The novel coronavirus SARS-CoV-2 has emerged as one of the most compelling public health challenges of our time. To address the myriad issues generated by this pandemic, an interdisciplinary breadth of research, clinical, and public health communities have rapidly engaged to find answers and solutions. One area of active inquiry is understanding the mode(s) of SARS-CoV-2 transmission. While respiratory droplets are a known mechanism of transmission, other mechanisms are possible. Of particular importance to global health is the possibility of vertical transmission from infected mothers to infants through breastfeeding or consumption of human milk. However, there is limited published literature related to vertical transmission of any human coronavirus (including SARS-CoV-2) via human milk and/or breastfeeding. There is a single study providing some evidence of vertical transmission of human coronavirus 229E, a single study evaluating presence of SARS-CoV in human milk (it was negative), and no published data on MERS-CoV and human milk. There are 9 case studies of human milk tested for SARS-CoV-2; none detected the virus. Importantly, none of the published studies on coronaviruses and human milk report validation of their analytical methods for use in human milk. These reports are evaluated here, and their implications related to the possibility of vertical transmission of coronaviruses  during breastfeeding are discussed. Fortunately, this pandemic has rapidly engendered a mobilization of the full range of expertise represented by research, clinical, and public health experts. While our understanding of the biology, clinical implications, and strategies for mitigation continues to evolve, one issue that has received limited attention is the implication of this pandemic for infant feeding practices. This lack of attention has resulted in mixed messages regarding guidance about optimal infant feeding practicesSeveral issues related to this topic demand immediate attention, the first and foremost of these being whether or not the virus is present in human milk. Of particular interest in this context are 1) the potential role that breastfeeding could play in vertical transmission of SARS-CoV-2 from women to infants via human milk; and 2) the potential protective effects of targeted antibodies and other immunoprotective components in human milk against COVID-19. The goal of this review is to evaluate the published evidence regarding the presence of this and other human coronaviruses in human milk.We used both Google Scholar and PubMed to identify relevant literature published as of April 4, 2020. Because some of the reports relating to SARS-CoV-2 have not yet been published in refereed journals , we also used a general Google search and a search of preprint servers bioRxiv and medRxiv. The list of search terms used can be found in 4 Notable examples include human immunodeficiency virus (HIV),6 cytomegalovirus (CMV),7 and human T-cell lymphotropic virus type 1 (HTLV-1).8 Perhaps the most prominent example of mother-to-child viral transmission via breastfeeding is HIV, where higher maternal milk and serum viral loads are associated with an increased risk of transmission.11 The risk of postnatal infection for breastfed infants of HIV+ mothers is ~10–20% over the first 2 years of life.13 However, compared to mixed feeding, exclusive breastfeeding is associated with lower risk of transmission of HIV infection to infants.15 In many high-income nations, breastfeeding is contraindicated in the case of maternal HIV infection.17 However, in low-and-middle-income nations, infant mortality from malnutrition and infectious disease may outweigh the risk of acquiring HIV.18It is well established that viral transmission through human milk can occur.20 The risk of CMV infection in neonates is highest in preterm or very low birthweight (<1500 g) infants;22 in a small percentage of infections, infants develop a severe complication known as CMV sepsis-like syndrome, which can be fatal.23 Nonetheless, breastfeeding is not contraindicated in CMV-seropositive women with healthy, term infants.25With respect to CMV, it is estimated that ~60–70% of breastfed infants of CMV-seropositive infants become infected with CMV.26 HTLV-1 infection is lifelong, and while most infected individuals remain asymptomatic, ~10% develop severe disease, including adult T-cell leukemia, a highly aggressive and usually fatal malignancy.27 Some organizations and agencies list maternal HTLV-1 as a contraindication for breastfeeding,28 while others do not.25For HTLV-1, breastfeeding is considered the major route of infection for infants.29 There are 7 identified strains known to infect humans. Four of the strains  are ubiquitous in humans and cause the common cold. There is limited evidence that one of these (229E) may be vertically transmitted from mothers to infants, although the mechanism remains unclear.30 The presence of 229E in neonatal gastric samples suggests that one possible mechanism for infection is through human milk, although this study30 did not specifically evaluate the presence of 229E in human milk.Human coronaviruses are enveloped, positive-sense, single-stranded RNA viruses first described in 1965.31In addition to those that cause the common cold, more virulent strains of human coronaviruses have emerged zoonotically since the early 2000s, the first being SARS-CoV in 2003. The first reports of SARS-CoV were from China, although the disease  quickly spread globally. SARS is clinically manifested by fever, dry cough, headache, muscle aches, and difficulty breathing. No treatment exists except supportive care, but there have been no reports of SARS-CoV transmission since 2004. The case fatality rate of SARS is estimated at 10%.32 and two reports of human milk being tested for SARS-CoV antibodies.33 The former is a case study of a woman infected during the second trimester of pregnancy (19 wk). A single milk sample was collected 131 days after the onset of symptoms, but no additional detail on the collection methodologies was provided. Milk was submitted to the US Centers for Disease Control and Prevention (CDC), where it was analyzed using reverse transcriptase polymerase chain reaction (RT-PCR) for viral nucleic acids, and enzyme immunoassay and indirect immunofluorescence to evaluate antibody presence. No additional details on analytical methods were provided. While no viral RNA was detected, antibodies to SARS-CoV were identified in the milk. The infant in this study was never tested for SARS-CoV infection. The latter study was a case report of a 38-yr-old infected in the first trimester of pregnancy (7 wk). She recovered fully and delivered a healthy male infant at 36 wk of gestation. Milk samples were collected at 12 and 30 d postpartum and tested for SARS-CoV antibodies; all were negative. No details on the collection or analysis of the milk were provided. The infant in this study tested negative for SARS-CoV. In both of these studies, it is possible that the women had stopped shedding the virus before the milk samples were collected, as SARS-CoV shedding in other biological samples typically peaks 12–14 d after the onset of disease.34 There are no documented cases of vertical transmission of SARS-CoV between mothers and infants.35Currently, there is one report in which human milk was tested for SARS-CoV,31 There are no reports, to our knowledge, of the presence or absence of MERS-CoV in human milk. However, there are reports of the presence of MERS-CoV in the milk of dromedary camels (Camelus dromedaries).38 There is one report of a human likely infected through the consumption of raw (unpasteurized) camel milk.39 In camel milk samples spiked with MERS-CoV, viable virus could still be recovered after 48 hr.40 These two observations have resulted in recommendations against consuming raw, unpasteurized camel milk. 41 It is unclear if there is vertical transmission of MERS-CoV between camelid cows and their calves, and whether infection occurs as a direct result of lactation/nursing in this species. There are no data on vertical transmission of MERS-CoV between women and their infants.42A related virus, MERS-CoV, emerged in Saudi Arabia in 2012. The disease caused by MERS-CoV, Middle Eastern respiratory syndrome (MERS), is characterized by severe respiratory illness with symptoms of fever, cough, and shortness of breath. Like SARS-CoV, MERS-CoV is a betacoronavirus. The case fatality rate of MERS is 34%.43 and similar clinical characteristics. The first reported cases of SARS-CoV-2 infection emerged in late 2019 in China. While the case fatality rate for COVID-19 (the disease caused by the SARS-CoV-2 virus) is much lower than those of SARS and MERS at ~2%,31 the spread of this pathogen has been much more rapid and extensive.The novel coronavirus, SARS-CoV-2, was named after SARS-CoV due to its shared sequence homology (77.9%)49 or by women whose infants were infected.52 In total, 23 milk samples produced by 16 women have been tested; all were negative for the presence of the virus. A description of the relevant characteristics for the women and infants in these studies can be found in 52 For the infants born to women infected during pregnancy, most were immediately separated from their mothers post-delivery and were not breastfed for the duration of the period observed in their respective reports. Twelve of the 16 infants described in these reports were born via cesarean section, and only one was specified as a vaginal birth. Repeated milk samples were analyzed for 4 of the women, collected up to 16 days apart. All the studies were conducted in China52 or Singapore.50At the time of writing, there were 9 studies that reported direct testing of milk produced by women who were infected with SARS-CoV-244 described a healthy, 34-yr-old woman who acquired the infection in week 40 of pregnancy. She gave birth to a male infant via cesarean section. The infant and his mother both tested positive for SARS-CoV-2 using pharyngeal swabs within 36 hr of the delivery. The infant was separated from his mother at delivery and fed formula for the duration of the period described in the study. The mother’s milk was collected at 36 hr postpartum; it tested negative for SARS-CoV-2 via RT-PCR. No description of the collection or testing methods was provided. The authors stated that they recommended that the mother not breastfeed, but instead pump milk to avoid mastitis.Wang and colleagues45 reported on two women who became infected during the third trimester of pregnancy. Patient 1 was 34 yr old and in week 37 of gestation at the time of diagnosis. She delivered a female infant via cesarean section 6 d after testing positive for SARS-CoV-2 via nasopharyngeal swab. The infant was separated from the mother immediately after delivery, and serial tests of the infant’s nasopharyngeal swabs were negative. A milk sample was collected within 24 hr of delivery and 16 d later; both were negative for SARS-CoV-2. Patient 2 was 29 yr old and in week 36 of gestation at the time of diagnosis. Her infant was delivered 5 d after she was diagnosed via RT-PCR analysis of a nasopharyngeal swab. A single milk sample was collected within 24 hr of delivery; it tested negative for SARS-CoV-2. The authors of this report did not specify how the sample was collected, other than “breastmilk was obtained after the first lactation.”In another case series from China, Fan and colleagues46 have provided the most extensive report to date, including data on milk produced by 6 women infected during pregnancy. The women were 26–34 yr-of-age and between 36 wk 2 d and 39 wk 4 d of gestation at diagnosis. The authors did not provide details on the methods used for milk collection, other than “breastmilk samples from patients with COVID-19 pneumonia were collected after their first lactation” and that milk was collected following World Health Organization (WHO) guidelines, but they did not provide a citation for this collection method. All milk tested negative for the virus, but no information was provided on the methods used for analysis.Chen and colleague47 milk produced by two women was tested. One woman was 34 yr old and at 40 wk gestation tested positive via oropharyngeal swab. Milk was collected and tested from this women at d 1, 2, and 12 postpartum; all samples were negative. Her male infant was delivered via cesarean and tested for SARS-CoV-2 via oropharyngeal swab when he was 1 and 7 days old; both swabs were negative. The other woman was a 30 yr old and delivered an infant vaginally after testing positive for SARS-CoV-2. Her infant tested negative at birth using an oropharyngeal swab; milk was collected on d 2 postpartum, it was also negative. Details were provided for neither the methods of collection nor analysis.In a report by Liu et al.,48 information was provided related to a 30-yr-old woman at 35 wk gestation who was positive for SARS-CoV-2 and delivered a male infant via emergency cesarean section. The infant was tested immediately upon delivery via oropharyngeal swab, which was negative. After delivery, the infant was kept in isolation away from his mother. Milk was collected immediately after delivery and on d 2 and 3 postpartum; all samples were negative. Again, no information on the collection or testing methods for the milk sample is available in this report.In a research letter by Li and colleagues,49 report on a 29-yr-old woman at 34 wk of gestation who was diagnosed with COVID-19 via nasopharyngeal swab. Nearly a month later, the woman delivered a female infant via cesarean section. The infant was immediately separated from the mother with no contact. The infant consistently tested negative for SARS-CoV-2 via nasopharyngeal swab over the first 12 d of life. However, a blood sample at 2 hr of age was positive for IgG and IgM antibodies to SARS-CoV-2. A milk sample was collected from the mother at d 6 postpartum; it tested negative. No information on the collection or testing methods for the milk sample is included in this report.In another research letter, Dong and colleagues51 After being exposed to infected family members, the 55-d-old female was admitted to the hospital with symptoms of COVID-19 and diagnosed based on clinical data and exposure history. She was “mixed fed.” Her mother’s milk was collected on the first 3 consecutive days of her hospitalization; all milk samples tested negative for SARS-CoV-2. No information on the collection or testing methods for the milk sample is included in this report. Yuehua and colleagues52 reported on a 3-mo-old, breastfed female who was hospitalized and tested via throat swab for SARS-CoV-2; the swab was positive. A single milk sample was collected from the infant’s mother; it tested negative. The authors provided no information on the collection or testing methods for the milk. Importantly, this infant developed symptoms of COVID-19 7 d before her parents did. As such, one possibility is that she was infected first and passed the infection to them. Another case report on a mature milk sample comes from Singapore.50 This report is particularly interesting as the infant had no symptoms but was hospitalized and tested because his caregivers were all hospitalized with COVID-19 and there was no one to care for him. The infant was 6 mo old and presumably at least partially human milk fed as a sample of milk was successfully collected from his mother. Despite being asymptomatic, a nasopharyngeal swab taken from the infant was positive for SARS-CoV-2. The authors reported that milk produced by the mother on a single day tested negative for the virus but do not specify how many samples were taken. This report provided no data on the methods used for the collection and analysis of these sample(s).While the previous reports focused on infected women, there are also three case studies focused on infected infants. In these studies, milk produced by the infants’ mothers was tested for SARS-CoV-2. The youngest of these infants was reported by Cui and colleagues.Despite the devastating clinical manifestations of SARS-CoV, MERS-CoV, and SARS-CoV-2, there remains much to be learned about their modes of transmission. Respiratory droplets are a documented source of the virus, but other sources such as human milk may exist. The primary purpose of this review was to examine the evidence  for the vertical transmission of SARS-CoV-2 from mother to infant via breastfeeding considering what is known about other human coronaviruses.49 This remains a critical area that must be addressed to fully understand the role, if it exists, of breastfeeding and the feeding of human milk in infant infection.There are currently 9 studies available on SARS-CoV-2 and human milk, collectively encompassing at least 23 milk samples, all of which tested negative for SARS-CoV-2. There are no comparable data for MERS, and a single case report for SARS, which yielded a negative result for the presence of the virus but positive results for antibodies specific to SARS-CoV. There have been no antibody tests in milk specific to SARS-CoV-2 in any of the reports to date, although one paper reported on SARS-CoV-2 antibodies in infant serum which were likely transplacental and thus maternal in origin.36 reported that milk was not collected from camels aseptically; rather, samples were obtained according to local milking customs. As such, it is possible that the presence of MERS-CoV in camel milk could be due to contamination from the milker, the calf, or the environment, rather than milk representing an endogenous source of the virus. However, the limited data available on all three of these viruses  leave many questions unanswered with respect to the role, if any, of human milk in vertical transmission of coronaviruses.The presence of viable MERS-CoV in camel milk is suggestive of the possibility that SARS-CoV-2 could be present and viable in human milk . Notably, Reusken and colleagues53 and RNases,56 and other PCR inhibitors.59 Thus, validation of methods using human milk is needed. In addition, other than general statements about the timing of collection  and brief descriptions of the RT-PCR assays used for nasal and throat swabs, none of the studies to date has described the methods of collection and how the milk was handled and stored in any detail. Of note is the fact that commonly used silica column-based RNA isolation methods are designed for a limited sample volume, and as such are not suitable for more voluminous liquid samples. In addition, nothing is known about stability of SARS-CoV-2 in human milk and how quickly (or at what temperature) it must be frozen to preserve fidelity. Information on sample collection, handling, and storage is critical to evaluating whether the negative results described in these studies could be due to inadequate methods used.One possible reason that the RT-PCR results for all the milk samples tested were negative is that the methods used were neither designed nor validated for human milk. Milk is a complex matrix containing substantial fat, DNases,30 159 maternal-infant dyads were tested . In this report, 229E was present in both maternal and infant samples in only 2 dyads. Additionally, in the milk of dromedary camels, MERS-CoV appears to be present at very low abundance.36 This suggests the possibility that very low viral load in milk might also lead to an inflation of false negatives.Another possibility is that there is low abundance of the virus in human milk, and it has simply not been captured in the limited samples tested so far. For example, in the report on other human coronaviruses by Gagneur and colleagues,49 who reported that both IgG and IgM antibodies to SARS-CoV-2 were present in the serum of an infant at 2 hr of age, despite multiple negative RT-PCR tests of nasopharyngeal swabs over the first days of life. The presence of antibodies at such an early stage of life could indicate transfer of SARS-CoV-2-specific antibodies from mother to infant during gestation. It is noteworthy that IgM antibodies49 present in the serum of SARS-CoV-2 negative infants cannot cross the placental barrier.49 Together, these observations suggest infant infection in utero, but that the virus may simply be absent from the upper respiratory tract immediately after birth.From the limited data on SARS-CoV, it appears that the presence of antibodies in milk could be influenced by timing of infection, where antibodies to SARS-CoV were detected only in milk produced by a woman who acquired the infection later in pregnancy. While the methods used to test this milk were not fully described, this observation could have impacts on the clinical management of infants born to women diagnosed with COVID-19 during pregnancy and/or lactation. This observation is also supported by the findings of Dong and colleagues60 angiotensin-converting enzyme 2 (ACE2) is one of the receptors used by SARS-CoV-2 to enter host cells.62 ACE2 is expressed across many body sites and tissue types, including the oral cavity  and in mammary tissue.63 If mammary epithelial cells express this receptor, then it follows that viable virus could exist in milk. If it does, then the introduction of virus-containing human milk could represent a mechanism of entry for SARS-CoV-2 and COVID-19 infection for infants.Very recent work has demonstrated that, like SARS-CoV and human coronavirus NL63,52 the infant was infected and symptomatic 7–8 days prior to the infant’s parents. This suggests the possibility that a “reverse” vertical transmission from infant to mother could occur, a phenomenon which has been observed for other pathogens, such as HIV65 and Ebola virus.66 One possible mechanism for maternal infection in this case is through retrograde flow, where milk and saliva move back into the mammary gland from the infant’s mouth during suckling.67 While this mechanism is speculative, it represents a possible route whereby an infant could theoretically transfer a pathogen it has encountered in the environment to the mother. It is also possible that maternal infection could occur through other mechanisms, such as infant respiratory droplets68 or via fecal matter.69Another observation worth considering is that in at least one of the reports71 Substantial interdisciplinary research on this topic is requred and should be performed rigorously and rapidly to best inform policies regarding early feeding choices and clinical management of breastfeeding mothers infected with SARS-CoV-2 and their infants.Human milk is the gold standard for infant feeding. However, confidence with regard to its safety and best practices around breastfeeding during maternal COVID-19 infection has been compromised by the lack of evidence as to whether SARS-COV-2 can be vertically transmitted in milk and/or during breastfeeding. As such, there exists an immediate need to rapidly generate rigorous evidence for the role (if any) of human milk and breastfeeding in vertical transmission of COVID-19 from mothers to infants. To accomplish this, validation of analytical methods for the human milk matrix, viability testing, and evaluation of other immune components in milk will all be critical to this effort, especially given the known protective effects of breastfeeding in other infant respiratory infections.


South Asia is the hotspot of beta-thalassemia, with an estimated 200,000 patients whose lives depend on regular blood transfusion. Due to COVID-19 pandemic, many countries have adopted unprecedented lockdown to minimize the spread of transmission. Restriction of nationwide human mobility and fear of COVID-19 infection has put thalassemia patients in a life-threatening situation because of an acute shortage of blood supply. As a public health preparedness strategy during a crisis like COVID-19 pandemic, the plights of thalassemia patients should be considered. Government-sponsored community blood-banks needs to be established or coverage expanded as a safety net for the thalassemia patients in lower- and middle-income countries. South Asia has been confronting a silent epidemic of thalassemia- a life-threatening inherited hemoglobin disorder. Over 200,000 estimated thalassemia patients (45–70 million carriers) live in the Indian Subcontinent whose lives depend on regular blood transfusion with chelation therapy –3. ThereWhile access to blood remains a persistent challenge, the coronavirus pandemic  has exacerbated the circumstances. To minimize the spread of SARS-CoV-2, the majority of the South-Asian countries have adopted lockdown measures. The lockdown has impacted people's lives in unprecedented ways—for example, the lack of public transportation to restrict people's movement. It is likely that such restrictions will also make it difficult, or even impossible at times, for both the blood donors and recipients . Data do not exist to measure how this would impact the lives of thalassemia patients. Understandably, the ongoing lockdown is likely to put a significant portion of the patients into a life-threatening condition due to an acute shortage of blood supply at the community level. Community clubs and NGOs have to postpone blood donation programs. In addition, friends and relatives are not coming forward to donate blood due to panic of COVID-19 infection , 5. For Uncertainty around COVID-19, followed by lockdown, may impact Bangladesh significantly. Here 10–12% of the 160 million population are thalassemia carriers, and the issue is largely neglected at the policy level [Another hospital-based study found that over 62% of HbE beta-thalassemia patients are transfusion-dependent, requiring transfusion 1 to 4 times every month [The current lockdown has exposed a severe weakness in the supply chain of blood, particularly for thalassemia patients. Recruiting voluntary donors is a big challenge in developing countries due to a lack of awareness. In Bangladesh, according to a WHO report, over 600,000 units of blood were collected against an estimated demand of 800,000 in 2016 . It is lIn future public health preparedness strategy during a crisis like COVID-19 pandemic, the issue of transfusion-dependent patients must be prioritized, particularly in thalassemia prone countries in South Asia. In this perspective, the community-based blood banking needs to be encouraged and expanded with government sponsorship to keep the vulnerable patients within a safety net.


Performing tracheotomy in patients with COVID-19 carries a risk of transmission to the surgical team due to potential viral particle aerosolization. Few studies have reported transmission rates to tracheotomy surgeons. We describe our safety practices and the transmission rate to our surgical team after performing tracheotomy on patients with COVID-19 during the peak of the pandemic at a US epicenter.Retrospective cohort study.Tertiary academic hospital.Tracheotomy procedures for patients with COVID-19 that were performed April 15 to May 28, 2020, were reviewed, with a focus on the surgical providers involved. Methods of provider protection were recorded. Provider health status was the main outcome measure.Thirty-six open tracheotomies were performed, amounting to 65 surgical provider exposures, and 30 (83.3%) procedures were performed at bedside. The mean time to tracheotomy from hospital admission for SARS-CoV-2 symptoms was 31 days, and the mean time to intubation was 24 days. Standard personal protective equipment, according to Centers for Disease Control and Prevention, was worn for each case. Powered air-purifying respirators were not used. None of the surgical providers involved in tracheotomy for patients with COVID-19 demonstrated positive antibody seroconversion or developed SARS-CoV-2–related symptoms to date.Tracheotomy for patients with COVID-19 can be done with minimal risk to the surgical providers when standard personal protective equipment is used . Whether timing of tracheotomy following onset of symptoms affects the risk of transmission needs further study. In the setting of COVID-19, the decision to proceed with tracheotomy poses new questions due to the unclear risk-benefit ratio. One major consideration is the potential risk of transmission from the patient to the health care team, as this procedure can be a source of aerosolized virus during and after tracheotomy tube placement.The 2019 coronavirus pandemic (COVID-19), caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), has affected >5 million Americans and claimed the lives of >161,000 as of August 10, 2020.3 Entering and manipulating the airway of a patient harboring active SARS-CoV-2 virus poses obvious risks. In an effort to protect personnel involved in the procedure, protocols to decrease transmission during tracheotomy were created, and recommendations were published and widely circulated on the appropriate level of personal protective equipment (PPE).24 While some guidelines recommended the use of powered air-purifying respirators for any aerosol-generating procedure, others recommended standard airborne precautions, relying mostly on eye protection and N95 respirators. At the current time, literature has not materialized demonstrating that enhanced PPE offers improved protection from SARS-CoV-2. We report our experience performing tracheotomy with standard PPE for airborne precautions  in patients diagnosed with SARS-CoV-2. We describe the specific protective equipment used and safety measures taken and report the exposures and rate of infection among the tracheotomy surgical providers.Previous reports documented an increased risk of SARS-CoV-2 transmission when operating on mucosal surfaces of the head and neck due to high viral loads in the upper aerodigestive tract.A retrospective chart review was completed of all patients requiring prolonged intubation due to SARS-CoV-2 infection who underwent tracheotomy by the otolaryngology service at Montefiore Medical Center. The research was approved by the Institutional Review Board at the Albert Einstein College of Medicine. Following consent, providers in our department involved in tracheotomy were surveyed regarding the protective measures taken during tracheotomy, presence of COVID-19 symptoms, results of SARS-CoV-2 testing, and antibody status before and after involvement in tracheotomy procedures.Institutional guidelines describing safe practices and PPE were created and circulated at the beginning of the COVID-19 pandemic . The otolaryngology team adhered to these guidelines throughout the study period. Tracheotomy was performed in the operating room (OR) or at the bedside in the intensive care unit (ICU) or general medical floor. Bedside procedures were generally favored to limit the risk of transmission during patient transport, to decrease the need for dedicated surgical staff, and to lower burden on OR time and personnel during the pandemic. OR procedures were carried out if the patient required other surgical procedures  or if the operating theater setting was deemed more appropriate by the attending surgeon. On the general medical floor, the procedural team included an attending surgeon, a resident surgeon, an anesthesiologist, and the patient’s nurse. In the ICU, the procedural team consisted of an attending surgeon, a resident surgeon, a respiratory therapist, the ICU nurse, and covering critical care staff, as needed, to administer medications. The number of people in the room was limited to only those necessary to execute the procedure, manage the endotracheal tube, and administer medications.Our consultation team advocated for repeat SARS-CoV-2 polymerase chain reaction (PCR) testing prior to tracheotomy to assess risk of transmission. However, this was not mandated and was not consistently performed, as it did not change medical or procedural decision making. Standard PPE for airborne precautions were maintained for every tracheotomy performed during the pandemic, regardless of repeat test results. According to guidelines from the US Centers for Disease Control and Prevention, this included a surgical cap, face shield/eye protection, N95 mask, and surgical gown and gloves (some physicians used 2 sets of gloves). Protection was donned prior to entry into the room and removed prior to exiting. All surgeons had been educated via institutional information sessions and electronic notifications regarding proper donning-and-doffing procedures for PPE. Tracheotomy procedures were carried out in routine open fashion. The surgical team preferred an open approach over percutaneous methods due to theoretical safety advantages secondary to the degree of control of the airway throughout the procedure, without the need for confirmatory bronchoscopy. Specific to the COVID-19 pandemic, all patients were routinely paralyzed throughout the procedure, and suction and cautery were minimized to avoid aerosolization of viral particles. Some providers also used anticholinergics to decrease secretions and thus minimize suctioning requirements. At entry into the trachea, the ventilator was placed on stand-by to eliminate positive-pressure aerosolization of tracheal secretions. Positive-pressure ventilation was resumed after placement of the tracheotomy tube with inflation of the cuff. Placement was confirmed with the usual methods, including adequate inspiratory and expiratory tidal volumes and/or end tidal carbon dioxide monitoring.We completed a retrospective chart review of all tracheotomies performed by the otolaryngology service on patients admitted with symptoms of COVID-19 and a positive SARS-CoV-2 PCR test result. Dates of admission, intubation, and tracheotomy were obtained to assess time to tracheotomy. Dates and results of SARS-CoV-2 PCR testing were collected with attention paid to the timing in relation to tracheotomy. The involved surgeons (based on operative notes) were surveyed to assess the safety practices utilized, the presence of COVID-19 symptoms, and the result of SARS-CoV-2 antibody testing.The primary outcome was SARS-CoV-2 antibody positivity or symptomatic illness of the attending and house staff surgeons involved in the tracheotomy procedures. Antibody testing was made available by our institution and offered to all providers. It was performed according to the presence of SARS-CoV-2 symptoms or desire of the provider to undergo testing; there were no specific guidelines or mandates to obtain testing at our institution during the study period. Data were not collected for respiratory therapists, anesthesiologists, nurses, or ICU staff involved, as these providers were highly varied across dates, locations, and operative settings.5Data for this study are largely descriptive and presented as whole numbers, proportions or percentages, and mean estimates, where appropriate, with ranges included as necessary. A swimmer’s plot was constructed to graphically represent the number of COVID-19 tracheotomy exposures for each surgeon. To estimate the likelihood of transmission of SARS-CoV-2 based on the data in this study, we constructed 95% CIs using the exact formula (Clopper-Pearson).Thirty-six tracheotomies in patients diagnosed with SARS-CoV-2 were identified and reviewed for this study. The locations were at the bedside in 30 of 36 (83.3%), of which 6 (20%) were done on the medical floors and 24 (80%) in the ICU. One of the tracheotomies done in the OR was in conjunction with thyroidectomy for compressive goiter. The average time from hospital admission for COVID-19 symptoms to tracheotomy was 31 days, with a range of 20 to 61 days. For patients with an immediate perioperative positive SARS-CoV-2 test result, the average time from hospital admission for COVID-19 symptoms to tracheotomy was 28 days, with a range of 20 to 38 days.All patients included in this review tested positive for SARS-CoV-2 during hospitalization. Ten patients (27.8%) had an immediate preoperative positive test result. This included any patient with a positive test result both before and after tracheotomy or within 72 hours prior to tracheotomy. Ten patients (27.8%) had a negative preoperative test result. The remaining 16 did not undergo SARS-CoV-2 PCR testing in the immediate preoperative setting. Two patients had a positive test result 5 days prior to tracheotomy; 1 patient, a positive test result 10 days prior; 3 patients, a negative test result 3 to 5 days after tracheotomy; 4 patients, a negative test result 7 to 10 days after; and 6 patients, no test result within 10 days before or after tracheotomy. The location of tracheotomies and the immediate perioperative testing status for these procedures are summarized in All tracheotomies were performed by 1 of 4 attending surgeons, who performed 16, 10, 9, and 1 tracheotomies. There were 11 resident surgeons involved; 2 of them were seropositive after developing COVID-19 prior to performing any tracheotomy for patients with SARS-CoV-2. Because they were hypothetically not at risk of contracting SARS-CoV-2 from the tracheotomy exposure, these 2 individuals were excluded from the final analysis. Overall, 4 attending surgeons and 9 house staff were involved in tracheotomies and included in the study. After exclusion of the previously mentioned providers, there were 65 exposures during tracheotomy included in our study: 19 to patients with an immediate preoperative positive SARS-CoV-2 test result, 18 to patients with an immediate preoperative negative SARS-CoV-2 test result, and 28 to patients with unknown SARS-CoV-2 testing status at time of tracheostomy. All surgeons followed the institutional recommendations for PPE and safety measures listed in 5 Based on 36 tracheotomy procedures, if the real population rate of transmission was 0.05 and the outcome was defined as any transmission occurring in any of the surgical providers present at the tracheotomy, the 95% CI would be 0.005 to 0.179. If the real population rate was 0.03, the 95% CI would be 0.001 to 0.149. We repeated this analysis using 65 surgical provider exposures, with a potential outcome of 1 transmission per exposure. Based on hypothetical population transmission rates of 0.05 and 0.03, the respective 95% CIs were 0.011 to 0.134 and 0.004 to 0.106. These results suggest that whether the tracheotomy procedure is used as the unit of analysis or the surgical provider exposure, there would be roughly a <2.5% probability of observing zero transmissions if the true population rate was as low as 0.03.To estimate the likelihood of transmission given our results, we constructed a 95% CI using the exact formula (Clopper-Pearson).6 There were also reports of providers being infected despite proper PPE, which led to some recommendations for enhanced PPE, including helmets, hoods, and powered air-purifying respirators.7 Early experience with SARS-CoV-2 in China indicated that health care workers involved in tracheotomy had a greater risk  of viral contraction than those who did not.8 This led to further recommendations regarding enhanced PPE when performing tracheotomy. In addition, recommendations against performing tracheotomy at all in patients who were SARS-CoV-2 positive were also widely accepted due to the risk of contraction by providers.2 As the otolaryngology community adjusted to these recommendations, the number of SARS-CoV-2 patients at our institution dramatically increased and persisted for several weeks. As a result, a careful approach to tracheotomy was necessary to optimize patient care and disposition and to properly allocate resources. After review of the existing literature, the decision was made to proceed with tracheostomy with standard PPE instead of powered air-purifying respirators. Despite initial concern, no transmission events were documented from patients to providers via standard SARS-CoV-2 PPE.At the onset of the COVID-19 pandemic in the United States, there were varying recommendations regarding the performance of tracheotomy given the risk of transmission to providers and the lack of evidence on which patients would benefit from it. Based on data from prior experience with highly communicable respiratory diseases, such as SARS, it was known that health care providers involved in endotracheal intubation or tracheotomy had a higher risk of viral contraction.9 The SARS-CoV-2 viral particle diameter is 0.06 to 0.12 µm,10 and it travels within aerosol particles, which are >1 µm. The N95 mask therefore protects providers against 99.5% of viral particles of SARS-CoV-2. Data regarding the variance in efficacy among brands are inconclusive. There was no standardized brand, style, or type worn by providers in our study. The masks worn were distributed by the institution, which varied throughout the study period. Based on our experience, an infectious transmission of virus from affected patient to provider during tracheotomy did not occur. Thus, we support the use of standard PPE during tracheotomy for patients testing positive for SARS-CoV-2.All providers in our study wore N95 masks, face shields, surgical caps, surgical gowns, and gloves. Arguably, the N95 mask was the most important piece of PPE among these items. N95 masks are effective at filtering 99.5% of particles >0.75 µm.One element that may have protected the providers in this study was the time from initial diagnosis to tracheotomy. The average time to tracheotomy in our cohort from hospital admission for COVID-19 symptoms was 28 days, with a range of 20 to 38 days in patients with an immediate perioperative positive SARS-CoV-2 test result. The timing of tracheotomy should balance the risk of transmission to the surgical team and the benefit of tracheostomy for the patient. Patient outcomes are not reported in this study; therefore, we are unable to comment on the ideal timing of tracheotomy.11 The correlation between positive results by quantitative PCR viral testing and infectivity is still being investigated. Recent studies showed that positive viral cultures were more common among patients with a positive SARS-CoV-2 PCR test result who had lower cycle threshold values , suggesting that patients who test positive have higher viral loads and high virulence. This study deduced that patients who test negative on PCR with higher cycle threshold values may not be excreting infectious viral particles and are thus less likely to put those in contact with them at risk of contraction.12 Further data suggest that patients affected by SARS-CoV-2 are infectious beginning 2.3 days prior to symptom onset, with infectivity peaking at 0.7 days before symptom onset and rapidly declining within 8 days following symptom onset. The majority of our patients underwent tracheotomy outside this window. However, these data were derived from patients with moderate forms of the disease. In addition, we concede that this is likely affected by the use of antivirals, anti-inflammatories, antibiotics, and immunomodulators,13 which were not measured in our review. However, all patients in our study did receive hydroxychloroquine and at least 3 days of steroids during admission prior to tracheotomy, per standard protocols recommended by the infectious disease service at our institution during the early COVID-19 surge in New York in March and April 2020.The virulence of SARS-CoV-2 and the natural history of viral loads and viral shedding among patients with severe COVID-19 disease are not fully understood. The majority of patients in our study tested negative for SARS-CoV-2 on immediate perioperative PCR or were not tested at the time of tracheotomy. Recent data suggest that even after upper respiratory tract samples become negative, lower respiratory tract samples may remain positive for up to 39 days.14 We believe that our report is representative of the exposure risks at most high-volume centers in the midst of the COVID-19 epidemic.The previously discussed data support decreased risk of transmission in patients with negative viral swabs. However, several patients in our study had a SARS-CoV-2–positive PCR test result at the time of tracheotomy, and most providers performed at least 1 tracheotomy on 1 of these patients . Despite this, no providers exhibited COVID-19 symptoms or seroconverted. In addition, all of the patients included in our study had the severe form of the disease, requiring intubation and mechanical ventilation. Data suggest that patients with the severe form of the disease have a higher viral load that decreases more slowly than those with mild disease.Despite our detailed report, our study has several limitations. Since we do not have precise data of SARS-CoV-2 viral load in the respiratory tracts at the time of tracheotomy for each case, we cannot clearly quantify the risk in our cohort. We also were not able to collate data for risk of transmission to other patients and health care staff on the basis of these procedures or posttracheostomy care. Furthermore, we were not able to assess risk of transmission to the nursing staff, respiratory therapists, or anesthesia staff, as these providers varied at each operation, though anecdotally these rates were very low based on ongoing discussions with our hospital staff. The majority of procedures were done in single-patient rooms, not all of which were negative-pressure rooms. Negative-pressure rooms were preferred to prevent the airborne particles inside the infected patient’s room from entering the rest of the unit and infecting other patients or providers. However, because of the extensive number of patients with COVID-19 at our institution, negative-pressure rooms were often allocated to nonintubated patients who were at risk of spreading virus to others, and in large part negative-pressure rooms were not available for tracheotomy procedures. Future studies may aim to evaluate whether negative-pressure rooms decrease the risk of transmission to other health care workers, as we are not able to comment on this in this study. In addition, now that we better understand the role of tracheotomy in the setting of the COVID-19 pandemic, future studies should consider assessing the risk to all health care providers during posttracheostomy care.All of our providers continued to wear standard PPE for routine tracheostomy care and tracheostomy tube changes. We performed tracheostomy tube changes only in the setting of air leak causing respiratory distress or when downsizing tracheotomy tubes for decannulation. The providers in our cohort were relatively young without significant comorbidities; this may have affected the presence of symptoms related to the exposure during tracheotomy. While several of these providers had antibody-negative testing after involvement, some reported only the lack of symptoms as the outcome measure. The CIs calculated for the transmission rate with 65 surgical staff exposures did not account for lack of statistical independence of those exposures. A larger study across multiple institutions would help to definitively conclude that our findings are widely generalizable.Despite these limitations, we believe that this review provides data to support minimal risk of contraction to the surgical team when performing tracheotomy in patients with SARS-CoV-2, using standard PPE for airborne precautions. Further studies are needed to quantify this risk, and we recommend routine review and adherence to the most up-to-date safety measures.Our data support that tracheotomy for patients with SARS-CoV-2 can be done with minimal risk to the surgical providers when standard PPE is used . In a large-volume setting with a relatively high number of tracheotomies, there was zero rate of transmission as a result of involvement in the procedure. Each center should adopt the safest measures available when preparing for high-risk procedures in the COVID-19 era, and we recommend corroborative reports before these measures are widely accepted.


Due to the rich vitamin content in citrus fruit, citrus is an important crop around the world. However, the yield of these citrus crops is often reduced due to the damage of various pests and diseases. In order to mitigate these problems, several convolutional neural networks were applied to detect them. It is of note that the performance of these selected models degraded as the size of the target object in the image decreased. To adapt to scale changes, a new feature reuse method named bridge connection was developed. With the help of bridge connections, the accuracy of baseline networks was improved at little additional computation cost. The proposed BridgeNet-19 achieved the highest classification accuracy (95.47%), followed by the pre-trained VGG-19 (95.01%) and VGG-19 with bridge connections (94.73%). The use of bridge connections also strengthens the flexibility of sensors for image acquisition. It is unnecessary to pay more attention to adjusting the distance between a camera and pests and diseases. Pests and diseases are major causes of huge economic loss in agricultural production. Timely detection and identification of these pests and diseases is essential to control their impact. Farmers used to rely on experienced experts to perform these tasks; unfortunately, this is a time-consuming, error-ridden, and costly process. To improve recognition efficiency, image processing and computer vision techniques are widely implemented. Bashish et al.  proposedThe above research all required pretreatment to select features for classifiers. In contrast, deep convolutional neural networks (CNN) are representation-learning models . They caFeature reuse is an indispensable component of many deep network architectures. It was first employed in the Highway Network  which deA new citrus pest and disease image dataset was recently created . The imaThe rest of this paper is organized as follows: Compared with traditional machine learning methods, deep learning is an end-to-end model which does not require the design of handcrafted features for classifiers . In addiNetwork architecture has a considerable effect on model performance. In general, a deeper network constructed results in a higher accuracy being delivered . HoweverImageNet pre-training enables people to easily access encouraging results . For thiOur dataset has 12,561 images. It covers 17 species of citrus pest and seven types of citrus disease. Each class contains over 350 images. Image samples used in earlier studies were gathered under laboratory conditions ,31, whicComputer vision competitions greatly promoted the development of deep learning. Many advanced design methods were proposed to improve network performance, which changed the situation of simply stacking convolutional layers. We followed the strategy of SqueezeNet  to desigAttention mechanisms usually produce an attention map to highlight the important features, which brings an additional computation overhead and increases the optimization difficulty. We followed the micro-construction of the Network in Network  to enhanIn order to address the degradation problem, He et al.  developeIt is well known that CNN models are very sensitive to translations and rotations . We obseThere are many mid-level and high-quality features in the intermediate layers. However, direct reuse of them will increase the computational complexity of the classification layer. In addition, the proportion of low-level features in shallower layers was greater than in deeper layers . Based oChannel compression: To reduce the number of useless features, the number of output channels from the 1 × 1 convolution is fewer than the number of input channels. This function is similar to the transition layer of DenseNet.Feature retention: Unlike a 3 × 3 convolution, a 1 × 1 convolution performs a simple linear transformation, which can largely preserve input feature information. To further strengthen output feature quality, two 1 × 1 convolutions were stacked after the concatenation operation.i-th feature map of the n-th layer, (n−1)-th layer.Conventional feature reuse strategies (addition and concatenation) do not consider the discrepancy between features from different layers. More specifically, the difference between shallow features and deep features is not only in the distribution characteristic, but also in the representation complexity. This complexity difference between adjacent layers can be measured by Equation (1). As the distance between layers increases, the difference mentioned will be more significant. The progressive feature reuse method shown in The overall architecture of BridgeNet for citrus pests and disease recognition is shown in It is of note that the use of deformable convolutions does not improve VGG-16 performance. In contrast, the application of bridge connections considerably increased validation and test accuracy. As for additional computational cost, the bridge connection created much less of a computational burden than deformable convolution; the use of bridge connections in the model costed 5.8 MB and the cost of using deformable convolutions was 40.8 MB. Weakly DenseNet-19 performed better than CBAMNet, which indicates that the two 1 × 1 convolutional layers used for feature enhancement are more effective than the attention mechanism. The accuracy of Weakly DenseNet-19 was further increased by using features from the middle layers for classification. In terms of additional computational cost, BridgeNet-19 spent 12.3 MB and MSN-19 consumed 10.3 MB. However, BridgeNet-19 obtained better performance than MSN-19, proving the higher parameter efficiency of using bridge connections. The smaller-size models took less training time per batch except for MSN-19, which had a slower training speed than BridgeNet-19. To validate the effectiveness of the new feature reuse method for adapting to object scale changes, the confusion matrices of Weakly DenseNet-19 and BridgeNet-19 were compared . Using tWe considered the number of bridge connections as a hyper-parameter and explored its impact on network performance. Bridge connections were introduced from top to bottom as the overall number of connections was increased. Weakly DenseNet-19 was used as the backbone architecture. In this study, a new CNN model was developed to identify common pests and diseases in citrus plantations. Each building block of the network contained two 1 × 1 convolutions that were used to enhance the features generated by 3 × 3 convolutional layers. Concatenation operations were used for feature reuse. To reduce redundancy, only features from adjacent layers were concatenated. We observed that, as the size of the target object in the image decreased, the use of mid-level features by the classification layer increased. Using this insight to adapt the model to scale changes, a new feature reuse method called bridge connection was designed. Experimental results show that the proposed BridgeNet-19 achieved the highest classification accuracy (95.47%). Compared with pre-trained models, our network also presented higher parameter efficiency; its model size (68.9 MB) was half of the pre-trained VGG-16 and VGG-19 networks.Training of deep CNN models usually requires a large-scale image dataset. However, it is very difficult and expensive to collect so many high-quality, close-up sample images in some specific fields such as medicine and biology. Although ImageNet pre-trained models can allow researchers to achieve satisfactory results on different types of datasets quickly and easily, they are designed too bulky and ill-suited to fit small datasets. We hope to find a better solution to solve this problem in the future.


Immune evasion is a major challenge for the development of successful cancer treatments. One of the known mechanisms is the expression of immune checkpoints (ICs)—proteins regulating the immune cells activation. The advent of immunotherapy using monoclonal antibodies (mAbs) to block the immune checkpoint receptor-ligand interaction brought about a landslide improvement in the treatment responses, leading to a prompt approval of such therapeutics. In recent years, it was discovered that a subset of patients receiving IC blockade treatment experienced a previously unknown pattern of treatment response called hyperprogression (HP), characterised by rapid deterioration on initialisation of the therapy. HP represents an urgent issue for clinicians and drug developers, while posing questions about the adequacy of the current clinical trial process. Here, we briefly summarise the state of knowledge and propose new directions for research into HP mechanisms, focusing on tumour-intrinsic signalling of IC proteins malignantly expressed by cancer. We also discuss the potential role of spontaneously occurring canine cancer in the assessment of immunotherapeutics, which can provide the missing link between murine and human studies. Cancer is an urgent problem facing the biomedical field. Its hallmark ability to modulate the host immune system and evade destruction represents a major challenge for the development of successful treatments. One of the key discovered mechanisms of immune evasion is based on the expression of proteins belonging to the immune checkpoint (IC) group. These ligands interact with receptors of the host immune cells to regulate their activation state. The increasingly common use of immunotherapy in cancer treatment, particularly the implementation of the IC blockade (ICB), preventing previously mentioned interaction, has proven a breakthrough treatment in some cancer types. While not all patients respond to this line of therapy, a substantial subset experiences rapid disease progression—a recently described phenomenon called hyperprogression (HP) or Hyperprogressive Disease (HPD). While the clinical data and some biological explanations have been comprehensively described before ,15,16,17Immunotherapy is a treatment modulating the activity of the host immune system. The ratio of improved survival to the extent of adverse effects is favourable for immunotherapy when compared to classic cancer therapies ,19. The Owing to a different mechanism of action as compared to the conventional chemotherapy or targeted therapies, ICB immunotherapy frequently induces atypical response patterns such as long-term remissions observed in multiple cancers. Melanoma is a prime example, with close to 50% of patients experiencing durable response. Such clinical trial results allowed for a breakthrough status and fast-track introduction of this immunotherapeutics class. The speed of approval did not however lend itself to a careful interpretation of the treatment responses in the non-responding groups. Two other patterns have been described that were not commonly observed in the past: pseudoprogression and hyperprogression.Upon initialisation of ICB, in several patients the disease progressed rapidly as measured by volumetric or 2-dimensional assessment of solid tumour size. Subsequently, the tumour volume decreased, leading to a successful treatment response. This phenomenon is considered to stem from inflammation and accumulation of tumour-infiltrating lymphocytes (TILs) that increase the tumour mass temporarily. Hodi et al. defined pseudoprogression as a tumour burden increase by at least 25% that turns out to not represent progressive disease during the following assessment. Interestingly, pseudoprogression can appear either within the first 12 weeks of treatment or in a more delayed form . It becaThe concept of hyperprogression (HP) or Hyperprogressive Disease (HPD) was introduced by Champiat et al. in 2016, based on the observation that a subset of patients receiving ICB experiences extremely rapid disease progression that leads to fast patient deterioration . ImportaIt is essential to detect and distinguish between progression, pseudoprogression and hyperprogression early. Since in most cases the tumour growth rate (TGR) data from before the immunotherapy is not available, the distinction is not possible by basic imaging of changes in the tumour size. Berge et al. demonstrated that in the presence of such data, TGR is a clinically relevant predictor of overall survival . AttemptCancers’, Kim and colleagues found that only five factors can be considered statistically significant in the general patients’ pool. HP was associated with elevated serum LDH concentration, presence of more than two metastatic sites, liver metastases and Royal Marsden Hospital (RMH) prognostic score equal 2 or more. Strong expression of PD-L1 was inversely associated with HP [Associations were made with increased age , higher  with HP . The aut with HP .Multiple mechanisms have been proposed to be involved in development of hyperprogression .+), which expanded upon PD-1/PD-L1 blockade, resulting in a strongly immunosuppressive microenvironment, allowing for rapid cancer cell proliferation. The clinical results were confirmed in vitro and validated in mouse models, strongly supporting the role of T-reg cells in hyperprogression. Disabling PD-1 with Cre-Lox system enabled authors to show that PD-1-deficient T-regs exhibited enhanced proliferation and immunosuppressive mechanisms. The results were analogous when PD-1 was blocked with a mAb. On the other hand, Nair et al. described inhibition of peripheral FoxP3 T-reg cells differentiation and a FoxP3 down-regulation through mTOR pathway under pembrolizumab-mediated PD-1 blockade [Kamada et al. described a rapid expansion of FoxP3 T-regulatory (T-reg) cells in gastric tumour patients with HP . FoxP3 iblockade . The treblockade ,54.+ and CD8+ cells overexpressed another inhibitory IC receptor TIM-3 (T-cell immunoglobulin and mucin-domain containing-3). TIM-3 was predominantly detected on ICB mAb-bound, intratumoral T-cells. The abundance of TIM-3+ cells was proportional to the length of the ICB treatment. Increase in TIM-3+ cells was not observed in cases which responded to the treatment. The up-regulation of TIM-3 is one of the mechanisms of resistance to PD-1-based therapies [Similarly, as previously hypothesised by Rauch et al. in their study on adult T-cell leukaemia/lymphoma under Nivolumab treatment , Koyama herapies . There aherapies . This ex+ T-cells that become activated and interact with breast cancer cells but are incapable of lysing them elicit dedifferentiation cascade in the target tumour cells, inducing cancer stem cell (CSC) formation [Stein et al. demonstrated that CD8ormation . They hyKudo-Saito et al. observed that in the murine model and in cell culture studies chemo- and immunotherapy activated putative cancer stem cells (CSCs) leading to aggressive proliferation and resistance. They suggested that HP may depend on the proportion of cancer cells elimination and dormant cells activation . While tIn 2017, Arlauckas et al. performed an in vivo imaging experiment by tracking fluorescently labelled anti-PD-1 mAbs, MC38 tumour cells, and tumour-associated macrophages (TAMs) injected into mice and following the interactions between those components . They obUntil recently, the mechanism of action of PD-1/PD-L1 blocking antibodies was considered to base solely on their ability to block the interaction between PD-1 receptor localised on the surface of T-cells and PD-L1 ligand expressed by cancer cells. In fact, success rates of PD-1/PD-L1 therapy vary between 20% and 90%, depending on the tumour type ,67,68. TMultiple recent publications reported that cancer cells express not only PD-L1 but also PD-1 in tumour types such as melanoma, hepatocellular carcinoma, ovarian cancer, and NSCLC ,70,71. UPD-L1 expression by cancer cells has been studied extensively and was associated with poor prognosis and metastatic disease . Intereshigh cells revealed activation of genes responsible for cell migration and motility. It was proposed that PD-L1 binds to Ras, then triggers the Ras/Raf/MAP/Erk cascade that regulates endothelial to mesenchymal transition, widely associated with enhanced tumorigenesis of GBM [PD-L1 tumour-intrinsic signalling was also studied in glioblastoma (GBM). mRNA sequencing of PD-L1s of GBM . Besidess of GBM . While ts of GBM ,85. Thers of GBM . The exas of GBM . Changess of GBM . VEGF was of GBM . In resuAs mentioned earlier, Kim et al. note that the elevated LDH serum level significantly associated with HP has well established links with several mechanisms of tumorigenesis and immune evasion. High LDH indicates intratumoral hypoxia as well as extracellular environment acidification. The authors concluded that the link to HP is unknown . It is kThe knowledge of HP incidence in different patient populations is lacking due to small cohorts and no data was found on HPD incidence between human races due to mostly Caucasian cohorts . CalifanBinding of PD-1 by an ICB antibody blocks the PD-L1 binding site; however, it is known that the second ligand PD-L2 uses a different binding site and mechanism . While tThe microbiome is a well-known driver of inflammation and immune response. There is a body of evidence that gut microbiota impacts the outcomes of ICB treatment in melanoma patients ,109,110,+ T-cells.Reports on the differences in the rate of HP between different ICB antibodies are contradictory ,49. SuchAny mechanism by which ICB could help cancer cells decrease neoantigen expression and presentation, up-regulate alternative IC proteins or the production of immunosuppressive enzymes and cytokines, can be an important part of HP genesis. Finally, in such a complex disease, there may be no single mechanism or marker of HP applicable to all the cancer types and patient populations.The crossing of Kaplan–Meier survival curves of the ICB arm and chemotherapy control arm in the early treatment period during ICB clinical trials such as Checkmate-057  has beenThere is no denying that the murine model has brought insight into human cancer immunology and a wide spectrum of human diseases. However, only a fraction of therapeutics effective in mice enter human trials and of those, approximately 8% will pass the I phase of clinical trials ,117. TheAs a matter of fact, the murine model bears several bottlenecks that may contribute to failures in resembling human diseases accurately . LaboratNumerous factors act in favour of comparative oncology research in dogs as a preclinical model for human disease. Cancer is the leading cause of fatality in dogs, affecting approximately one in four, and as many as 50% of dogs in certain predisposed breeds . DespiteOne of the most prominent diseases of unmet clinical need in both humans and dogs is osteosarcoma (OS), a fatal malignancy with poor prognosis in both species . In the While the concept and existence of HP remain controversial and occasionally questioned, dialogue around it is increasing. Based on a PubMed search for “hyperprogression”, after the introductory publication by Champiat et al. in 2016 there were nine search hits the following year, then 27 in 2018, 45 in 2019 and just six in January 2020. The topic was also discussed in depth during a dedicated panel at the AACR 2019 meeting in Atlanta. The exploration of causative mechanisms behind HP and development of prediction/detection methods is urgent if we consider the increasingly complex landscape of registered trials applying multiple ICB together or in combination with other treatment approaches. Here we propose that factors such as tumour-intrinsic IC signalling, the impact of pH in the tumour microenvironment on mAb functionality, gut and tumour microbiome composition, patients race and endocrine status, ICs and FcRs polymorphisms and the impact of PD-1/PD-L1 blockade on the PD-1 interaction with its other ligand/s deserve attention in the process of elucidating HP mechanisms. We further suggest that the canine model of human cancer could naturally mimic the characteristics of human disease, including the heterogeneity of the patient population, and offer an advantage over the murine model. Increased understanding of hyperprogression will facilitate the development of methods to correctly predict personalised treatment responses, stratify patients in regard to the expected benefit and to detect an early need for therapy change. Targeted use of ICB could benefit all patients irrespectively of their response to immunotherapy. This would improve safety and efficiency profiles of current and future therapeutics. As pointed out by Houot and others , HP is a


Coronavirus disease 2019 was declared a pandemic on 11th March 2020. All non-urgent surgical procedures have been postponed indefinitely. The British Association of Head and Neck Oncology state that only those with treatable head and neck cancer unsuitable for alternative treatment should undergo surgery. This paper details our management of a patient who tested positive for severe acute respiratory syndrome coronavirus-2 days before curative surgery for laryngeal cancer.3 transglottic squamous cell cancer was pre-operatively identified as an asymptomatic carrier of severe acute respiratory syndrome coronavirus-2. Following 14-day isolation and laboratory proven viral clearance, he underwent successful major surgery. He was managed throughout the peri- and post-operative phases without complications or adverse effects on staff.By following British Association of Head and Neck Oncology guidance, a 49-year-old male scheduled for total laryngectomy and bilateral neck dissection for a TWith careful planning, previous coronavirus disease 2019 positive status should not prevent an individual from undergoing successful total laryngectomy and bilateral neck dissection in a safe and timely manner during the pandemic. The disease is associated with severe morbidity and mortality in the elderly, the immunocompromised, and those with co-morbidities such as hypertension, diabetes and obesity. Coronavirus disease 2019 has claimed the lives of 852 758 people as of 2nd September 2020.3 During the Covid-19 pandemic, all non-urgent surgical procedures have been postponed indefinitely. Life and limb saving surgery, including operations for cancer, should go ahead.4 With regard to head and neck cancer, guidelines published by the British Association of Head and Neck Oncologists state that only those with treatable disease not suitable for alternative treatment (i.e. radiotherapy) should undergo laryngectomy for laryngeal or hypopharyngeal squamous cell carcinoma (SCC) given the high risk of viral transmission during surgery.5Although the exact transmission route of the virus remains unclear, it has been well documented that ENT surgeons and intensivists are at greatest occupational risk given the requirement of examinations, instrumentation and aerosol-generating procedures (AGPs) involving the nose and throat, which are known to harbour a high viral load.3 transglottic SCC who tested positive for SARS-CoV-2 in the pre-operative period. We detail his pre- and peri-operative management, post-operative course, and safe discharge.Although all cancer centres have been operating as appropriate throughout the Covid-19 pandemic, there are no descriptions of patients having been previously infected with SARS-CoV-2 and successfully undergoing major surgery. Here we present the case of a 49-year-old male who underwent successful total laryngectomy and bilateral neck dissection for a T3 transglottic lesion with no evidence of distant disease; biopsy of this lesion confirmed a moderately differentiated SCC. His case was discussed at the regional head and neck multidisciplinary team meeting, and a total laryngectomy and bilateral neck dissection was proposed, to which he subsequently agreed.A 49-year-old male was referred to the head and neck service with a 6-week history of hoarseness. His past medical history included anxiety and hypertension. He lived alone, smoked 15 roll-up cigarettes daily and consumed a moderate volume of alcohol per week. He had a World Health Organization performance status score of 1 and a Malnutrition Universal Screening Tool score of 0. Direct examination and subsequent computed tomography of the head and neck confirmed a THis operation was planned for the end of March 2020 for social reasons; however, two weeks prior to his operation date the Covid-19 pandemic was declared. The patient underwent standard pre-operative investigations including blood work, electrocardiography and an echocardiogram, the findings for all of which were normal. British Association of Head and Neck Oncology guidance for laryngectomy during Covid-19 was followed; the patient was advised to self-isolate for 14 days and asked to attend his local ENT department for two Covid-19 swab tests within the week prior to surgery. His first swab was negative; however, the second swab, performed 4 days later, was positive for SARS-CoV-2. His surgery was postponed and he was asked to self-isolate for a further 14 days. He was asymptomatic at the time of testing and remained so throughout his isolation period.During the 14-day self-isolation period, he underwent Covid-19 swab testing on days 7 and 14. Both swabs were negative and therefore his operation was rescheduled to take place 2 days following his most recent negative swab test. The day before surgery, he was admitted to a ‘clean’ tertiary centre with no accident and emergency or dedicated Covid-19 ward. He was again swab tested on admission and found to be negative.The patient successfully underwent total laryngectomy and bilateral neck dissection following clearance of Covid-19, only two weeks later than initially planned. Despite his negative swab results, enhanced personal protective equipment (PPE) was worn throughout the surgical procedure by operating theatre staff, including a filtering facepiece code 3 (FFP3) mask, face shield, disposable cap, fluid-resistant gown and gloves. There were no surgical or anaesthetic-related peri-operative complications.The patient progressed well on the ward following surgery. Healthcare professionals wore enhanced PPE, including FFP3 masks, face shield, fluid-resistant gowns and gloves when carrying out AGPs (e.g. suctioning and stoma care). On morning ward rounds, he was reviewed by a senior member of the medical team who wore a fluid-resistant surgical mask, face shield, apron and gloves. There were no post-operative complications and the patient was safely discharged two weeks after his surgery.3N0  classification). Following discussion with the oncology department, the patient decided against adjuvant radiotherapy. The British Association of Head and Neck Oncology advises against same-day tracheoesophageal puncture and speech valve insertion during the pandemic, to reduce the length of hospital stay and associated complications; he therefore remains on the waiting list for this.The patient was reviewed in clinic one month post-operatively; his wounds were healing well and he was eating and drinking without issue. His final pathology report revealed a completely excised, moderately to poorly differentiated laryngeal SCC, pathologically confirmed as tumour–node stage T5 Coronavirus disease 2019 testing must be performed before tracheostomy and certainly before laryngectomy; two Covid-19 tests over an undefined interval period provide more definitive results. A laryngectomy must not be performed in a Covid-19 positive patient. In cases of airway obstruction, intubation or tracheostomy must be performed as an alternative, and definitive treatment performed once the patient is Covid-19 negative. All patients, regardless of Covid-19 status, must be managed peri-operatively on the assumption they are infected; enhanced PPE and FFP3 masks must be worn by all operating theatre staff. Primary tracheoesophageal puncture is not recommended, to avoid associated risks in the immediate and short-term recovery period. Surgery with greater post-operative risks, such as microvascular free flaps and pedicled flaps, should be avoided if possible.As a result of the declaration of the Covid-19 pandemic immediately following the decision to proceed with this patient's curative surgery, the standard pre-operative assessment required alteration, to ensure both patient and staff safety. British Association of Head and Neck Oncology guidance for laryngectomy during the Covid-19 pandemic was adhered to.By following British Association of Head and Neck Oncology guidance, we were successful in identifying an asymptomatic Covid-19 carrier in the pre-operative phase. We were then able to advise a 14-day self-isolation period, to ensure his safety and that of other patients and healthcare staff.In this case, two consecutive negative tests separated by 7 days during the 14-day self-isolation period facilitated prompt and safe operating theatre scheduling for a patient who required urgent major surgery during the pandemic. We found that one further negative swab on the day prior to surgery provided additional reassurance of a low transmission risk. Within our institution, we have also implemented a Covid-19 pre-operative safety checklist to be completed by a member of the surgical and anaesthetic teams on the morning of surgery . Patient6 In the case of the post-laryngectomy patient, the minimum of a surgical fluid-resistant face mask, face shield, apron and gloves should be worn for daily post-operative examination, performed by the most senior medical staff member, to allow the identification of complications at the earliest opportunity. Irrespective of Covid-19 status, enhanced PPE including FFP3 masks should be worn when performing AGPs, including suctioning of the laryngectomy stoma. All clinical staff should perform thorough hand hygiene before and after any patient contact, and at regular intervals throughout the working day.The enhanced PPE protocol in the peri- and post-operative periods should be adhered to by all involved clinical staff, regardless of known Covid-19 status, given the unknown false negative Covid-19 polymerase chain reaction testing rates.During the coronavirus disease 2019 (Covid-19) pandemic, all non-urgent surgical procedures have been postponed indefinitelyOnly those with treatable head and neck cancer unsuitable for alternative treatment should undergo surgery given the high-risk nature of the procedureBritish Association of Head and Neck Oncology has produced guidance on managing patients undergoing laryngectomy during the pandemicThis is the first reported case of a patient identified as Covid-19 positive pre-operation successfully undergoing total laryngectomy and bilateral neck dissectionThis treatment was conducted following a 14-day self-isolation period and viral clearanceIf guidance is followed, previous Covid-19 positive status should not prevent laryngectomy and neck dissection in a safe and timely manner during the pandemicCoronavirus disease 2019 testing in the post-operative period should be based on clinical suspicion for SARS-CoV-2 infection. Our local practice requires swab testing every 4 days for all in-patients aged over 70 years.7 In this case, the two-week delay for surgery did not appear to affect outcome in terms of pathology and therefore patient prognosis. Despite this good outcome following a two-week delay, surgery of this nature should be performed as soon as is safely possible.It has been previously described that delays of four weeks in those with moderately to poorly differentiated laryngeal SCC risked worsening TNM stage and prognosis.This is the first report in the literature of a patient having been identified as Covid-19 positive in the pre-operative period and subsequently undergoing successful total laryngectomy and bilateral neck dissection after viral clearance and a 14-day isolation period. This case therefore proves that if the appropriate steps are taken in the pre-, peri- and post-operative phases, then previous Covid-19 positive status should not prevent an individual from undergoing successful surgery for laryngeal cancer in a safe and timely manner during the Covid-19 pandemic.


The COVID-19 pandemic is marked by a wide range of clinical disease courses, ranging from asymptomatic to deadly. There have been many studies seeking to explore the correlations between COVID-19 clinical outcomes and various clinical variables, including age, sex, race, underlying medical problems, and social habits. In particular, the relationship between smoking and COVID-19 outcome is controversial, with multiple conflicting reports in the current literature. In this study, we aim to analyze how smoking may affect the SARS-CoV-2 infection rate. We analyzed sequencing data from lung and oral epithelial samples obtained from The Cancer Genome Atlas (TCGA). We found that the receptor and transmembrane protease necessary for SARS-CoV-2 entry into host cells, ACE2 and TMPRSS2, respectively, were upregulated in smoking samples from both lung and oral epithelial tissue. We then explored the mechanistic hypothesis that smoking may upregulate ACE2 expression through the upregulation of the androgen pathway. ACE2 and TMPRSS2 upregulation were both correlated to androgen pathway enrichment and the specific upregulation of central pathway regulatory genes. These data provide a potential model for the increased susceptibility of smoking patients to COVID-19 and encourage further exploration into the androgen and tobacco upregulation of ACE2 to understand the potential clinical ramifications. COVID-19 is the respiratory illness caused by the SARS-CoV-2 virus that is currently affecting millions of people worldwide and putting extraordinary pressure on public health systems everywhere. Understanding the mechanism of this viral infection is important for developing preventative measures against infection and treatments for the disease. SARS-CoV-2 is a novel coronavirus which is part of the coronavirus family. It is related to the SARS-CoV and MERS-CoV viruses, which cause Severe Acute Respiratory Syndrome (SARS) and Middle East Respiratory Syndrome (MERS), respectively [In SARS-CoV-2, host recognition is carried out by the spike protein on the surface of the viral envelope. The spike protein binds to the ACE2 protein in human cells. After this, the spike protein can be cleaved by the serine protease TMPRSS2 ,4. Next,2 and Mas receptors [1 receptor, which promotes inflammation in the lungs [1 on immune cells prevents the polarization of macrophages and T-cells into pro-inflammatory types [COVID-19 also has a strong immunological component, where poor outcomes have recently been associated with cytokine storms and a hyperinflammatory immune system . Case steceptors . Angiotehe lungs . Thus, the lungs . Howeverry types . Furtherry types . Since Sry types .Cigarette smoke has been associated with an increased susceptibility to COVID-19 , althougRNA-sequencing data for lung epithelial solid tissue normal samples were obtained from The Cancer Genome Atlas (TCGA). Sequencing data were used to identify significant differences in gene expression between groups, namely smoking and nonsmoking patient samples. To investigate the possible effect of smoking in SARS-CoV-2, we compared the expression of ACE2 in current smokers versus past smokers (no data were available for never-smokers), as shown in Using the enrichment scores assigned to the significant pathways by GSEA, immune cell population sizes were inferred from gene expression data, and ratios of relevant cell populations were calculated, as shown in Specifically, the impairment of antigen-processing and -presenting cells may decrease the recognition of the foreign pathogen. In order to further explore this relationship, differential expression was performed on the TMPRSS2 in current smoker vs. past smoker patients, as TMPRSS2 is critical for SARS-CoV-2 entry into the cell. TMPRSS2 was also upregulated in current smokers compared to past smokers, as shown in In order to elucidate a mechanism for smoking-mediated susceptibility to SARS-CoV-2 infection, we utilized GSEA to compare the enrichment of pertinent pathways to ACE2 and TMPRSS2 expression. Of these relevant pathways, those involved in androgen signaling were emphasized. It was previously found that androgen signaling increases TMPRSS2 expression , and smoThe primary entry route of the SARS-CoV-2 virus is through inhalation, with the virus eventually gaining access to the respiratory epithelium. However, there are several reports that this virus can also gain entry through other means, including through the oral route. For this reason, we investigated the expression of ACE2 and TMPRSS2 in oral epithelial cells. RNA-sequencing data for oral epithelial solid tissue normal samples were obtained from TCGA and were used to compare the gene expression between smoking and nonsmoking patients. ACE2 and TMPRSS2 were significantly upregulated in smokers versus nonsmokers, as shown in p < 0.05). Of the core enriched genes with the most interactions with other core enriched genes in androgen pathways, ARRB2, CDK6, and ID2 were all upregulated in smokers. The knockdown of ARRB2 inhibits AR in androgen-dependent cells [To further investigate the hypothesis in oral epithelial cells, GSEA was used to compare ACE2 and TMPRSS2 expression to androgen pathway activity in smoker oral epithelial cells. ACE2 expression was correlated to the enrichment of many core androgen pathway genes, including EP300, CDK6, SGK1, HDAC1, and NCOR1, as shown in nt cells . CDK6 hant cells . Generalnt cells . The uprCOVID-19 has been proven to be a dangerous and far-reaching disease as the number of infections and deaths continues to rise worldwide. Although the majority of patients eventually recover, the world has not seen this magnitude of devastation in such a short period of time in recent memory. The long-term effects of this virus are also unknown at this time, although it is believed that many patients will have significant sequelae from this infection. Therefore, aside from prevention from being infected, investigating the factors that determine susceptibility to COVID-19 infection and the mechanisms underlying these factors is vital to keeping SARS-Cov-2 in check. This study investigated one of these factors—smoking—and suggested a possible mechanism for smoking-mediated susceptibility to COVID infection.We first analyzed lung epithelial tissue sequencing data, as this is the most common site of COVID infection. Our initial analysis revealed an upregulation of ACE2 alongside TMPRSS2 in smoking patients. Additionally, ACE2 expression in smoking patients was correlated to significant immune modulation. Together, these results suggest a possible role of smoking in SARS-CoV-2 infection, as the host cells show an upregulation of these two genes as well as decreased immune surveillance by antigen-presenting and -processing cells. It is already known that cigarette smoke alters androgen pathway signaling, and that androgens increase the expression of TMPRSS2. Taken together, we hypothesized that a smoking-mediated increase in androgen pathway signaling causes an increase in TMPRSS2 expression and activity alongside ACE2 upregulation, thereby increasing host cell susceptibility to SARS-CoV-2 entry. To investigate this hypothesis further, we analyzed the expression of androgen pathway genes in smoking patient samples. This analysis revealed that the enrichment of androgen pathways corresponded with the upregulation of ACE2 and TMPRSS2. To extend our model to a wider range of sites that may be the point of entry for SARS-Cov-2, we performed a similar analysis using oral epithelial tissue sequencing data. Similar to the lung epithelium, the oral epithelium exhibited an overexpression of ACE2 and TMPRSS2 in smoking patient samples. An analysis of androgen pathway expression in smoking patients showed an upregulation of key genes that are central regulators of the pathway. Although this effect is not as drastic as it is in the lung epithelium, our data show that our hypothesized mechanism for increased SARS-CoV-2 susceptibility may also be applied to the oral epithelial cells. In summary, our findings demonstrate a model for smoking-induced SARS-CoV-2 infection via the dysregulation of ACE2 and TMPRSS2 levels. This model may be useful in identifying at-risk patients and further our understanding as to why COVID-19 is such a dangerous and variable disease. Further investigation is necessary to investigate the interactions among tobacco smoke, androgens, SARS-Cov-2, ACE2, TMPRSS2, and their associated genes, including the validation of our computational data using in vitro and in vivo experiments. https://portal.gdc.cancer.gov/legacy-archive/search/f) on 5 August 2018. Clinical data, such as smoking history, for each patient were downloaded from the Broad Institute Firehose website (https://gdac.broadinstitute.org/). Genomic alteration data for each patient were obtained from the Broad Institute TCGA Genome Data Analysis Center’s  analysis report (2016).RNA-sequencing data were obtained for 49 adjacent normal tissue samples of lung squamous cell carcinoma and for 22 adjacent normal tissue samples of head and neck squamous cell carcinoma from the Cancer Genome Atlas (TCGA)  was used to analyze all gene sets (pathways and signatures) for enrichment with respect to gene expression. Biological pathways and signatures were obtained from the Molecular Signature Database (MSigDB). The gene expression data for each of the five genes were inputted as continuous variables in the phenotype file. The gene expression dataset had expression values of all genes in counts per million (CPM). Pearson correlation was used to correlate gene expression with the expression of genes in a pathway to produce a ranked list.