H2) branch of the immune system, which decreases ACE2-receptor expression (i.e. receptors SARS-CoV-2 uses to infect host cells), balances the inflammatory TH1/TH17 branches of the immune system triggered by SARS-CoV-2 infection, and reduces inflammation through the release of anti-inflammatory/regulatory cytokines. Because STHs are common and affect the most vulnerable and marginalized members of society, it is especially important to consider how these parasites may impact COVID-19 outcomes. Areas experiencing endemic STH infections are often characterized by a lack of preventative infrastructure and medical care, which may further exacerbate risk of SARS-CoV-2 infection and COVID-19 development. For this reason, we also explore biocultural factors that contribute to disease outcomes for both SARS-CoV-2 and STH infections. Biocultural and Evolutionary Medicine perspectives on COVID-19 are crucial for understanding the global impact of the disease.The novel virus, Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2), and the associated Coronavirus Disease 2019 (COVID-19) represent a pathogen to which human beings have limited to no evolved immune response. The most severe symptoms are associated with overactive inflammatory immune responses, leading to a cytokine storm, tissue damage, and death, if not balanced and controlled. Hypotheses within Evolutionary Medicine, including the Hygiene/Old Friends Hypothesis, provide an important lens through which to understand and possibly control this overactive immune response. In this article, we explore the role that infection with soil-transmitted helminths  may play in dampening SARS-CoV-2 symptoms and mitigating the worst COVID-19 outcomes. Specifically, STHs stimulate the immunosuppressive and regulatory T-helper 2  may suppress inflammatory immune activity, thereby potentially reducing COVID-19 disease severity. Structural and lifestyle factors shaping coinfection patterns are also discussed. The novel virus, Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2), and the associated Coronavirus Disease 2019 (COVID-19), spread rapidly among the global population. By September 6, 2020, an estimated 26 962 795 people have tested positive for infection and 880 955 have died ; howeverIn this theory-based review, we consider both proximate mechanisms and ultimate causes of COVID-19 virulence, arguing that an Evolutionary Medicine framework should be used to examine how mismatches between our evolved biology and current lifestyles may shape COVID-19 disease outcomes. Because COVID-19 is associated with a hyperinflammatory immune response, we focus this paper on the immunoregulatory effects of certain parasitic species, so-called ‘old friends’, that share long coevolutionary histories with humans  and are Specifically, we highlight the need for an evolutionary perspective to clarify how variation in parasitic disease exposure throughout the life course and across populations may affect immune system regulation, influencing immune-reactivity to SARS-CoV-2 and possibly altering COVID-19 severity. We use the mismatch paradigm to consider how evolutionarily novel environments that alter parasite exposure and subsequent immune development may result in more robust inflammatory immune responses, possibly increasing the risk of severe COVID-19 symptoms. This approach allows for the application of a biocultural perspective to identify shared socio-ecological factors influencing both parasite and SARS-CoV-2 infection patterns, as well as COVID-19 disease outcomes. We end with a discussion of future work needed to elucidate the relationship between parasite infection and COVID-19 disease outcomes.An exploration of potential interactions between macroparasite infection and immune responses to SARS-CoV-2 is timely and of great importance. Coinfection between parasites and COVID-19 is occurring in many locations around the world, especially in regions with limited access to medical intervention, and coinfection prevalence will likely increase as basic medical services and government resources needed for anti-helminth public health campaigns are depleted by the ongoing fight against COVID-19. This information may help healthcare workers and researchers better understand and track various possible outcomes of SARS-CoV-2 infection.The disease COVID-19 affects multiple organ systems, resulting in major respiratory distress, as well as potentially impacting the blood vessels, gastrointestinal tract and central nervous system . CurrentRecent evidence has clearly linked obesity, cardiovascular disease, autoimmune diseases, and other chronic diseases with more severe COVID-19 symptoms and an increased risk of hospitalization and death from infection , 4, 16. Age also appears to be an important player in COVID-19 disease outcomes, such that mortality rates are highest among older adults . ChildrePopulation-level differences in disease patterns are also apparent. While political and cultural factors certainly shape the effectiveness of pandemic control measures, underlying differences at the local level  and subsequent variation in immune system development early in life may also play important roles in shaping chronic inflammatory risk and immune response to SARS-CoV-2. An evolutionary perspective is consequently required to clarify why certain individuals and populations may be at a greater risk of experiencing severe COVID-19 outcomes.The field of Evolutionary Medicine offers an important lens through which to study COVID-19 patterns. Evolutionary (or Darwinian) Medicine addresses how natural selection has shaped the human body, leading to beneficial traits, trade-offs and seemingly suboptimal physical designs . In otheH2] immunity), which evolved specifically in response to macroparasite infection [A counterpart to the Hygiene Hypothesis, the Old Friends Hypothesis, more narrowly focuses on the coevolutionary relationship humans share with certain commensal bacteria and macroparasite species, pointing to a specific branch of our immune system  due to their global prevalence and coevolved immunoregulatory effects. STHs are the most prevalent neglected tropical disease  worldwide . NotablyTania solium  cysts in the brain (i.e. cysticercosis), infection results in seizures and death; yet, while dampened immune activity decreases immunopathology, increased host tolerance allows the cysts to proliferate [H2 response [H2 response is triggered by parasites, immune cell profiles become more anti-inflammatory. Intracellular microbial and viral infections, on the other hand, trigger inflammatory TH1 and TH17 responses.Like other parasites, STHs survive in their host through balanced parasitism in which they actively manipulate host immune activity to tolerate the parasite’s presence , 38. Thiliferate , 40. Thuresponse , 41, 42.H1 pathway [H1 immune cell types [More specifically, helminth antigens are able to actively suppress inflammation by suppressing the production of interleukin (IL)-12 and by blocking the induction of the T pathway . This he pathway , 43. Altll types . Interlell types .Soil-transmitted helminths are important to consider in conjunction with COVID-19 because they affect many of the same organ systems. For instance, many STH species affect the lungs, with some migrating through lung tissue during their larval stage , 38. FurH2/Treg profile, tolerance for commensal bacteria and certain food types may be lost and conditions like inflammatory bowel disease and Crohn’s disease may occur [Trichuris trichiura (whipworm) and its close relative Trichuris suis (pig whipworm) have been shown to reduce intestinal inflammation due to excreted products that trigger a localized TH2/Treg response [Many common chronic inflammatory conditions  are considered mismatch diseases prevalent in higher-income countries. This pattern is likely due to multiple lifestyle factors , includiay occur . Trichurresponse , 55, 56.H2 immune pathway is associated with increased allergic sensitivity, suggesting that the immune pathway that initially evolved in response to macroparasites becomes dysregulated and pathogenic when triggered by non-parasitic antigens [H2 response is activated without the immunosuppressive and anti-inflammatory cells that helminths also trigger. For example, when helminths bind to dendritic cells and trigger the TH2 response, it is balanced by the production of IL-10 and Treg, which ultimately reduce lung inflammation [Importantly, evidence suggests that STH-related immune activity is linked with allergy risk. An overactive Tantigens . In the ammation . This baH2-dominated immunity, balancing both TH1 immunity and the innate inflammatory response [Beyond localized anti-inflammatory effects, immune priming from STH infection during immune system development in infancy and early childhood may provide systemic protection from inflammation, likely by skewing future immune responses toward adaptive Tresponse . Exposurresponse . Still, response , 59.The potential for STH-associated immune responses to shape the risk of developing chronic inflammatory conditions may prove important in countering COVID-19. While STHs are ‘old friends’, coevolving with humans throughout our long evolutionary histories, novel and more virulent pathogens have emerged in recent human history. Humans have no specific evolved immune responses to these new health challenges, allowing them to readily infect individuals and spread rapidly among crowds , 61, as H1 immune response, which is dominated by proinflammatory cytokines  [H1 and TH2 responses are important and act to balance one another, hypothetically preventing immune dysregulation [H2 response are being targeted in COVID-19 vaccine development because of the TH2 response’s ability to counter the hyperinflammatory TH17 response [Unlike macroparasite infections, viruses trigger the T, IL-1β) . In healgulation . These rgulation  suggestiresponse .H1 and TH17 pathways [H2 response, which balances and regulates the proinflammatory TH1 and TH17 responses through the release of regulatory and immunosuppressive cytokines  and Treg cells [pathways , which rpathways , 17. Beceg cells , 44, 63,eg cells   due to the lack of TH2 priming during development and altered base-line inflammatory activity. Without STH exposure and TH2 priming to balance these inflammatory responses, the overactive TH1 and TH17 responses may plunge those infected with SARS-CoV-2 into cytokine storms. If true, this suggests that populations with endemic STH infections (and fewer pre-existing chronic conditions) should exhibit better COVID-19 disease outcomes, including fewer cases of fatal cytokine storms.In addition to reducing viral load and mitigating COVID-19-related hyperinflammation, STH infection may also decrease the prevalence of preexisting chronic inflammatory conditions known to be risk factors for COVID-19 morbidity and mortality , 28–34. While very few studies have tested possible links between COVID-19 disease outcomes and co-existing parasitic infection, limited early evidence suggests there may be a meaningful association. For example, one preliminary study, which has not yet been peer-reviewed, compared WHO data on COVID-19 prevalence around the world to data on prevalence of various parasitic infections. The researchers documented inverse correlations between incidence of symptomatic COVID-19 and STH prevalence . They alH2-associated reduction in ACE2-receptors) and lung damage associated with COVID-19. Helminth-associated immunosuppression has been shown to reduce the likelihood of fighting off infection quickly, as is the case with coinfections between helminths and the bacteria Mycobacterium tuberculosis [For instance, it is important to recognize that parasites, including STHs, are a major health problem globally. While we hypothesize that the immunosuppressive effects of STH infection may mitigate COVID-19 outcomes, it is possible that the STH-associated immunoregulation may increase SARS-CoV-2 susceptibility  [H2 response and higher eosinophil levels at the start of infection, resulting in comparatively less tissue damage and lower viral loads. However, to the best of our knowledge, no study has directly tested relationships between STH infection and COVID-19 outcomes. Clearly, more work is needed to understand how parasite infection affects COVID-19 disease outcomes, especially across biologically and culturally diverse populations [Another concern is that the immune response to STH infection could make certain COVID-19 outcomes worse. For instance, elevated eosinophil levels caused by STH infection could have proinflammatory affects in host tissue, which could lead to increased lung tissue damage, although pulmonary eosinophilia has not been linked with lung damage in COVID-19 patients to date . This ald IL-10) ; althougd IL-10) . Importaulations , 88.While we hypothesize that coinfection with STHs and SARS-CoV-2 may reduce the risk of severe inflammatory COVID-19, it is important to consider other factors associated with both infection types that may also affect disease outcomes. For instance, STHs tend to be most common in low-income countries that already experience high infectious disease burdens due to limited access to medical care or infrastructure that protects from infection. Thus, variation in disease exposure and subsequent immune activity is likely due to a mix of factors, including individual immune system development (as discussed above), but also local conditions  appear to result in reduced diversity , 107. A llaceae, . Furtherllaceae, . BecauseSeveral intersecting biocultural factors influence infectious disease risk (including both STH infections and SARS-CoV-2). These complex interactions are currently understudied in low-income countries and regions, where populations are most vulnerable to rampant infectious and parasitic disease spread. Further, COVID-19 disease prognosis and presentation may differ from the symptoms typically exhibited in patients from high-income nations because of the immunoregulatory effects of STH coinfections. Healthcare workers and researchers should consequently consider how local conditions may influence SARS-CoV-2 spread and COVID-19 symptomology across diverse populations. Clarifying various possible manifestations of COVID-19 is crucial for the correct identification and containment of cases, especially among individuals with comorbidities (e.g. parasitic infection) not typically seen in high-income countries.Given the known effects of STH infection on host immune function, it seems probable that pre-existing STH infection may influence SARS-CoV-2 infection risk, as well as COVID-19 disease severity. Still, given the negative health effects associated with heavy STH infection  , 37, we Work needs to be done to understand if COVID-19 symptom severity is significantly reduced among individuals coinfected with STHs compared to those without a concomitant STH infection (i.e. by comparing rates of diagnosed symptomatic COVID-19 cases between areas with and without endemic STH infection). In addition, within a population experiencing endemic STH infection, links between current STH infection and the likelihood of severe COVID-19 symptomology should be considered in association with deworming programs, and studies could be designed to test and monitor outcomes between individuals enrolled in deworming programs and those who have not recently received anthelminthic treatment. If STH infection appears to mitigate COVID-19 severity, the immune mechanisms accounting for this difference should be explored, including a hypothesized reduction in ACE2 receptors and altered cytokine profiles in STH-infected versus uninfected COVID-19 patients. Research also needs to address if the immunoregulatory effects of STH infections vary across individuals with other pre-existing comorbidities  that may be related to their immune state.The STH categorization contains multiple species that occupy different parts of the digestive tract, interact with their host in different ways and may be associated with species-specific cytokine profiles . This meThe mechanisms by which parasites regulate host immune function and immune responses appear to vary based on when individuals are exposed . If STHsLikewise, are the anti-inflammatory effects of STH infection in response to COVID-19 only apparent for those currently infected with helminths, or are these same immunoregulatory effects evident in those with a history of STH infection (even if they are not currently infected)? How recently does an individual need to be exposed to STH infection to exhibit anti-inflammatory immunoregulation? To examine the immune effects of past STH exposure, future studies can examine COVID-19 symptom severity in groups of individuals (e.g. immigrant communities) who have transitioned at various ages from regions with widespread STH disease to areas with no endemic STH infections.Evidence suggests that STH infections are immunosuppressive. Do these immune dampening effects of STH infections increase host susceptibility to SARS-CoV-2 infection? If so, are the anti-inflammatory effects discussed above enough to outweigh the negative outcomes that this immunosuppression might cause? Will STH infection reduce the efficacy of any eventual COVID-19 vaccine? Further, some have argued that the excessive immune activity linked with severe COVID-19 may also predispose individuals for infection with other pathogens, including parasites . Do we tIf any relationship is evident between STH and SARS-CoV-2 infection, how does this association vary between distinct populations? What lifestyle differences  may account for this variation? What underlying biological factors  may influence these interactions? How do interactions between STHs and the intestinal microbiota affect COVID-19 outcomes? And finally, how do the metabolic and nutritional costs associated with both STH infection and the microbiome affect nutritional status and disease prognosis?In conclusion, while little is definitively known given the novelty of the virus in humans, few studies have tested links between COVID-19 outcomes and co-existing parasitic infection. Very preliminary research suggests meaningful relationships between endemic STH infection and local SARS-CoV-2 risk/prognosis, but the effect of parasite infection on COVID-19 patterns likely varies by parasite species and based on host characteristics  , 80, 83.


Nature by Zou et al. reported that peroxisomes, membrane-bound oxidative organelles, contribute to ferroptosis through the biosynthesis of plasmalogens for lipid peroxidation  responsible for lipid peroxidation.4 More specifically, the oxidation of polyunsaturated fatty acids (PUFAs) appears to be a key driver of ferroptosis. Distinct types of PUFAs are associated with different risks of pathological conditions and diseases. Previous studies have shown that PUFAs are esterified into membrane phospholipids, especially phosphatidylethanolamine (PE)-containing phospholipids (arachidonic acid and adrenic acid), and then generate lipid peroxides, leading to ferroptosis in various cells or tissues.5 The acyl-CoA synthetase long-chain family member 4 (ACSL4)-lysophosphatidylcholine acyltransferase 3 (LPCAT3) pathway is responsible for the production of most PUFAs during ferroptosis. In contrast, the lipid peroxidation repair enzyme glutathione peroxidase 4 (GPX4) and the endosomal sorting complexes required for the transport (ESCRT)-III membrane repair machinery can prevent ferroptosis. Now, Zou et al.1 report that peroxisome-dependent plasmalogen production constitutes yet another, ACSL4/LPCAT3-independent phospholipid resource susceptible to ferroptotic oxidative damage in cancer cells, neurons, and cardiomyocytes -induced ferroptosis: the clear cell renal cell carcinoma 786-O cell line and the human ovarian carcinoma OVCAR-8 cell line. This procedure led to a marked enrichment of peroxisome-related genes, the deletion of which increased cellular viability. Peroxisomes play multiple roles, including the production or elimination of hydrogen peroxide, the synthesis of certain lipids, and the degradation of long-chain and branched-chain fatty acids. Through the depletion or overexpression of peroxisome biogenesis genes , the authors found that the number of peroxisomes was positively correlated with susceptibility to ferroptosis. Therefore, peroxisomes may be added to the list of organelles that can initiate the ferroptotic cell death.Subsequently, the author explored how peroxisomes affect the sensitivity of cells to ferroptosis. Peroxisomal enzymes involved in the synthesis of plasmalogens, such as alkylglycerone phosphate synthase (AGPS), fatty acyl-CoA reductase 1 (FAR1), and glyceronephosphate O-acyltransferase (GNPAT), were significantly enriched among the CRISPR targets that confer cytoprotection. Lipidomic analysis revealed that the production of plasmalogens was diminished in PEX3- or PEX10-deficient cells. These experiments indicate that peroxisomes affect the sensitivity of cells to ferroptosis by synthesizing plasmalogens, a subclass of ether phospholipids that are abundant in cell membranes in the cardiovascular, immune, and nervous systems. At difference with common phospholipids, plasmalogens use an ether (instead of an ester) bond to connect the glycerophospholipid. Their sn-1 chains are non-hydrolyzable ether-linked chains, while their sn-2 chains are connected by conventional ester bonds. Due to this special chemical structure, the synthesis of plasmalogen precursor needs to be carried out independently in the peroxisome. Here, the precursor 1-O-alkyl-glycerol-3-phosphate (AGP) is synthesized by FAR1 and AGPS and then transported to the endoplasmic reticulum (ER) for further biosynthetic reactions involving 1-acylglycerol-3-phosphate O-acyltransferase 3 (encoded by AGPAT3). Finally, the ER-resident enzyme plasmanylethanolamine desaturase 1  mediates the production of PUFA-plasmalogen. Importantly, the provision of liposomal nanoparticles composed of purified plasmalogens to cells was sufficient to confer sensitivity to ferroptosis by lipid peroxidation, further confirming the implication of plasmalogens in ferroptosis.GPX4−/− cancer cells developed tumors that proliferated as quickly as GPX4-expressing parental cancer cells. Further lipidomics and genetic analysis showed that such GPX4−/− cells had not reacquired GPX4 expression and instead downregulated the synthesis of plasmalogens to become resistant against ferroptosis. In contrast, the differentiation of neuronal SY5Y precursors or cardiomyocytes derived from human induced pluripotent stem cells was associated with an increased sensitivity to ferroptosis induced by GPX4 inhibition, correlating with an increase in PUFA plasmalogens.Finally, the authors investigated the pathophysiological role of plasmalogen-dependent ferroptosis in disease-related scenarios. Unsurprisingly, human GPX4 knockout cancer cells inoculated into immunodeficient mice initially failed to strive, presumably due to an increase in the ferroptotic demise of the cells. However, after a latency period, mice carrying In summary, the results presented by Zou et al. revealed a previously unrecognized role for peroxisome-mediated plasmalogen synthesis in promoting ferroptosis. Like any pioneering studies, the current work raises many interesting questions for the future. What particular advantages do peroxisome-synthesized plasmalogens confer to cells beyond rendering them susceptible to ferroptosis? During the induction of ferroptosis, what structural changes need to occur in peroxisomes? Do peroxisomes contribute to ferroptosis by other mechanisms than those related to lipid synthesis? What is the role of ferroptosis in peroxisome biogenesis disorders, such as Zellweger syndrome? Do cells employ pexophagy, the selective autophagic degradation of peroxisomes, as a strategy to regulate their susceptibility to ferroptosis?


We implemented universal masking  as a strategy to preserve our workforce and to protect patients by reducing the risk of SARS-CoV-2 transmission from HCW to HCW, from patient to HCW, and from HCW to patient during asymptomatic or presymptomatic exposures. We aimed to measure the effect of universal masking on coronavirus disease 2019 (COVID-19) acquisition within the healthcare setting.Atypical presentations of severe acute respiratory coronavirus virus 2 (SARS-CoV-2) infection along with its ability to be transmitted from asymptomatic and presymptomatic individuals pose unique infection prevention challenges.https://www.cdc.gov/coronavirus/2019-ncov/hcp/guidance-risk-assesment-hcp.html) fully-recommended Personal Protective Equipment (PPE) or reported a breach in PPE.Duke Health consists of a tertiary care academic hospital, 2 community hospitals, 21,014 HCW, and more than 180 primary care and specialty clinic practices in 10 counties in North Carolina, providing approximately 70,000 inpatient hospitalizations and 2.4 million outpatient visits annually. We prospectively recorded incident SARS-CoV-2 infections among HCW across our healthcare system to determine the impact of universal masking on nosocomial acquisition of SARS-CoV-2 within this population. We defined HCW to include all staff working in the inpatient or outpatient healthcare setting, regardless of the provision of direct patient care. Incident cases of HCW-associated SARS-CoV-2 cases were reported to the hospital system’s infection prevention team by Employee Health (EH). A team of case tracers interviewed all HCW patients to review potential community and occupational exposures. Based on the interview findings, each case was adjudicated by a panel of the authors  into the following categories: community-acquired, healthcare-acquired, or an unknown acquisition route. Community-acquired SARS-CoV-2 cases were defined as HCWs who had an unmasked exposure to a known positive person such as a family member, friend, or coworker outside of the hospital for greater than 10 minutes at less than 6 feet. Healthcare-acquired SARS-CoV-2 cases were defined as a HCW who had an unmasked exposure for greater than 10 minutes at less than 6 feet to another HCW who was symptomatic and tested positive for SARS-CoV-2 or a HCW who had an exposure to a patient with a positive SARS-CoV-2 test and was either not wearing the Centers for Disease Control and Prevention (CDC)  in North Carolina.From March 15, 2020 to June 6, 2020 we assessed all HCWs who tested positive for SARS-CoV-2. Based on the panel adjudication, 38% cases were community-acquired, 22% were healthcare-associated, and 40% did not have a clear source of acquisition. Of note, 80% of HCWs did not work on COVID-19 units.Of the healthcare-associated cases, 70% were related to unmasked exposure to another HCW for more than 10 minutes less than 6 feet apart and 30% were thought to be secondary to direct care of SARS-CoV-2 positive patients.One week following the implementation of universal masking on March 31, 2020, we observed a significant decrease in the cumulative incidence rate of healthcare-acquired SARS-CoV-2 infections among HCWs (Figure Universal masking of all HCWs significantly reduced the rate of healthcare-acquisition of SARS-CoV-2, thereby flattening the healthcare-associated SARS-CoV-2 epidemiologic curve in our healthcare system. HCWs with community-acquired SARS-CoV-2 or who had an unknown route of acquisition SARS-CoV-2 at the same incidence rate as other community members. We attribute the lower rate of healthcare-acquired infections in part to providing universal source control via masking, thereby mitigating the spread from asymptomatically infected or minimally symptomatic individuals. Mask etiquette, defined as wearing a mask at all times when physical distancing is not possible around anyone outside of your household contacts, limiting unmasked exposures indoors, and performing hand hygiene before and after touching the face mask, needs to be reinforced both inside and outside the workplace to help preserve the HCW workforce. Mask etiquette must also be performed alongside other infection prevention measures including following standard and transmission-based precautions, hand hygiene, physical distancing, and self-isolation coupled with immediate testing and contact notification when symptomatic. Finally, the recent changes to the CDC guidance that call for masking all inpatients and outpatients while direct care is provided and the addition of a face shield to our pandemic PPE outfit will hopefully lead to further reduction in healthcare-acquired SARS-CoV-2. HCWs will need ongoing reminders to follow recommended public health guidance to protect themselves from community acquisition of SARS-CoV-2 as the pandemic continues.


The COVID-19 pandemic highlights the substantial public health, economic, and societal consequences of virus spillover from a wildlife reservoir. Widespread human transmission of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) also presents a new set of challenges when considering viral spillover from people to naïve wildlife and other animal populations. The establishment of new wildlife reservoirs for SARS-CoV-2 would further complicate public health control measures and could lead to wildlife health and conservation impacts. Given the likely bat origin of SARS-CoV-2 and related beta-coronaviruses (β-CoVs), free-ranging bats are a key group of concern for spillover from humans back to wildlife. Here, we review the diversity and natural host range of β-CoVs in bats and examine the risk of humans inadvertently infecting free-ranging bats with SARS-CoV-2. Our review of the global distribution and host range of β-CoV evolutionary lineages suggests that 40+ species of temperate-zone North American bats could be immunologically naïve and susceptible to infection by SARS-CoV-2. We highlight an urgent need to proactively connect the wellbeing of human and wildlife health during the current pandemic and to implement new tools to continue wildlife research while avoiding potentially severe health and conservation impacts of SARS-CoV-2 "spilling back" into free-ranging bat populations. The threat of emerging infectious diseases (EIDs) to wildlife health and biodiversity conservation is recognized , but croRhinolophus affinis) sampled from Yunnan province, China, in 2013 . Pu. PuSarbeequinum) .Hibecovirus and Nobecovirus also have been reported mostly from Old-World bat families Rhinolophidae, Hipposideridae, Rhinonycteridae, and Pteropodidae, except for novel viruses of the latter subgenus detected in four species of the vespertilionid genus Scotophilus in Asia and Africa  occur in a greater diversity of bat families and across more global regions than the other subgenera  and one that is globally distributed (Molossidae). Distinct CoV lineages in the subgenus Merbecovirus were described from three species of Pteronotus (family Mormoopidae), four species of Artibeus, and Seba’s short-tailed bat  from tropical regions of Mexico . Th. Th80]. f Mexico , 81. Novn Mexico . In vitr disease . Similar disease , availabSarbecovirus spp.), a focus of many surveillance efforts, have been almost exclusively documented in Old-World Yinpterochiroptera. SARSr-CoVs were only found in the ultra-diverse and globally distributed bat suborder Yangochiroptera under conditions with plausible transmission from co-roosting Rhinolophus sp. bats [Our inference of true patterns of CoV occurrence and distribution in bat populations is limited by uneven global sampling. Yet SARSr-CoVs  infected with Marburg virus down-regulate immune-stimulatory pathways and maturation of cells targeted by the virus while up-regulating pathogen-sensing pathways [Except for n health –105. In n health . For exapathways . Unique pathways . Egyptiapathways . Our potpathways . SARS-Copathways , 118, thpathways . CoVs capathways . SpillovEffectively managing risks of human disease caused by emerging zoonotic pathogens and ensuring the health and conservation of wildlife species that are potential reservoirs of those disease agents can be synergistic goals under a One Health framework. Spillover risk (from or to wildlife) is often greatest in disturbed ecosystems where there is an elevated frequency of human–wildlife interactions or disruption of ecological patterns , 120–124Conservation-compatible surveillance of bat viruses has demonstrated the potential for mutually beneficial collaboration between public health scientists and conservation stakeholders , 129. DiAdopting a precautionary approach in the face of global COVID-19 transmission among human populations, national and international wildlife organizations have advised limiting capturing and handling of bats in the field to minimize the risk of humans infecting wild bats with SARS-CoV-2 until further assessment can be made , 138. ThMerbecovirus (like those known to occur in the Americas) are not known to target ACE2 cell receptors, instead using as a receptor DPP4/CD26 or possibly other receptors [Multiple research strategies that do not involve close contact with free-ranging bats already exist for addressing critical gaps in understanding CoV diversity, distribution, evolution, and potential health effects in temperate-zone bats. For example, a combination of host-cell receptor analyses and in vitro and in vivo experimental infections across a diversity of bat and other mammalian species have helped inform potential host range expansion for SARS-CoV-2. The receptors that many CoVs use to gain access to host cells, such as angiotensin-converting enzyme 2 (ACE2) and dipeptidyl peptidase-4 (DPP4/CD26), have undergone positive selection in bats, resulting in diverse and recombinant CoV strains , 143. Theceptors , 144. Cueceptors , 72, 145www.nabatmonitoring.org) [Examples of other “hands-off” methods applicable to both bat disease and conservation research include the following: virus discovery and characterization focused on existing specimens archived in scientific museums or through partnerships and collaboration with established national bat disease monitoring or surveillance programs , 148; moing.org) , 156, asing.org) . DevelopMany questions remain about the risk of SARS-CoV-2 to naïve wildlife populations, the influences of human behavior on those risks, and the potential for establishment of new CoV reservoirs. Cross-species virus transmission events are relatively rare, requiring an infectious reservoir host to be in contact with a recipient host when conditions concurrently favor susceptibility and onward transmission , 114. ThS1 Tablehttps://doi.org/10.5066/P9U461PJ.The table lists bat species in which betacoronaviruses (β-CoVs) were detected, organized by viral subgenera and clade (for Sarbecorviruses), bat family, bat suborder, and general global region where the species of bat occurs. Reference to the published literature sources of information for each row are listed in the last column. Provided in comma-separated value (.csv) format at (XLSX)Click here for additional data file.


Engineered mammalian cells for medical purposes are becoming a clinically relevant reality thanks to advances in synthetic biology that allow enhanced reliability and safety of cell-based therapies. However, their application is still hampered by challenges including time-consuming design-and-test cycle iterations and costs. For example, in the field of cancer immunotherapy, CAR-T cells targeting CD19 have already been clinically approved to treat several types of leukemia, but their use in the context of solid tumors is still quite inefficient, with additional issues related to the adequate quality control for clinical use. These limitations can be overtaken by innovative bioengineering approaches currently in development. Here we present an overview of recent synthetic biology strategies for mammalian cell therapies, with a special focus on the genetic engineering improvements on CAR-T cells, discussing scenarios for the next generation of genetic circuits for cancer immunotherapy. Cancer immunotherapy has the goal of improving anti-tumor immune responses reducing off-target effects typical of chemotherapies and other state-of-the-art treatments. Since cancer cells often evade the surveillance of the immune system, immunotherapies have the function of priming the immune response to make it more efficient. Different classes of immunotherapies have already been approved for cancer treatment and some others are in clinical trials  intendinCytokines act by actively limiting tumor growth with a direct anti-proliferative or pro-apoptotic action or by enhancing tumor recognition and cytotoxicity of the immune system against cancer cells . NeverthLastly, cancer vaccines represent another promising strategy to increase the immune response against cancer. They are divided into (i) cell vaccines, obtained from the tumor or immune cells, (ii) protein/peptides vaccines based on tumor-associated antigens (TAAs), and (iii) genetic vaccines, which use viral, plasmid vectors, or mRNA from autologous tumor tissue to deliver the antigen. Cancer vaccines can be either prophylactic or therapeutic. Examples of prophylactic vaccines include the one against hepatitis B virus which accounts for liver cancer, and the one against human papillomavirus, responsible for the most cervical cancers . Therapeutic vaccines have the goal to increase the tumor-specific adaptive response. Cancer vaccines have shown reduced toxicity and autoimmunity issues, although their efficacy is, for now, lower than other immunotherapeutic strategies, due to (i) an inappropriate activation of effector cells, (ii) limited accumulation of these cells in the tumor, and (iii) to the immunosuppressive tumor microenvironment. Advances in synthetic biology allowed engineering safer and more effective vectors, DNA or RNA–based implemented on bacterial or viral backbones, carrying genetic components, and functional resources . + T cells may undergo impaired activation in the TME. The isolation of these cells from excised tumors and the subsequent reinvigoration in vitro before reintroducing them back to the patient is one of the strategies currently used in clinics to increase T cells response against tumor .,62.59,62Several strategies that implement AND logic gates on engineered T cells have been developed up to date. One model consists of splitting intracellular activation domains into two different CARs recognizing diverse antigens  have been shown to prevent T cell activation against cells expressing two antigens while killing the ones expressing only the antigen recognized by activating CARs. iCAR design consists of a ligand-binding extracellular domain fused to an immune checkpoint protein endodomain , thus transmitting inhibitory signals upon antigen encounter, and preventing CAR-T cells activation against cells expressing two TAA C. SpecifStandard TIL treatment protocols usually require treating patients with repeated high doses of IL-2 which however can also induce the expansion of immunosuppressive Tregs and trigger severe side effects on treated patients ,80. HencOther approaches trying to confine cytokine signaling to engineered cells involve IL-12 expression. For instance, stably transduced T cells to express IL-12 driven by NFAT synthetic promoter  allow ILThis strategy has been also developed for CAR-T cell models. Multiple cytokine-producing CARs, namely “armored” CAR-T cells , includiAs CAR-T cell therapies can induce a large array of severe, life-threatening side effects in patients, tight spatio-temporal control of CAR-T cell viability represents a fundamental objective to make these therapies safer ,86,87,88The counterpart of these strategies is irreversibility. Alternatively, more dynamic ON-OFF strategies were developed to temporarily inactivate engineered T cells in cases of severe side effects. To achieve this, TET-ON and TET-OFF systems  have demLastly, optogenetics provides a means of tight spatio-temporal control of CAR-T cell activation. Briefly, optogenetics are based on the engineering of target cells with light-inducible sensors which, upon activation, can trigger the expression of actuator proteins in a tight spatio-temporal manner. By using optogenetic devices based on CRY2 (cryptochrome 2)–CIB1 (cryptochrome-interacting basic-helix-loop-helix 1) system, fused to transcription factors involved in target gene expression, scientists successfully induced IL-2, IL-15, and TNFα production , as wellWe have discussed how bioengineering can aid the design of more effective CAR-T therapies by targeting several shortcomings. Recently, a new constraint has emerged as a limiting factor in CAR-T  long-term action. T cell exhaustion (TCE) is a physiological phenomenon that occurs when T cells are stimulated during long periods of time . ExhaustOther potential targets arose from functional screenings abrogating the expression of candidate genes, as well as transcriptomic analysis of T cells at different maturation stages. By this means, recently published data showed that downregulation of key proteins involved in T cells terminal differentiation and exhaustion such as NR4A, TOX/TOX2, TET2, Regnase1, or PTPN22 ,99,100 iApart from strategies counteracting T cell exhaustion/favoring T cell stemness, modulation of TCR downstream signaling mediators represents an emerging approach aiming to boost T cell activation and effector functions. CISH, a negative regulator of T cell activation, whose expression is induced upon activating signals such as TCR stimulation or IL-2 engagement, can inhibit T cell activation by inducing PLC-γ1 proteasomal degradation . SeminalSynthetic biology has demonstrated as a powerful engine in the scenario of cancer immunotherapy, especially with the advent of CAR-T cells therapy in recent years. However, several efforts are still needed to improve the design of genetic devices with long-term stability and efficacy. It has been recently shown that exogenous payloads can impact cellular physiology by using intracellular resources . This waIn parallel, modularity-based strategies could substantially reduce the costs and time of T cell expansion into antigen-specific CAR-T cells and therefore significantly accelerate the development of new approaches applied to the clinic. As the biology of T cells is complex as any other mammalian cells, circuit design should be precisely adjusted to prevent or revert non-expected behavior as it is observed within some cells becoming exhausted. To engineer the next generation of CAR-T cell therapy and increase long-term efficacy, associated sensor-actuator strategies should be considered to promote T cell stemness and prevent dysfunction. The increasing knowledge in T cell biology associated with the modular and controllable approaches will allow us to develop innovative synthetic immunology therapeutic strategies to fight cancer and other immune diseases.


ACVRL1 gene. Her clinical course did not involve mechanical ventilation or worsening epistaxis, and she was successfully discharged after two weeks. The endothelial damage and the consequent angiogenic process in COVID-19 patients deserve further investigation.Hereditary hemorrhagic telangiectasia (HHT) is a rare autosomal-dominant disease characterized by pathologic angiogenesis that provokes vascular overgrowth. The evidence about the influence of Acute Respiratory Syndrome Coronavirus-2 (SARS-CoV-2) in patients with rare diseases is scarce. We aimed to know the prevalence of coronavirus disease 2019 (COVID-19) in HHT patients. The HHT pathogenic angiogenesis and endothelial injury in COVID-19 are discussed using data from RiHHTa (Computerized Registry of Hereditary Hemorrhagic Telangiectasia) registry. RiHHTa is an open, multicenter, prospective, observational registry including adult patients with HHT. A 27-item survey that captured clinical data of admitted HHT patients for COVID-19 was distributed to all RiHHTa investigators from June 8th to June 24th 2020. Only one out of 1177 HHT patients was admitted for COVID-19 pneumonia. She is a 74 years-old woman with a pathogenic variant in  Dear Editor:ENG; encoding endoglin) and activin A receptor type II-like 1  genes. Both encoded proteins highly impact in angiogenesis as their loss-of-function provokes vascular overgrowth, mainly due to over-activation of phosphatidylinositol 3-kinase signalling [Hereditary hemorrhagic telangiectasia (HHT) is a rare autosomal-dominant disease characterized by vascular malformations. Disease-causing variants are mostly detected in endoglin (The Severe Acute Respiratory Syndrome Coronavirus-2 (SARS-CoV-2) cause the coronavirus disease 2019 (COVID-19), that has spread worldwide . Since thttps://rihhta.healthincode.com) available in Spanish or English with individual encoded access for each researcher and counted on the nonremunerated collaboration of the genetic studies company Health in Code . The design of the RiHHTa registry was approved by the Ethics Committee of the Hospital Universitari de Bellvitge. The rationale and methodology of RiHHTa have been published elsewhere [ACVRL1 gene and hepatic vascular involvement. Her clinical course did not involve mechanical ventilation or worsening epistaxis, and she was successfully discharged after 2 weeks.RiHHTa (Computerized Registry of Hereditary Hemorrhagic Telangiectasia) is an open, multicenter, prospective, observational registry including adult patients with HHT and developed within the Rare Diseases Working Group of the Spanish Society of Internal Medicine . RiHHTa lsewhere . For thiThis low prevalence of COVID-19 pneumonia among HHT patients may be due to several reasons. First, HHT patients could have self-isolated more strictly than non-HHT patients due to fear of COVID-19 therapeutic measures that might have worsened their recurrent epistaxis. Another possibility could be related to HHT pathogenesis. Observational data on lymphocytic endotheliitis suggest that SARS-CoV-2 provokes endothelial injury . A recen


Platinum-resistant ovarian cancer is characterized by its poor prognosis and limited treatment options. Angiogenesis plays a fundamental role in the development of drug-resistance in ovarian cancer. Anlotinib, a novel oral multi-targeted tyrosine kinase inhibitor which targets a board spectrum of angiogenesis-associated growth factor receptors, has shown promising anti-tumor efficacy in clinical trials. Herein, we report a case of ovarian cancer treated with anlotinib plus etoposide after secondary cytoreductive surgery.A 45-year-old female with primary platinum-resistant ovarian cancer who progressed rapidly after the first cytoreductive surgery, the second cytoreductive surgery, and several lines of treatment. The patient refused to receive intravenous chemotherapy any more.Primary platinum-resistant ovarian cancer.The oral combination treatment of anlotinib  and etoposide  were delivered.Finally, the patient was responsive to the orally treatment of anlotinib combined with etoposide. The patient has been alive with no evidence of disease progression for 18 weeks.Our case suggests that oral treatment of anlotinib combined with etoposide which is acceptable and convenient, may be an additional option for the management of platinum-resistant ovarian cancer. Although the majority of patients with ovarian cancer are responsive initially to the standard first-line platinum-based chemotherapy, 20% to 30% of patients never achieve a response in the primary setting who are considered as primary platinum-resistant. Effective treatment strategies are limited for patients who progressed or had persistent disease on primary platinum-based chemotherapy. For platinum-resistant ovarian cancer, non-platinum-based agents are recommended, such as docetaxel, etoposide, liposomal doxorubicin, and paclitaxel. However, the abnormal tumor vasculature may hinder the delivery of therapeutic agents into the inside of large tumor masses. Therefore, removal of large chemo-resistant tumors could exert favorable impact on the following treatment of recurrent disease. Although it is generally accepted that secondary cytoreductive surgery can be considered for patients with recurrent platinum-sensitive disease, retrospective studies also indicated that secondary cytoreductive surgery could provide clinical benefit in patients with platinum-resistant disease with a longer post-relapse survival.,4Ovarian cancer is the leading cause of mortality in malignant gynecological tumors. Bevacizumab, a FDA-approved monoclonal antibody targeting vascular endothelial growth factor (VEGF), and other compounds targeting tyrosine kinases are two major types of anti-angiogenesis agents. Anlotinib (AL3818) is a novel oral multi-targeted tyrosine kinase inhibitor on tumor angiogenesis and growth. Anlotinib has a broad spectrum of inhibitor effects on targets such as vascular endothelial growth factor receptors 2/3 (VEGFR2/3), fibroblast growth factor receptor 1-4 (FGFR1-4), platelet-derived growth factor receptors α/β (PDGFR α/β), c-kit, and Ret. The phase II/III randomized, double-blind ALTER-0303 trial has demonstrated that patients treated with anlotinib exhibited significantly longer overall survival compared with patients treated with placebo . Anlotinib has been approved in China as a single drug therapy for patients with locally advanced or metastatic non-small cell lung cancer (NSCLC) who have progressed after the second line therapy or beyond. In addition, several clinical trials are ongoing to evaluate the efficacy of anlotinib in a variety of malignancies such as gynecologic tumors, colorectal cancer, and gastric cancer. NCT02558348 is an ongoing phase I/II clinical trial assessing anti-tumor efficacy of anlotinib single treatment in patients with ovarian cancer, endometrial cancer, or cervical cancer. Another phase I/II clinical trial, NCT02584478, is designed to evaluate the efficacy of anlotinib plus standard platinum-based chemotherapy in recurrent or metastatic endometrial, ovarian, fallopian, primary peritoneal, or cervical carcinoma. However, there is no report of the efficacy of anlotinib in ovarian cancer. Herein, we present a case of a female patient with primary platinum-resistant ovarian cancer treated with anlotinib combined with etoposide after secondary cytoreductive surgery in our hospital.Anti-angiogenesis is also considered as promising target for the treatment of platinum-resistant ovarian cancer.2A 45-year-old female was diagnosed with ovarian serous papillary cystadenocarcinoma after right salpingo-oophorectomy in August 2015. Informed consent was obtained from the patient. After neoadjuvant chemotherapy with two cycles of paclitaxel and carboplatin, she had undergone an interval cytoreductive surgery consisting of left salpingo-oophorectomy, omentectomy, and pelvic lymphadenectomy. She was considered as optimal debulked stage IIIc disease according to the findings during the surgery. Following this, she received seven cycles of adjuvant chemotherapy with paclitaxel and carboplatin. Seven months after the initial treatment, she experienced the first relapse with the elevated serum CA125 level  of liver diagnosed by CT scan and she had undergone trascantheter arterial chemoembolization of the hepatic lesion. The treatment of apatinib was discontinued due to the side effects such as hand-foot skin reaction, oral ulcer, and pain. Then, intravenous chemotherapy with pemetrexed was delivered. After three cycles, she refused to receive intravenous chemotherapy any more. Then, the patient progressed rapidly with the markedly elevated CA125 and CT image of new metastasis in left supraclavicular lymph nodes.The patient was referred to our institution  in August 2017. After the discussion by our multi-disciplinary team (MDT), chemotherapy with liposomal doxorubicin was recommended and a secondary cytoreductive surgery should be considered. Molecular testing was also recommended by the MDT, but no pathogenic alterations of Three months after the last treatment of pemetrexed, the patient began to take anlotinib 12 mg, qd) and etoposide  orally. Informed consent was provided by the patients for the treatment of anlotinib. Eleven weeks after the orally combined treatment, the CT scan indicated partial response (PR). Although the liver metastases was similar compared with the previous evaluation, the involved lymph nodes in mesenterium and right inguinal region were shrinking compared with the previous evaluation  for such special population and highly individual basis should be taken into consideration.Ovarian cancer is characterized by its high mortality and advanced diagnosis.,4 A retrospective study have demonstrated a significantly prolonged median time to first progression  in platinum-resistant ovarian cancer receiving secondary cytoreductive surgery compared with patients receiving chemotherapy alone. Several case reports also indicated a survival benefit in women with platinum-resistant disease treated with secondary cytoreductive surgery combined with hyperthermic intraperitoneal chemotherapy (HIPEC).101110 In our case who was heavily treated and progressed rapidly after several lines of chemotherapy, the secondary cytoreductive surgery plus sequential maintenance treatment provided a progression-free survival (PFS) of 5.8 months.For platinum-resistant ovarian cancer, non-platinum-based agents are recommended, such as docetaxel, etoposide, liposomal doxorubicin, and paclitaxel. However, the abnormal tumor vasculature may hinder the delivery of therapeutic agents into the inside of large tumor masses. Therefore, removal of large chemo-resistant tumors could exert favorable impact on the following treatment of recurrent disease and the goal of complete cytoreduction has extended to the setting of recurrent ovarian cancer. However, the role of cytoreductive surgery in management of recurrent ovarian cancer remains controversial. Only retrospective studies have demonstrated the survival benefit from secondary cytoductive surgery in recurrent setting.,13 The microenvironment of tumor including blood vessels, inflammatory cells, stromal cells, and other components plays essential roles in the process of tumor progression and the development of drug resistance. Angiogenesis-associated growth factor receptors are most widely studied and considered as promising treatment targets.,14 A retrospective study revealed that higher angiogenesis-associated growth factor receptors such as PDGFR-beta and VEGFR-2 levels were associated with the platinum resistance status of ovarian cancer. Several studies also demonstrated that the increased levels of angiogenesis-associated growth factor receptors were associated with inferior prognosis in ovarian cancer.161716 FDA has approved bevacizumab, a monoclonal antibody targeting VEGF, for the treatment of platinum-resistant, recurrent ovarian cancer.,19 Although bevacizumab could prolong PFS in patients with platinum-resistant disease, no significant benefit of overall survival (OS) was achieved. Multi-target tyrosine kinases compounds have also exhibited promising anti-tumor efficacy in clinical trials. The phase II trial MITO 11 demonstrated a significant improved progression-free survival in the treatment of pazopanib which inhibits VEGFR1-3, PDGFRα/β, and PDGFR 1-3 plus paclitaxel arm (6.35 months) relative to paclitaxel arm (3.49 months) in platinum-resistant, recurrent ovarian cancer . Other clinical trials evaluating platinum-based chemotherapy in combination with multi-target tyrosine kinases inhibitors such as nintedanib and cediranib also achieved therapeutic efficacy in platinum-sensitive ovarian cancer.,22Multiple mechanisms may contribute to platinum resistance in ovarian cancer.,23 Anlotinib has a broad spectrum of inhibitor effects on targets such as vascular endothelial growth factor 2/3 (VEGFR2/3), fibroblast growth factor receptor 1–4 (FGFR1-4), platelet-derived growth factor receptors α/β (PDGFR α/β), c-kit, and Ret. It has been reported that the anti-angiogenesis efficacy of anlotinib is stronger than several anti-angiogenesis regimen, including sunitinib and sorafenib, because anlotinib can target more targets than these drugs.,24 The anti-angiogenesis activity of anlotinib has been illustrated in vitro and in vivo.,25,26 In addition, anlotinib can highly bound to plasma and has a lower incidence of toxic effects in terms of grade 3 or higher. Anlotinib has shown promising efficacy in the treatment of advanced NSCLC, metastatic renal cell carcinoma, soft tissue sarcoma, and advanced medullary thyroid cancer.,2829302829 Anlotinib has been approved in China as a single drug therapy for patients with locally advanced or metastatic non-small cell lung cancer (NSCLC) who have progressed after the second line therapy or beyond. Thus, anlotinib may contribute to overcome the chemo-resistant status of ovarian cancer. NCT02558348 is an ongoing phase I/II clinical trial assessing anti-tumor efficacy of anlotinib single treatment in patients with ovarian cancer, endometrial cancer, or cervical cancer. Another phase I/II clinical trial, NCT02584478, is designed to evaluate the efficacy of anlotinib plus standard platinum-based chemotherapy in recurrent or metastatic endometrial, ovarian, fallopian, primary peritoneal or cervical carcinoma.Anlotinib (AL3818) is a novel oral multi-targeted tyrosine kinase inhibitor which could highly selectively target several angiogenesis growth factor receptors. In addition, etoposide was also effective in patients with paclitaxel-resistant ovarian cancer.Etoposide, a cost-effective oral agent, has a long history in the treatment of platinum-resistant ovarian cancer. The reported median response duration of etoposide single drug treatment was 4.3 months and the median progression-free interval was 5.7 months in patients with platinum-resistant ovarian cancer.We used anlotinib in combination with etoposide to control the platinum-resistant refractory ovarian cancer after the secondary cytoreductive surgery. This oral treatment is acceptable, convenient, and cost-effective without the need for hospital admissions or infusion pumps. Hand-foot skin reaction, hypertension, proteinuria, and triglyceride elevation were the most common adverse events in the treatment of anlotinib. In our case, the patient only experienced grade 2 hand-foot skin reaction which was well managed.4In conclusion, the therapeutic strategy of anlotinib plus etoposide may provide an additional treatment options for patients with platinum-resistant ovarian cancer. Further investigations are warranted to optimize the efficacy of anlotinib plus etoposide in the treatment of patients with platinum-resistance ovarian cancer.Thank for the patient for agreeing to the publication of the case.Conceptualization: Li Sun, Shangli CaiData curation: Meng Yang, Xuan Zhang, Hua Li, Lingying WuSupervision: Li SunWriting – original draft: Li Sun, Yuzi ZhangWriting – review & editing: Li Sun, Shangli Cai, Meng Yang, Xuan Zhang, Lingying Wu, Hua Li


In this review, we discuss recent developments in the rapidly accelerating field of CRISPR-mediated genome engineering. We begin with an overview of the canonical function of the CRISPR platform, followed by a functional review of its many adaptations, with an emphasis on its applications for genetic interrogation of the normal and diseased nervous system. Additionally, we discuss limitations of the CRISPR editing system and suggest how future modifications to existing platforms may advance our understanding of the brain.The multitude of neuronal subtypes and extensive interconnectivity of the mammalian brain presents a substantial challenge to those seeking to decipher its functions. While the molecular mechanisms of several neuronal functions remain poorly characterized, advances in next-generation sequencing (NGS) and gene-editing technology have begun to close this gap. The clustered regularly interspaced short palindromic repeats (CRISPR)-associated protein (CRISPR-Cas) system has emerged as a powerful genetic tool capable of manipulating the genome of essentially any organism and cell type. This technology has advanced our understanding of complex neurologic diseases by enabling the rapid generation of novel, disease-relevant  Complex behavior is driven by extensive structural and genetic interactions in the mammalian CNS. Historically, neuroscientists have examined these interactions using a variety of histological, electrophysiological and pharmacological techniques. While indispensable, these techniques lack the specificity of targeted genetic approaches to dissect neuronal function. Recent advances have allowed the coupling of high-throughput next-generation sequencing (NGS) technologies with the cell-type specificity of modern molecular genetics to interrogate complex network interactions and behaviors at unprecedented scale and resolution. The ability to read, write, and manipulate genomes with cell-type specificity is critical, especially considering the cellular heterogeneity of various CNS structures .Early attempts at targeted gene editing were performed with zinc finger nucleases (ZFNs) and transcription activator-like effector nucleases (TALENs), both of which rely on programmable DNA-binding proteins coupled to active endonucleases to cleave specific DNA sequences . While sSince the initial adaptation of CRISPR, novel variants continue to be discovered in diverse microbial species, differing in endonuclease size, substrate preference and target recognition requirements . MoreoveStreptococcus pyogenes, the Type II CRISPR-Cas9 system (spCas9) was the first enzyme repurposed from its native role as a bacterial adaptive immune system for genome editing applications in eukaryotic cells  and a transactivating crRNA (tracrRNA) for its activity A. CRISPR utility .Prevotella and Franciscella 1), is a related DNA targeting enzyme that departs mechanistically from Cas9 in ways that may be advantageous. For example, unlike Cas9, Cas12a processes its own CRISPR array (crRNA precursors) into mature crRNAs, independent of any ancillary enzymes and a tracrRNA. Cas12a also recognizes a different PAM sequence , generates staggered cuts and requires a much shorter guide RNA than Cas9 . Cas12a’s compact guide RNA architecture and self-crRNA processing ability make it well suited for multiplexed gene-targeting, particularly through the use of custom arrays encoding multiple crRNAs. Recently, these properties were optimized and harnessed for large scale gene-editing, with Campa and coworkers reporting the ability to deliver and express 20 crRNAs and Cas12a from a single vector, simultaneously  achievable in neurons transduced with AAV-sgRNAs. Swiech and coworkers sought to expand the applicability of CRISPR for broad in vivo use by adapting Cas9 for packaging into popular viral vectors for gene delivery into the brain , sgRNA cassette and other necessary expression components into a single vector is infeasible. To circumvent this, Swiech and coworkers developed an AAV-CRISPR system that expresses spCas9 and its respective sgRNA from separate AAV vectors. Applying AAV-CRISPR to target various genes in vitro and in vivo recapitulated the substantial editing efficiency observed in transgenic Cas9 mice. For example, targeting methyl CpG binding protein 2 (MeCP2) in cultured neurons produced morphologic defects concurrent with MeCP2 loss of function. Furthermore, multiplexed targeting of several DNA methyltransferase genes within the dentate gyrus was capable of producing context-specific freezing deficits in mice that received contextual fear conditioning, while sparing behavioral performance in other tasks .The earliest studies that implemented CRISPR-Cas for neuronal gene editing delivery . Using the brain . The adein vivo construct delivery  KO mice, which significantly reduced exaggerated stereotypies (excessive digging and jumping). Analysis revealed 14.6% of striatal mGluR5 genes contained loss of function (LOF) mutations, while mGluR5 mRNA and protein levels decreased by roughly 50%. Despite modest editing efficiency, these results highlight the potential of nanoparticle-based systems to deliver CRISPR and therapeutically edit genes in the brain. While CRISPR-Gold administration was sufficient to reverse the behavioral phenotype, additional optimization of nanoparticle entry into neurons will likely expand the use of non-viral, nanoparticle-based methods for genome editing in neuroscience.Recently, the nanoparticle-based CRISPR-Gold system was used to target mGluR5, a metabotropic NMDA receptor involved in autism spectrum disorder (ASD)-related hyperexcitation . CRISPR-in vivo stability and longevity, sustaining high levels of expression for over a week, which declined below detection thresholds after three weeks. Unlike virally delivered CRISPR transgenes that remain stably expressed for extended periods, nanocomplex-delivered RNPs possess limited opportunity to perform their gene targeting functions. Remarkably, in vivo targeting of β-secretase 1 (Bace1) in the hippocampal CA3 region of 5XFAD transgenic mice produced an editing efficiency of 45% which significantly reduced Aβ plaques and Aβ42 secretion. Surprisingly, a single hippocampal injection of Bace1-targeting nanocomplexes elicited persistent improvements in contextual and associative memory three months after treatment , these Cre-dependent Cas9 rat lines present a significant advancement for future gene studies in behavioral neuroscience.While the broad availability of genetically modified mice has contributed to their widespread use in biomedical science, rats remain the preferred animal model in behavioral neuroscience research. The paucity of available transgenic rat models has left an unmet demand for additional transgenic rat lines . Germlin2 phases of the cell cycle, which may restrict harnessing HDR’s full potential in postmitotic cells such as neurons , Mikuni and coworkers targeted neural progenitors at embryonic days (E)12 and E15, when HDR should be active. Embryonic brains were subjected to in utero electroporation (IUE), to deliver sgRNAs, Cas9 coding plasmids, and a hyperactive Piggyback transposase system to allow the stable integration of these transgenes and donor templates consisting of single stranded oligonucleotides (ssODNs). This approach enabled the modification of targeted genes to possess N-terminal or C-terminal epitope tags. Remarkably, SLENDR was also capable of inserting large sequences into endogenous loci, such as the GFP-coding region, facilitating protein localization studies. The authors reported modification frequencies as high as 7.5% of neurons when targeting was performed at E12, and slightly lower levels when performed at E15. It is important to note that indel formation will occur at a much higher frequency compared to HDR-mediated sequence insertion using this system. Additionally, the authors specifically targeted the beginning and end of the protein coding regions, to reduce the chance that indel formation would have a consequence on protein structure and function.Although Cas9’s canonical function is to cleave DNA, it can also be used to introduce foreign transgenes and new sequences using HDR B. Low nein vivo , was shown to allow HDR-mediated gene modification of neurons in the mouse adult brain B. They oin vivo  as high in vivo HMEJ produced knock-in frequencies of ∼50%. Although HD strategies ensure locus specificity through extensive donor template homology, unique template production is generally restrictive for high-throughput experimentation. Therefore, unrestricted by locus homology, HI systems have gained more traction. The HITI and more recently developed HiUGE systems also exploit NHEJ repair to introduce DNA payloads. Both HITI and HiUGE incorporate similar components and mechanisms to achieve targeted transgene integration. For example, the use of a non-homologous donor vector with sgRNA recognition sequences is ubiquitous among NHEJ-mediated systems. However, HITI and HiUGE depart as HiUGE donors contain self-targeting sgRNAs, while HITI donors require sgRNA recognition sequences to be manually matched between the target and donor. The addition of a self-targeting guide RNA to HiUGE vectors permits the development of “all-in-one” donor libraries that may function complimentarily with large-scale CRISPR genetic screens.While broadly considered an inherently error-prone process, various NHEJ-dependent DNA-editing tools have been developed that demonstrate the remarkably high editing frequency and precision of NHEJ repair C. These Germline editing with CRISPR-Cas9 has proven remarkably useful for genetically modifying animals . Howeverin vitro, prompting the development of regulable sgRNA expression vectors, which successfully regulated gene-editing events in a Dox-dependent manner. To determine whether this Dox-regulable CRISPR-Cas9 system was suitable for in vivo applications, AAV vectors encoding Cas9 and Dox-inducible sgRNAs were infused into the basolateral amygdala (BLA). In vivo genome-editing analysis revealed that only animals receiving Dox contained indels at the target locus. Additionally, Dox-inducible and constitutively expressed systems exhibited near identical levels of gene editing, demonstrating that spatiotemporally precise editing is achievable in the brain without significant loss of efficiency. Additional Cre and Dox-inducible CRISPR systems have been developed based on the smaller SaCas9 endonuclease. For further discussion of the SaCas9 orthologue and these inducible tools, we direct the reader to (Some of the first inducible CRISPR systems were regulated by components of the popular tetracycline-dependent promoter (Tet) system , which ceader to .de novo transcription and translation involved in transcriptionally mediated responses, permitting a more rapid response to dynamic cellular environments. Additionally, reducing the permissible window for gene-targeting events could significantly reduce the off-target modifications reported with constitutively active Cas9. Several inducible Cas9 enzymes have been developed whose activities are post-translationally regulated with small molecules , the NF-κB trans-activating subunit p65 (p65), or the Krüppel-associated box domain of Kox1 (KRAB) to dCas9 ; Fig. 3E studies . Althoug studies . This ob studies . These sEarly second-generation systems employed an epitope-based scaffolding strategy to increase activator recruitment known as SUperNova SunTag; . The SunAnother study  examinedA separate group screened putative activator domains for gene activation potency, identifying VP64, p65 and the Epstein–Barr virus R transactivator (Rta) as the most potent transcriptional activators. However, dCas9-p65 and dCas9-Rta both exhibited lower transcription rates than the original dCas9-VP64 chimera. To overcome this, combinations of activators were fused with the aim of cooperatively inducing higher gene expression . Using dAscl1 and Neurod1 genes in HEK293T cells, revealing SAM  and sgRNA arrays targeting multiple genes (eight coding genes and two long noncoding RNAs), Zhou and coworkers successfully overexpressed several targeted genes simultaneously. When coupled with existing genome wide CRISPRa sgRNA libraries, these SPH mice provide a useful tool for endogenous gene overexpression and genome wide screening in the brain.In order to circumvent the difficulties associated with viral delivery of large, multicomponent systems to the nervous system, the authors generated a transgenic mouse line harboring a Cre-dependent SPH system. Considering that cell-type and circuit-specific multiplex strategies will likely be required to successfully interrogate gene networks in vivo. By examining VPR expression under the control of several promoters, they were able to identify and produce a LV system that permitted robust VPR expression in vitro and in vivo under the control of the neuron-specific Synapsin promoter. This neuron-optimized LV VPR system was applied in various neuronal contexts and was notably capable of potent, isoform-specific induction of various BDNF transcripts in vivo  and small interfering RNA (siRNA) has accumulated . AlternaSyt1), vesicle associated membrane protein I (Vamp1), syntaxin 1A (Stx1a), and synaptosome associated protein 25 (Snap25), genes responsible for vesicular neurotransmitter release, were targeted in cultured hippocampal neurons. To compare the efficiency of CRISPRi and RNAi-mediated knock-down, sgRNAs and shRNAs were tested for each target gene. CRISPRi produced ∼90% reduction in mRNA and protein levels of all genes targeted, compared to a modest reduction produced by RNAi. Additionally, whole-cell patch-clamp recordings of CRISPRi-targeted hippocampal neurons revealed significant reductions in EPSCs, as expected from disruption of the neurotransmitter release pathway  or GABAergic (VGAT-dCas9-KRAB) neurons. LV infusion into the dentate gyrus revealed a roughly 20% transduction rate of neurons confined to the granule layer. Analysis of dCas9-KRABon IPSCs .Syt1 within glutamatergic and GABAergic neurons enables altering of the inhibitory-excitatory (I-E) ratio within the hippocampus. As the hippocampus is implicated in various forms of learning and memory -induced FXS neurons retained high levels of FMRP expression months after their engraftment into live mouse brains.As dynamic DNA methylation has been proposed to regulate activity-dependent gene expression, Liu and coworkers sought to determine whether their LV dCas9-TET1 system could induce brain-derived neurotrophic factor (BDNF) expression by targeting the BDNF IV promoter for demethylation in cultured primary neurons . Neuronaome FXS; . dCas9-Tcis-regulatory elements to DNA binding proteins  and the trans-acting spliceosome (MAPT gene could decrease the accumulation of pathogenic (isoform 4R) tau, cortical neurons differentiated from patient-derived iPSCs were transduced with AAV encoding dCasRx-hnRNPa1 and three exon 10 targeting sgRNAs. dCasRx-hnRNPa1 mediated exon-skipping was shown to reduce 4R/3R ratios by 50%, a level similar to unaffected controls  makes it highly suited for use in AAV vectors. As described above, the CasRx fusion and three sgRNAs fell below AAV packaging limitations, a characteristic that may inspire the future development of CasRx-based effectors that are capable of elucidating RNA function in the brain.in situ, or more recently, prime editors that can faithfully install edits through reverse-transcription of an RNA template  in primary neuronal cultures. Although a promising development, the frequency of genome-wide off targets and unintended reverse transcription products remain unknown. This, in concert with the modest editing frequency achieved with the latest prime editor, may preclude its immediate use in vivo. Nonetheless, this technology presents an exciting new development towards achieving high-fidelity, corrective gene editing with CRISPR.Prime editors present the latest advance in precision gene editing. Anzalone and coworkers introduced a Cas9-nickase (Cas9n)-based system that couples the DSB-free editing strategy pioneered with base-editors to an sgRNA-based RNA donor template , a stratThe recent exponential advances in NGS technologies and the easy design and production of large numbers of unique sgRNAs has facilitated the high-throughput investigation of various psychiatric and neurodegenerative disorders, cancer, and essential gene functions through large-scale CRISPR screens . CRISPR-in vitro or ex vivo, two CRISPR-Cas9 mediated in vivo screens have recently been reported in the brain  that were previously identified in tumor samples taken from human glioblastoma multiforme (GBM) patients  administration to laboratory mice . Host ann humans . Howevern humans . While Sn humans  may theon humans , may limex vivo, clonally selected efficiency, expanded and then re-implanted, essentially bypassing the issue of inefficient HDR mediated precision editing. In another example it was recently demonstrated that AAV-CRISPR could be delivered via tail injection in a mouse model of hereditary tyrosinemia Type 1 (HTI) to correct genetic mutations within the liver , in neurons is another significant hurdle for the use of CRISPR for neuroscience research and human therapy. The available data indicate that precise editing occurs at relatively low levels in neurons, limiting the utility of these methods and currently making them unlikely to have any benefit clinically. Although precise editing occurs relatively infrequently in most cell types compared to NHEJ-mediated indel formation, disorders that afflict mitotically active cell populations may be more amenable to HDR-based therapies. For example, hemopoietic progenitor cells can be genetically modified he liver . AlthougThe CRISPR-Cas system has emerged as a highly adaptable platform with extensive utility in multiple areas of biomedical and basic science. Given its ability to target nearly any gene or RNA transcript, alter gene expression and modify epigenetic states with high specificity, CRISPR-Cas represents an invaluable tool helping drive the rapid pace of discovery in biological sciences. While early studies only demonstrated its use in peripheral tissues, recent efforts have produced CRISPR-Cas systems amenable for use in the CNS. Additionally, the development of CRISPR-expressing animals, as well as the discovery of AAV-compatible orthologues, have provided substantial tools for probing neuronal function at multiple levels of analysis. While newly developed CRISPR-transgenics may be crossed with existing Cre-driving lines, novel CNS-optimized tools will likely require viral vector encoding and delivery. Challenges associated with viral vectors such as packaging constraints, low virus infectivity and low gene editing efficiencies remain limiting factors for using CRISPR in the brain. In order to maximize the therapeutic and research potential of available systems, existing delivery methods must be optimized and new, more effective ways of introducing these systems must be developed. Undoubtedly, future improvements and applications of CRISPR-Cas technology will surface. Despite these challenges, recent advances in CRISPR-Cas technology have provided researchers with powerful new tools for engineering the neuronal genome.


Very little is known about the risk that severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) viral infection poses to cancer patients, many of whom are immune compromised causing them to be more susceptible to a host of infections. As a precautionary measure, many clinical studies halted enrollment during the initial surge of the global Novel Coronavirus Disease (COVID-19) pandemic. In this case report, we detail the successful treatment of a relapsed and refractory multiple myeloma (MM) patient treated with an anti-B cell maturation antigen (BCMA) chimeric antigen receptor (CAR) T cell therapy immediately following clinical recovery from COVID-19.The 57 year old Caucasian male patient had a 4-year history of MM and was considered penta-refractory upon presentation for CAR T cell therapy. He had a history of immunosuppression and received one dose of lymphodepleting chemotherapy (LDC) the day prior to COVID-19 diagnosis; this patient was able to mount a substantial immune response against the SARS-CoV-2 virus, and antiviral antibodies remain detectable 2 months after receiving anti-BCMA CAR T cell therapy. The recent SARS-CoV-2 infection in this patient did not exacerbate CAR T-associated cytokine release syndrome (CRS) and conversely the CAR T cell therapy did not result in COVID-19-related complications. One month after CAR T cell infusion, the patient was assessed to have an unconfirmed partial response per International Myeloma Working Group (IMWG) criteria.Our case adds important context around treatment choice for MM patients in the era of COVID-19 and whether CAR T therapy can be administered to patients who have recovered from COVID-19. As the COVID-19 global pandemic continues, the decision of whether to proceed with CAR T cell therapy will require extensive discussion weighing the potential risks and benefits of therapy. This case suggests that it is possible to successfully complete anti-BCMA CAR T cell therapy after recovery from COVID-19.CRB-402 study registered 6 September 2017 at clinicaltrials.gov (NCT03274219). The global COVID-19 pandemic represents a worldwide public health crisis and directly impacts cancer care. Patients with multiple myeloma (MM) have cellular and humoral immune dysfunction causing them to be more susceptible to infections . Anti-B A 57-year-old Caucasian male patient with a 4-year history of IgG-kappa MM was referred to Mount Sinai Hospital in New York City in early February 2020 because of disease progression. He was penta-refractory  and had previously received nine lines of therapy.2)/fludarabine (30 mg/m2)], the patient returned to clinic with fever, cough, and diarrhea. Nasopharyngeal polymerase chain reaction (PCR) test confirmed SARS-CoV-2 infection.In early February 2020, approximately 3.5 weeks prior to the first confirmed case of COVID-19 in New York City, the patient was enrolled in a clinical study (NCT03274219) of bb21217, an investigational BCMA-directed CAR T cell therapy. The study was conducted in accordance with the Declaration of Helsinki and International Conference on Harmonisation guidelines for Good Clinical Practice, and the protocol was approved by local or independent institutional review boards (IRB) at each study center. Informed consent was obtained from each patient. The patient received bridging therapy with melphalan and bortezomib while awaiting CAR T cell manufacturing. He was asymptomatic and screened negative by PCR test for SARS-CoV-2 two days prior to a planned 3-day course of lymphodepleting chemotherapy (LDC). Approximately 24 hours after receiving the first dose of LDC [cyclophosphamide  for grade 1 neutropenia and discharged with instructions to self-isolate at home after 3 days of hospitalization.The patient was monitored weekly, and further therapy was held until SARS-CoV-2 clearance was confirmed by nasopharyngeal PCR test, 39 days after COVID-19 diagnosis. Upon approval by the sponsor and our IRB, the patient reinitiated the full 3-day course of LDC in preparation for CAR T cell administration. At this point, inflammatory markers were normal, and SARS-CoV-2 antibodies were detected at a titer of 1:2880 using an IgG assay developed by Mount Sinai [Our case adds important context to the evolving literature about treatment of MM patients in the era of COVID-19 . This isThe patient experienced Grade 2 CRS, an expected toxicity associated with CAR T therapy, which resolved within 6 days, and the patient is currently in partial response (unconfirmed), per IMWG assessment , 12. InfInterestingly, this patient experienced a robust humoral response to viral infection, despite his history of immune suppression. Two months after treatment with a BCMA-directed CAR T therapy, which can ablate normal BCMA expressing plasma cells, the antibody titer decreased from 1:2880 to 1:320 but remained clearly detectable approximately 4 months after infection. Whether this patient developed and retained long-term immunity against COVID-19 remains to be determined.With the initial surge of COVID-19, many clinical trial sites paused trials of CAR T cell therapy for MM patients. As trials re-start, investigational sites should ensure access to critical care and availability of appropriate drugs to manage CAR T cell-associated toxicities and carefully weigh the potential risks and benefits of therapy prior to proceeding with CAR T cell therapy. Our report, although limited to a single patient experience, suggests that patients who have tested antibody positive for SARS-CoV-2 can proceed with CAR T cell therapy without flare-up of COVID-19-related symptoms. High antibody titers can be generated in myeloma patients; SARS-CoV-2-specific antibodies were retained despite effective anti-BCMA CAR T cell therapy in this patient. Additional studies to determine the effect of BCMA targeting agents on the risk of SARS-CoV-2 re-infection are warranted. As COVID-19 immunity after recovery has not been well characterized, appropriate precautions such as social distancing, facial mask, and good hygiene are recommended to prevent re-infection.The information available regarding management strategies for MM patients during the COVID-19 pandemic is insufficient to provide evidence-based recommendations; however, several consensus statements have been published that provide guidance. The recommendations included within this report—securing access to supportive care, taking appropriate precautions to avoid SARS-CoV-2 infection, and carefully weighing the potential risks and benefits of CAR T cell therapy—align well with existing consensus statements , 15.


Influenza burden estimates help provide evidence to support influenza prevention and control programs at local and international levels.Through a systematic review, we aimed to identify all published articles estimating rates of influenza-associated hospitalizations, describe methods and data sources used, and identify regions of the world where estimates are still lacking. We evaluated study heterogeneity to determine if we could pool published rates to generate global estimates of influenza-associated hospitalization.2I>99%), we were unable to pool published estimates.We identified 98 published articles estimating influenza-associated hospitalization rates from 2007-2018. Most articles (65%) identified were from high-income countries, with 34 of those (53%) presenting estimates from the United States. While we identified fewer publications (18%) from low- and lower-middle-income countries, 50% of those were published from 2015-2018, suggesting an increase in publications from lower-income countries in recent years. Eighty percent (n = 78) used a multiplier approach. Regression modelling techniques were only used with data from upper-middle or high-income countries where hospital administrative data was available. We identified variability in the methods, case definitions, and data sources used, including 91 different age groups and 11 different categories of case definitions. Due to the high observed heterogeneity across articles (The variety of methods, data sources, and case definitions adapted locally suggests that the current literature cannot be synthesized to generate global estimates of influenza-associated hospitalization burden. Seasonal influenza epidemics are important contributors to the global burden of hospitalizations each year . The WorImprovements in laboratory and surveillance capacity in many middle- and low-income countries since the 2009 influenza A(H1N1) pandemic have allowed countries to better understand influenza virus transmission within their population and collect the requisite data to estimate influenza-associated disease burden . In 2015Through a systematic review, we aimed to identify all published articles estimating rates of influenza-associated hospitalizations, describe methods and data sources used, and identify regions of the world where estimates are still lacking. We evaluated study heterogeneity to determine if we could pool the current literature to generate global estimates of influenza-associated hospitalization. This review will inform future efforts to generate global estimates of influenza-associated hospitalization burden.We systematically searched six computerized literature databases  to identify articles published from January 1, 2007 - February 28, 2018. As one of our exclusion criteria for laboratory testing was the exclusion of publications using laboratory tests other than reverse transcription polymerase chain reaction (RT-PCR), we limited our search window to articles published in 2007 or later, as RT-PCR testing was rarely used before 2007 . Search Once we removed duplicate publications, two independent reviewers screened titles and abstracts for potential exclusion based on the criteria presented in For accepted articles, we used a standardized tool to extract data for analysis, including information about country, region, or sites where data were collected, case definitions, data sources, estimation methods, influenza virus laboratory testing methods, and rates of influenza-associated hospitalizations. When available, we also obtained confidence intervals (CIs) or other reported measures of error for rates of influenza-associated hospitalization. We classified age group-specific rates into the age grouping that most closely matched eight age groups used by WHO  [2I statistic [I2>75%), we were unable to complete a meta-analysis to generate pooled summary estimates [We assessed study heterogeneity to determine if rates could be logically pooled using meta-analysis. For this quantitative analysis, we included only a subset of the full data set: all nonzero, nonnegative rate estimates with a reported measure of error. We excluded zero and negative rates because the log rate variance was too great for the models to run. Only overall influenza-associated hospitalization rates were included; influenza type- or subtype-specific estimates were excluded. We used a three-level random effects meta-regression model to account for within article clustering. Study heterogeneity was assessed statistically using the Cochran Q test and the tatistic . As the stimates . AnalyseOur database search identified 3949 articles of potential interest, and we identified 30 additional articles through the review of relevant bibliographies  . The majSixty-four articles (65%) presented rates from high-income countries, as defined by the World Bank, whereas 18 (18%) presented rates from lower-middle-income or low-income countries -24. The All extracted articles presented a variety of different case definitions or discharge diagnoses to identify potential influenza-associated hospitalizations. Thirty-six articles (37%) defined hospitalizations based on laboratory-confirmation of influenza or a discharge diagnosis of influenza; the vast majority of these (n = 34/36) used a multiplier approach. Forty-one articles (42%) used a case definition of hospitalized acute respiratory infection (ARI); 20 focused on ARI with fever, 17 on ARI without a fever requirement, and four using both ARI with fever and ARI without fever case definitions. Certain case definitions, such as all respiratory, all circulatory, combined respiratory and circulatory, or all-cause diagnoses, which represent broad categories used to code administrative data, were only used in articles using regression modelling techniques. Overall, 17 (17%) articles estimated influenza-associated hospitalization rates using more than one case definition. Of these, nine (53%) articles used regression modelling techniques where authors compared estimates across different case definitions . The remaining eight articles (47%) used multiplier techniques with different case definitions for different age groups (n = 8) or different years (n = 1) following WHO case definitions . While wAll 19 articles that used regression modelling techniques identified hospitalizations using administrative databases. Forty-five percent (n = 35) of articles using a multiplier approach identified hospitalizations using administrative databases and all were describing high and upper-middle-income countries. Fifty-seven (58%) articles noted that the hospitalizations identified represented all hospitalizations in their study region (complete population coverage). All articles using a health care utilization survey (n = 9) or a hospital administrative survey (n = 13) to adjust the population denominator used a multiplier approach.A median of three years of data were presented in each article (interquartile range: 2-6 years), with estimates from 1991-2017. We observed variability in rates of influenza-associated hospitalizations by age group, region, and method. Across all study years, annual rates, per 100 000 population, ranged from -11 to 7543 for children aged <2 years, -12 to 3417 for children aged 2-4 years, -4 to 4763 for children aged <5 years, -2 to 999 for children aged 5-14 years, -3 to 325 for persons aged 15-49 years, -20 to 179 for persons aged 50-64 years, and -40 to 1437 for persons aged ≥65 years , and the I2 statistic attributed 99.97% of the variance in the estimates to study heterogeneity rather than chance. Therefore, we were unable to generate pooled summary estimates or conduct a meta-analysis. Even after stratifying the data by age group, method classification (multiplier vs regression modelling approach), case-patient identification method, World Bank income classification, or World Health Organization region, study heterogeneity remained high within each sub-group (>99%)  had no reported measure of error, an additional 12 had negative point estimates, and 109 rates were zero. These rates were all excluded from our evaluation of heterogeneity. After limiting the quantitative analysis to non-type or subtype specific  estimates, 1500 rates from 54 articles remained. The Cochran Q test indicated the presence of heterogeneity  precluding them from being able to generate population-based rate estimates. This may have limited the number of countries included in this study. To address this limitation, WHO published an approach in 2015 to assist countries conducting influenza virus sentinel surveillance to estimate hospital catchment populations . While oWhile we were not able to pool results from our systematic review of the literature, published estimates of global influenza-associated hospitalization burden do exist. Wang X et al.  and The Like all systematic reviews, the data on case definition and methodology we were able to capture was limited by the information provided in each publication. Many articles published influenza-associated hospitalization rates as a secondary objective of their study and often did not include detailed methods or measures of error for their estimated rates. We made an effort to extract additional information from references and reach out to authors directly, however, lack of information may have limited our review. Additionally, a wide variety of age groups and case definitions were used across publications, which limited direct comparisons. While these age groups or case definitions may be relevant to the local context, the variability between articles within each category made it challenging to categorize accepted articles. Finally, we were not able to capture and account for additional factors that may have impacted published estimates, such as population health, care seeking behaviors, access to care, hospitalization practices, laboratory specimen collection and handling, and the application of case definitions. The variability in determining if a patient should be hospitalized independent of disease severity, such as bed capacity, admitting physician, and ability to pay, could increase unmeasured variability in hospitalization burden estimates compared to similar death burden estimates.In conclusion, there is growing literature on seasonal influenza-associated hospitalization burden, though estimates from lower-middle- and low-income countries are still limited. Published influenza-associated hospitalizations rates were estimated using a variety of methods, data sources, and case definitions, which complicates the comparison and synthesis of these data on a global level. Due to the strong correlation between methodologic characteristics and high variability across estimates, we were unable to generate pooled estimates from the current published literature. Future collaborations and analyses between countries using standardized methods and case definitions across different populations or analyses applying different methods in the same study population could allow for a direct comparison of the impact of specific epidemiologic methods and help to understand the differences in rates of influenza-associated hospitalization.


Given the highly infectious nature of the coronavirus disease 2019 (COVID-19) virus and the lack of proven specific therapeutic drugs and licensed vaccines effective against it, early diagnosis of the disease is of paramount importance. The common chest CT imaging of confirmed COVID-19 cases is discussed here, which shows ground-glass opacity, crazy paving, and consolidation. In late December 2019 with the outbreak of coronavirus disease 2019 (COVID-19) and an exponentially growing death toll in Wuhan, China, a state of emergency was declared . As confCase 1Ground-glass OpacityGround-glass opacity (GGO) is frequently observed due to enhanced lung attenuation without obscuration of underlying vessels. In GGO, alveoli are partially filled with fluid which appears gray on CT images. The distribution of GGO lesions in COVID-19 tends to be peripheral, bilateral, and along the pleura and bronchovascular bundles -6.A 45-year-old woman from the Rasht provincial hospital presented three days after the onset of fever (38.2°C), cough, and chills. She did not reveal any history of sick contacts in the family or underlying lung disease. Unenhanced chest CT images at her presentation showed patchy ill-defined GGOs as an archetypal response to acute lung injury in the left and right lower lobes of the lung Figure .Case 2Crazy PavingFollowing lung involvement in COVID-19, the thickened interlobular septa and intralobular lines laid over a background of GGO known as crazy paving can be observed.A 51-year-old man presented in our clinic with fever (38.7°C), cough, dyspnea, and intermittent chest pain. In his physical examination, he exhibited coarse breath sounds during auscultation. Axial thin-section unenhanced CT images obtained on March 4, 2020 show crazy paving mostly in the left and right lower lobes of the lung , nonproductive cough, muscle aches, and dyspnea. In his lung examination, coarse breath sounds were heard during auscultation. His transaxial unenhanced chest CT images show dense consolidation  assays and the probability of false-negative rRT-PCR results, chest CT imaging as a noninvasive imaging modality can be a useful tool with high accuracy for early diagnosis of suspected COVID-19 cases . For thiInterestingly, Fang et al. found that COVID-19 rRT-PCR sensitivity may be as low as 71% compared to the 98% sensitivity of CT for COVID-19 infection ,9. As thWhen radiologists see the common chest imaging findings of this new strain of coronavirus, they can identify COVID-19 cases in a timely manner based on the appropriate epidemiologic and demographic features. In so doing, prompt reasonable preventive action can be implemented to curtail the transmission of this enveloped positive-sense RNA virus.