tools into plant genomes is still the most used approach. However, it can generate unintended transgenic integrations, while Cas9 prolonged-expression can increase cleavage at off-target sites. In addition, the selection of genetically modified cells from millions of treated ones, especially plant cells, is still challenging. In a protoplast system, previous studies claimed that such pitfalls would be averted by delivering pre-assembled ribonucleoprotein complexes (RNPs) composed of purified recombinant Cas9 enzyme and in vitro transcribed guide RNA (gRNA) molecules. We, therefore, aimed to develop the first DNA-free protocol for gene-editing in maize and introduced RNPs into their protoplasts with polyethylene glycol (PEG) 4000. We performed an effective transformation of maize protoplasts using different gRNAs sequences targeting the inositol phosphate kinase gene, and by applying two different exposure times to RNPs. Using a low-cost Sanger sequencing protocol, we observed an efficiency rate of 0.85 up to 5.85%, which is equivalent to DNA-free protocols used in other plant species. A positive correlation was displayed between the exposure time and mutation frequency. The mutation frequency was gRNA sequence- and exposure time-dependent. In the present study, we demonstrated that the suitability of RNP transfection was proven as an effective screening platform for gene-editing in maize. This efficient and relatively easy assay method for the selection of gRNA suitable for the editing of the gene of interest will be highly useful for genome editing in maize, since the genome size and GC-content are large and high in the maize genome, respectively. Nevertheless, the large amplitude of mutations at the target site require scrutiny when checking mutations at off-target sites and potential safety concerns. The development of technologies related to genetic improvement, such as transgenesis and more recently, genome editing, have changed the way humans have grown food for thousands of years. Today, the most promising tool to DNA manipulation is CRISPR , a gene-editing technology that has been adapted from the bacterial immune system against viral infections . CurrentAlthough much is already known about the principles of CRISPR-Cas9 genome editing, the likelihood of different outcomes in terms of resolution, efficiency, accuracy, and DNA modification structure has shown to be species-dependent. Various factors, including target site choice, gRNA design, the properties of the endonuclease, the type of DSB introduced, whether or not the DSB is unique, the quantity of the endonuclease and gRNA, and the intrinsic differences in DNA repair pathways in different species, tissues, and cells will result in differences in the mutation signatures generated in plant species .Zea mays L. is an ancient amphidiploid species with a duplicated chromosome number of n = 10  and filtered through a double filter (14 and 40 μM Nylon mesh). Protoplasts were collected after centrifugation at 100 g for 3 min and washed two times in 10 mL of W5 solution. Protoplasts were resuspended in cold MMG solution . Its viability and concentration were determined using Fluorescein Diacetate  dye in the hemocytometer. To confirm the internalization of the RNP complex inside cells, an assay was performed using fluorescent-labeled tracrRNA molecules  , and then transferred to multi-well plates for 24 h under gentle agitation (40 rpm) in the dark at 25 °C.Maize protoplasts were gene-edited by introducing CRISPR-Cas9 RNP complex (no integration of exogenous DNA) via PEG-mediated transfection . Protopl® Plant Mini Kit , followed by amplification of the target region by PCR using Taq Q5 High-Fidelity DNA Polymerase  and primers listed in TM-ThermoFisher Scientific, Sao Paulo, SP, Brazil).In order to characterize the spectrum and frequency of DNA changes at the target gene, genomic DNA was isolated using DNeasyCRISPR-Cas9 DNA changes were calculated based on the insertions and deletions (In/Dels) around the cleavage site (3 bp upstream of the PAM sequence) using the Inference of CRISPR Editing Software—ICE software v.2 . It has been previously shown that the ICE software results are comparable to the Next Generation Sequencing (NGS) results .Cleavage activity of gRNAs 1 to 5 was tested using 0.5 μg of crRNA and 1.5 μg of Cas9 enzyme to 300 ng of DNA. While all the designed gRNAs were able to cleave PCR products of the IPK gene in our study, the different gRNA sequences varied in their cleavage efficiency . gRNA2, Protoplasts viability was checked before and 1 h after transformation. The viability of protoplasts at the isolation step was on average 75% and on average more than 65% after transformation. Therefore, these protoplasts were considered stable after transformation, which would allow further cell culture propagation.Frequently, results indicating low efficiency of CRISPR-Cas9 editing using RNPs delivery cannot discriminate low transfection rates from poor DNA cleavage and repair activity. In order to overcome this limitation and confirm the internalization of Cas9-gRNA RNPs, we performed a fluorescent microscopy assay. Labeled tracrRNA molecules confirmed the internalization of the RNP complex . AlthougRNPs containing gRNAs 2, 3, and 4 were transfected into isolated protoplasts with PEG 4000 and the results are displayed as the percentage of indels detected at the cleavage site based on Sanger sequencing and analysis with the ICE software v.2 . The conDNA sequences from the universal primer used to amplify the IPK gene in maize (876 bp) from treated samples were compared to the same fragment in negative controls (no RNP delivered). Other negative controls  were also tested against the first negative control and showed no DNA changes. The obtained gene-edited sequences for each gRNA at 40 min of exposure are displayed in Results on the percentage of mutated sequences (technology efficiency), the size of the DNA change (number of base pair change), the type of DNA change (deletions or insertions), and a theoretical knockout (KO) score are summarized in CRISPR-Cas9 technology is a powerful tool for plant breeding and research. While still evolving as a technology to determine the rules for gRNA design and the algorithms to predict target and ‘off-target’ sequences, CRISPR applications still rely on empirical results to test the performance of new systems . NotablyBrassica napus and found that the transformation resulted in at least five independent insertions of the vector backbone sequences in the plant genome.Our experiments demonstrated the suitability of the PEG-delivered CRISPR-Cas9 RNPs system for gene-editing screening in maize. We showed that high-efficiency gene-edited maize cells could be obtained using less time-consuming (15 days) and labor-intensive procedures . In addition, the advantage of our system in relation to the use of vectors is that it prevents the integration and expression of exogenous DNA sequences, isolating the effect of gene-editing modification and avoiding transgene-introgressed side effects. Although non-integrating plasmids could be transfected into plant cells to deliver programmable nucleases, transfected plasmids are degraded in cells by endogenous nucleases, and the resulting small DNA fragments can be inserted at both on-target and off-target sites in host cells  . For exaIn the present report, we show a positive correlation between the time of exposure to RNPs and the efficiency of site-directed mutagenesis in maize, as ascertained with Sanger sequencing. In previous reports referring to the use of RNPs in plant protoplasts , the autArabidopsis protoplasts and found that the editing efficiency was not directly related to the Cas9 concentration but also dependent on the time course of the analysis. At 24 h after delivery, more efficiency was observed when applying 20 instead of 60 ug of Cas9 (71 in contrast to 54%). Opposite results were obtained at 72 h after delivery. Nevertheless, increasing the amount of Cas9  was consistent with a crescent indel frequency in brassica sp. protoplasts [The Cas9:gRNA ratio also influences target efficiency in a species-specific manner. Three different Cas9:gRNA ratios were tested in apple and grapevine protoplasts . While ttoplasts . The effAgrobacterium-mediated, and biolistics  neither presents mutations with a substitution of bp . AlthougWe have shown that RNPs could be used for targeted CRISPR-Cas9 via PEG delivery as a model system to screen for gene-editing outcomes in maize. Target insertion and deletion DNA changes (In/Dels) were induced using 45 ug of Cas9 and gRNA used at a 3:1 ratio, and a positive correlation between exposure time (20 and 40 min) and indel frequency was observed. By targeting preserved coding regions, we can anticipate that the model could be applied to several maize varieties by validation using IPK gene. However, in vivo In/Del frequencies differed among gRNA sequences. In addition, the proposed method for sequencing analysis is also restricted to a window of 30 bp deletions and 14 bp additions. Further studies will be focused on CRISPR-Cas9 off-target activity and on the regeneration of edited protoplasts.


As the novel coronavirus (COVID-19) has spread globally, a significant portion of women have undergone childbirth while possibly infected with the virus and also under social isolation due to hospital visitor restrictions. Emerging studies examined birth outcomes in COVID-19 positive women, but knowledge of the psychological experience of childbirth remains lacking. This study survey concerning childbirth and mental health launched during the first wave of the pandemic in the US. Women reporting confirmed/suspected COVID-19 during childbirth were matched on various background factors with women reporting COVID-19 negative. We found higher prevalence of clinically significant acute stress in birth in COVID-19 positive women. This group was 11 times as likely to have no visitors than matched controls and reported higher levels of pain in delivery, lower newborn weights, and more infant admission to neonatal intensive care units. Visitor restrictions were associated with these birth outcomes. COVID-19 positive women with no visitors were 6 times as likely to report clinical acute stress in birth than COVID-19 positive women with visitors. The findings underscore increased risk for childbirth-induced psychological morbidity in COVID-19-affected populations. As hospitals continue to revise policies concerning visitor restrictions, attention to the wellbeing of new mothers is warranted. The coronavirus (COVID-19) pandemic’s immense scope and duration has made clear the urgent need to better understand the virus’ physical and psychological impacts on vulnerable populations. From a generational health perspective, perhaps no population’s experience is more critical to understand and safeguard than that of delivering mothers. In the midst of a global public health crisis characterized by a potentially lethal and highly infectious virus, many non-emergency hospital-based health procedures were postponed. Nevertheless, delivering mothers all over the globe were among the very few populations that continued to be treated in hospital settings.A significant portion of them underwent childbirth when they were suspected or confirmed of the novel coronavirus disease. Some may have experienced mild but also severe physical symptoms  such as Emerging studies have focused largely on obstetrical and neonatal correlates of COVID-19 infection status. A body of research suggests that there is increased risk for adverse outcomes in pregnant women with COVID-19. In a recent systematic review of nine studies in China, the incidence of preterm births, low birth weight, C-section, and NICU admission were found to be higher in COVID-19 positive cases than in the general population . LikewisAn important issue to consider is the subjective experience of childbirth and potential heightened psychological adversity in birth for COVID-19-positive laboring women. Although childbirth is typically considered a happy event, a significant proportion of new mothers report their delivery as highly stressful in samples assessed before the pandemic , 11 and A salient factor that may influence the childbirth of COVID-19-affected mothers concerns the unique stressor of the pandemic: social isolation. With the goal of reducing infectious exposures to visitors, other patients, the community, and healthcare teams, and in the wake of uncertain and rapidly evolving situations, policies restricting visitors have been implemented to lower the number of people in hospitals. Accordingly, mothers who contracted COVID-19 have faced drastic restrictions on maternal visitors and supportive companions in the delivery room and during the postpartum stay. A significant number of COVID-19-affected mothers may have even experienced childbirth without the emotional support provided by having close friends or family in the room with them . They maAs the obstetric health risks and benefits in the face of a still poorly understood virus remain unclear  and hospTo this end, we studied a large sample of women who recently gave birth when COVID-19 was prevalent in the United States, among them 68 women reported suspected or confirmed COVID-19 positive. We matched this group on a wide range of background factors to 68 women who gave birth in the outbreak of the pandemic but were negative for COVID-19. There have been no studies to date that use a comprehensive matched-group analysis that could allow for better understanding of the contribution of COVID-19 positivity to childbirth outcomes while controlling for background factors that increase perinatal adversity. We examined whether being COVID-19 positive is associated with stressful psychological experiences of birth as well as obstetrical and neonatal outcomes and whether having no visitors during delivery hospitalization stay was associated with these outcomes.This study is part of a research project that was launched on April 2nd, 2020, in the midst of the COVID-19 pandemic in the United States, with the overarching goal of understanding the impact of COVID-19 on childbirth and maternal mental health. Women who had given birth in the last six months were recruited through announcements on our hospital’s research study platform as well as via social media and postpartum professional communities; they were asked to complete an anonymous survey and were informed that by agreeing to complete the survey they are implying their consent to participate in the study. Therefore, all subjects who took the survey consented to the study. Partners Healthcare  Human Research Committee approved the study measures and procedures and granted exemption for this study and the study was carried out in accordance with the approved protocol. The sample in this study was derived from 2,417 women who gave birth since COVID-19 was prevalent in their communities and provided the childbirth date; they were on average two months postpartum. We identified 68 women who reported being COVID-19 positive, suspected or confirmed, during pregnancy and/or childbirth. We then identified a matched control group of 68 women who reported being COVID-19 negative. The groups were matched on demographic factors, primiparity, prior trauma and childbirth history, and prior mental health.In this sample of a total of 136 postpartum women, the vast majority delivered a healthy baby at term (86.8%), had a vaginal delivery (71.3%), and around half (50.7%) were primiparas. The average age of participants was 32 years old. The majority were married (89%), had at least middle-class income , were employed (72.1%), and had at least a college degree (83.1%). Participants resided in the United States (80.0%), in Canada (4.4%), Europe (2.9%), Central/South America (2.9%), Asia (2.9%), and 2.2% in the Caribbean and Middle East. Four participants (2.9%) did not report their geographic location.Acute stress responses to childbirth were assessed with the commonly used Peritraumatic Distress Inventory (PDI) . The PDIObstetrical and infant factors concerning recent childbirth were measured with respect to gestational age, medical complications in labor and delivery (yes vs. no), degree of pain in labor and delivery , sleep deprivation (defined as less than six hours of sleep on the night before childbirth), use of pain medication (yes vs. no), use of induction medication (yes vs. no), and mode of delivery. Additionally, we measured newborn weight (lbs.), newborn biological sex, neonatal intensive care unit (NICU) admission of newborn, skin-to-skin contact after delivery (yes vs. no), rooming-in (yes vs. no) and breastfeeding habits .COVID-19-related restrictions were assessed in regard to visitor policy. Participants were asked whether there were “any visitor restrictions during your hospital stay?”. Response options  were classified as “No visitors” versus “Other”. We also asked participants whether they were separated from their infant.Background factors pertaining to demographics, prior mental health, and traumatic exposure were used for creating matching study groups. Theses variables included maternal age, education level, marital and employment status, income, race/ethnicity, and primiparity. We also assessed history of mental health problems , and the number of prior traumatic events (happened or witnessed) with the commonly used Life Events Checklist for DSM-5 (LEC-5)  , infant-related factors , COVID-19-related restrictions , and psychological experience of birth, namely, acute stress responses in birth. Kolmogorov-Smirnov and Shapiro-Wilktests were conducted to assess normality in quantitative outcome scores. Based on these tests, Mann-Whitney U tests were performed to estimate differences between study groups in quantitative measures, and chi-square test for independence of measures (with Fisher’s exact estimation of significance) for estimating differences in categorical measures. Finally, we conducted Kruskal-Wallis tests and chi-square test for independence of measures (with Fisher’s exact estimation of significance) to examine whether no visitors may account for the differences between COVID-19 positive and negative women.2χ(27) = 18.21, p = .90, such that each group comprised 68 women.An overall balance test  indicatePercentages of birth-related outcomes are presented in COVID-19 positive women reported significantly greater pain in delivery (weak-to-moderate in effect size) than COVID-19 negative women. No group differences were found in sleep deprivation, pain in labor, medical complications, medication for induction and/or pain, or mode of delivery.OR = 3.72). In addition, COVID-19 positive women gave birth to infants with lower  weights than COVID-19 negative women. Fewer COVID-19 positive women were with their newborn in the room during their stay at the hospital (OR = 0.18) than COVID-19 negative women. No group differences were found in gestational age, infant sex, breastfeeding, and skin-to-skin contact.A higher percentage of babies of COVID-19 positive women were admitted to the newborn intensive unit of care , with COVID-19 positive women who had visitors (n = 51) and those with no visitors (n = 17). Percentages are presented in To examine whether no visitors during delivery hospitalization account for the differences between the COVID-19 positive and negative groups , we compared COVID-19 negative women who had visitors (The analyses indicated that COVID-19 positive women who had no visitors reported significantly greater pain in delivery see  and deliThe COVID-19 pandemic offers a rare opportunity to examine the experience of childbirth under stressful conditions such as social isolation. As infectious disease outbreaks continue, it is critical that we generate new knowledge to inform preparations and guidelines of perinatal care during these outbreaks.Our study sought to examine the childbirth experiences of women who had delivered during peak infection rates of the pandemic and were suspected or confirmed to have contracted COVID-19, a population likely to have undergo labor and delivery while potentially being acutely ill and at the same time subject to drastic hospital restrictions concerning social isolation. We compared women who reported being COVID-19 positive, suspected or confirmed, to women who reported not having contracted the virus but who were similar on a range of background factors such as demographics, prior mental health, and even trauma history. This rigorous matched-control group approach has not been implemented in previous studies and allows for the generation of knowledge on the potential adverse influence of COVID-19 infection status of birth outcomes while controlling for background factors that are associated with COVID-19 infection and negative maternal outcomes.The main study findings show that nearly 50% of suspected or confirmed COVID-19 positive women reported clinically significant acute stress symptoms in birth. They were as much as two times more likely to experience acute stress than non-affected women and to perceive higher degrees of pain in childbirth even though no differences were found in factors such as obstetrical complications, medication for pain, or delivery mode between COVID-19 positive and negative cases. These findings underscore how childbirth can become a traumatic experience and evoke an acute stress response for women with the novel coronavirus.We further document increased exposure to salient social stressors surrounding childbirth in affected women. As might be expected, the results reveal that hospital policies enforcing visitor restrictions were frequently implemented with delivering women suspected or confirmed of COVID-19 infection. As much as 25% of COVID-19 positive women had no visitors during their delivery hospitalization stay. This group was 11 times as likely not to be permitted a support person to accompany them than women negative for COVID-19. COVID-19 positive women were also much more likely to experience physical separation from their newborn. In accord with previous studies , the newOur findings reveal that social isolation surrounding childbirth may increase risk for maternal morbidity. We found heightened clinically significant acute stress in COVID-19 positive women who had no visitors. They were 6 times as likely to report acute stress symptoms than COVID-19 affected women who were permitted visitors during their delivery hospitalization. We also found that COVID-19 positive women who did not have a support person experienced greater pain in delivery, delivered newborns with lower weight, and had elevated NICU admission rates. These findings accord with the evidence of emotional comfort and support in birth being associated with improved birth outcomes , and sugThis study’s findings may be useful in informing clinical policies during the COVID-19 pandemic. While much attention has been paid to the physical symptoms in mothers with COVID-19, our study emphasizes the importance of considering mothers’ psychological wellness. The findings suggest that increased awareness should be given in labor and delivery and postpartum units to the psychological symptoms surrounding childbirth that may arise in women who are sick with or suspected of having the virus; additionally, the potential emotional liability of not permitting a support person during hospitalization should be noted. While routine screening for traumatic childbirth does not exist in postpartum hospital units, our study suggests that assessment of acute stress responses in delivering mothers who are COVID-19 positive is warranted. Ongoing monitoring of mental health symptoms in this high risk group after hospital discharge is important as those with stable symptoms in accord with routine care are likely to be quickly discharged and face social isolation during the postpartum period, which is considered a time of heightened psychological vulnerability , 28.Shortcomings of this study include reliance on anonymous self-report measures that allowed for conducting a study swiftly during the initial heights of the pandemic but not for inclusion of patients’ medical records. We rely on respondents accurately reporting their COVID-19 infection status, and their receiving accurate information from COVID-19 testing protocols at the hospitals where they delivered. Additionally, we do not have information on the severity of respondents’ COVID-19 symptoms, only their infection status. We cannot rule out that acutely ill women were those not permitted visitors. Also, while we used a well-validated measure to assess acute stress which has shown good correspondence with clinician assessments, we did not include diagnostic measures. Retrospective assessments could be prone to recall bias and hence the importance of the use of matched controls. This convenient internet sample introduces a bias towards women from a certain socioeconomic class.In conclusion, we find that confirmed or suspected COVID-19 positive women experience increased psychological morbidity surrounding childbirth compared to delivering women without COVID-19. We find that COVID-19 positive women experience increased levels of pain during delivery and give birth to newborns of lower weight which are more likely to be separated from their mothers and sent to the NICU. This increased adversity appears especially heightened in cases where a support person is not allowed in the maternity unit. As hospitals around the world continue to update their delivery protocols for COVID-19 positive women and determine risk and benefits of visitor restriction policies, more research is needed to optimize maternal care during these unprecedented times.


Introduction: Hospital response to the COVID-19 outbreak has involved the cancellation of elective, deferrable surgeries throughout Europe in order to ensure capacity for emergent surgery and a selection of elective but non-deferrable surgeries. The purpose of this document is to propose technical strategies to assist the pediatric surgeons to minimize the potential aerosolization of viral particles in COVID-19 patients undergoing urgent or emergent surgical treatment using laparoscopic approaches, based on the currently available literature. The situation and recommendations are subject to change with emerging information.Materials and Methods: The Scientific Committee and the Board of the European Society of Pediatric Endoscopic Surgeons gathered together in order to address the issue of minimally invasive surgery during this COVID-19 pandemic. A systematic search through PubMed, Embase, and World Wide Web of the terms “COVID-19,” “Coronavirus,” and “SARS-CoV-2” matched with “pneumoperitoneum,” “laparoscopy,” “thoracoscopy,” “retroperitoneoscopy,” and “surgery” was performed. Non-English language papers were excluded. A PRISMA report was performed. Criticalities were identified and a consensus was achieved over a number of key aspects.Results: We identified 121 documents. A total of 11 full-text documents were assessed to address all concerns related to the adoption of minimally invasive surgery. All aspect of pediatric minimally invasive surgery, including elective surgery, urgent surgery, laparoscopy, thoracoscopy, retroperitoneoscopy, and pneumoperitoneum creation and maintainance were extensively addressed through systematic review. A consensus regarding urgent laparoscopic procedures, setting and operation techniques was obtained within the Committee and the Board.Conclusions: The ESPES proposes the following recommendations in case minimally invasive surgery is needed in a COVID-19 positive pediatric patients: (1) consider conservative treatment whenever safely possible, (2) dedicate a theater, columns and reusable laparoscopic instrumentation to COVID-19 pediatric patients, (3) prefer disposable instrumentation and cables, (4) use low CO2 insufflation pressures, (5) use low power electrocautery, (6) prefer closed-systems CO2 insufflation and desufflation systems, and (7) avoid leaks through ports. These recommendations are subject to change with emerging information and might be amended in the near future. The World Health Organization (WHO) recommendations to contain the coronavirus (COVID-19) outbreak include social distancing, avoidance of crowding and staying at home. Reducing the flow of people within countries also increases the likelihood of successful infection control. Restrictions implemented in Italy on the 7th of March 2020 included limitations on all personal activities. Similar limitations were implemented by other countries during the following weeks.Most hospitals have been involved in treatment of COVID-19 patients. In areas where the virus outbreak has been particularly severe, hospital infrastructure has been acutely remodeled to enable the direction of resources toward the care of COVID-19 patients. The need to contain the outbreak, treat cases and protect healthcare personnel have featured as prime considerations.In spite of this scenario, ordinary illnesses, trauma, and diseases requiring surgery continue to prevail alongside COVID-19. For reasons that are not yet understood, children appear to be less susceptible and generally the course of the disease is less severe  insufflation and electrocautery has been raised as a concern that is being closely reviewed worldwide.Patients suffering from these diseases will continue to be treated according to the COVID-19 infection prevention strategies of their local institutions. In procedures involving laparoscopy and thoracoscopy, the possibility of viral contamination through aerosolization during carbon dioxide  is safely possible and does not negate the outcome for the patient, this should be considered. Ideally, patients should be tested for COVID-19 before procedures, if this is practically feasible.Dedicated theater facilities (including laparoscopic column camera and cables) for suspected or confirmed COVID-19-positive patients should be arranged, if possible. Appropriate filtering and ventilation are important, and negative pressure rooms may also be useful. Unnecessary night-time operating should be avoided in order to decrease the workload of anesthesiologists and nurses likely to be needed to reinforce adult teams.In case of thoracoscopy, retroperitoneoscopy, or laparoscopy we recommend, whenever possible:Dedicate to COVID-19-positive patients a laparoscopic column (see above)2 and/or use of suspension techniquesMaintain low pressure COKeep power settings of electrocautery as low as possibleUse disposable insufflation cables with high-capacity filters2 evacuation systems with filtersUse customized or commercially available closed COAvoid leaks through trocarAvoid the use of trocars without valvesFavor the use of disposable trocars, best if with sealing balloonsPrefer staplers instead of loops (appendectomy) to minimize the risk of leaks through the portsUse disposable trocars in the context of a COVID-19-positive patient, if available. If reusable trocars are used, they should be cleaned separately from other surgical equipment2 at the end of the operation in a closed system with filters.Aspiration of the residual COStrict use of PPE and infection prevention strategies as for conventional open surgery and anesthesia for aerosol producing maneuvers should be followed. It is strongly recommended to follow the guidelines for droplet and airborne precautions of local institutions. This is particularly true for airway and endoscopic procedures. For specific detailed guidelines we recommend WHO as well as local institutional guidelines.In order to contain COVID-19 outbreak and to support the requirements of various European Health Systems in order to deal with this dramatic pandemic, it is necessary to adhere to a number of indications that apply to pediatric surgery and have been addressed by most governments.Postpone all elective surgery according to institutional or local guidelinesConsider a number of time-dependent diseases requiring elective non-deferrable surgeryPrepare to encounter COVID-19-positive patients requiring urgent surgical treatment.2 pneumoperitoneum creation during laparoscopy in case of Hepatitis  should be considered, as well as the capacity for ultrafiltration of any aerosolized particles that may also pose a similar or greater risk if an open approach is adopted , 6, 7, 9As a summary, as pediatric surgeons belonging to the European Society of Pediatric Endoscopic Surgeons (ESPES) we suggest the following concerning laparoscopic, thoracoscopic, and retroperitoneoscopic procedures in COVID-19 positive patients:Dedicate a theater to perform urgent procedures, avoid unnecessary night-time operatingConsider conservative treatments, whenever safely possibleDedicate minimally invasive columns and reusable instrumentationPrefer disposable instrumentation and cables2 insufflationUse low-pressure COUse low-power electrocautery2 insufflation and desufflation systemsPrefer closed-systems COAvoid leaks through ports .The aims of this document are to provide optimal patient care, maintain safety for all perioperative staff, and, as much as possible, help contain the COVID-19 pandemic. The situation and recommendations are subject to change with emerging information and might be amended in the near future.The original contributions presented in the study are included in the article/supplementary materials, further inquiries can be directed to the corresponding author/s.AP performed the Systematic Review. AP, AC, MA, and WV drafted the paper. MS, NK-D, ID, MM-S, CG, FC, and HS reviewed the draft and contributed to the consensus. All authors reviewed and approved the final version of the manuscript.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.