Influenza viruses pose significant disease burdens through seasonal outbreaks and unpredictable pandemics. Existing surveillance programs rely heavily on reporting of medically attended influenza (MAI). Continuously monitoring cause‐specific school absenteeism may identify local acceleration of seasonal influenza activity. The Oregon Child Absenteeism Due to Respiratory Disease Study  implements daily school‐based monitoring of influenza‐like illness‐specific student absenteeism (a‐ILI) in kindergarten through Grade 12 schools and assesses this approach for early detection of accelerated influenza and other respiratory pathogen transmission in schools and surrounding communities.Starting in September 2014, ORCHARDS combines automated reporting of daily absenteeism within six schools and home visits to school children with acute respiratory infection (ARI). Demographic, epidemiological, and symptom data are collected along with respiratory specimens. Specimens are tested for influenza and other respiratory viruses. Household members can opt into a supplementary household transmission study. Community comparisons are possible using a pre‐existing and highly effective influenza surveillance program, based on MAI at five family medicine clinics in the same geographical area.Over the first 5 years, a‐ILI occurred on 6634 (0.20%) of 3,260,461 student school days. Viral pathogens were detected in 64.5% of 1728 children with ARI who received a home visit. Influenza was the most commonly detected virus, noted in 23.3% of ill students.ORCHARDS uses a community‐based design to detect influenza trends over multiple seasons and to evaluate the utility of absenteeism for early detection of accelerated influenza and other respiratory pathogen transmission in schools and surrounding communities. The theoretical relationships between influenza infection in school‐aged children, K‐12 absenteeism, and influenza infection in the surrounding community, and study components and hypotheses are demonstrated in Figure 22.1www.oregonsd.org) encompasses the villages of Oregon and Brooklyn, decentralized subdivisions, and farms in Dane County, Wisconsin .The Oregon School District  is an absence for any reason. Absence due to illness (a‐I) is an absence due to any reported illness. Absence due to ILI (a‐ILI) is an a‐I for which ILI symptoms are reported.2.7We considered established definitions for ILI2.8OSD utilizes Infinite Campus®,2.9An automated process within Infinite Campus® extracts daily counts of absent individuals by school, grade, and absence type . Data contain no personal identifiers, are compliant with the Family Educational Rights and Privacy Act ,2.10Each school receives $4000 per year to defray costs associated with IT support and effort by the attendance staff.2.112.11.1Students are not required to be absent, and school does not need to be in session for home visits to occur. To comply with FERPA, interested parents/guardians call the study line to initiate a home visit. If a student meets the inclusion criteria, a 20‐min home visit occurs within 2 days of initial phone contact and within 7 days of symptom onset.student attends, or is eligible to attend , a school within the OSDhas an illness characterized by ≥2 of 6 acute respiratory infection (ARI)/ILI symptoms scores ≥2 points on the Jackson scaleInclusion criteria include:illness onset ≥7 days before anticipated time of specimen collectionanatomical defect for which nasal specimen collection is contraindicatedstudent participated too recently Exclusion criteria include:If criteria are met, family members are invited to participate in an optional household transmission substudy. Participation is allowed even if individual members opt out or are unable to complete the entire study.individuals of any age/gender residing in the same household as ORCHARDS participantable to provide appropriate consent/assentInclusion criteria include:anatomical defect for which nasal sampling is contraindicatedhousehold participated too recently Exclusion criteria include:2.11.2Research coordinators obtain written informed consent from parents/guardians and/or adult students, and assent from younger students using forms tailored to reading levels based on age.2.11.3Demographic, epidemiologic, and symptom data are collected on an Acute Respiratory Infection and Influenza Surveillance Form , based o2.11.4A nasal swab specimen is collected using a Puritan® Sterile Foam Tipped Applicator for rapid influenza diagnostic testing (RIDT). In addition, a nasopharyngeal (NP) or high oropharyngeal (OP) specimen is obtained using a Copan FLOQSwabs™ flocked swab. The NP/OP swab is placed into a 3.0‐ml Remel MicroTest™ M4RT® Transport viral transport medium (VTM) tube, placed into a small biohazard bag, and maintained at 2–8°C. Following processing for RIDT, the residual nasal swab is added to the VTM containing the NP/OP swab. A courier delivers the VTM tube to the Wisconsin State Laboratory of Hygiene (WSLH), usually within 24 h of collection.2.11.5Student participants receive a $20 gift card at the end of the home visit. Research coordinators call parents/guardians with RIDT results within 24 h  of the home visit. Laboratory confirmed results are mailed to families within 2 weeks of a home visit and are accepted as documentation for an excused absence by the OSD.2.122.12.1At the ORCHARDS home visit, recruited families receive a packet containing substudy information, instructions, consents/assents, and a collection kit for each household member. Each collection kit contains a data form, two nasal swabs, and two small biohazard bags, each of which contain a 3.0‐ml Remel MicroTest™ M4RT® VTM tube. VTM tubes and swabs for Day 0 and Day 7 are individually marked.The research coordinator explains components of the family packet to all present household members and reviews consent/assent forms. A designated adult assures that all interested household participants sign the consent/assent forms prior to specimen collection. Household members collect their own specimens on Day 0 (within 24 h of home visit) and Day 7 . The timing (Day 7) for follow‐up data and specimen collection is based on prior influenza transmission studies.2.12.2Household composition: the number and characteristics of people sharing the household of an ORCHARDS participant. Information includes relationship to the ORCHARDS participant, age, gender, number of bedrooms in home, employment outside of home, school attendance, and daycare attendance.Household member illness assessment on Day 0 (“Today” section): influenza vaccine status for the current season, and information about any ARI/ILI illnesses occurring over the past 7 days.Household member illness assessment on Day 7, 1 week after the initial visit (“Follow‐up” section): information about any ARI/ILI illnesses occurring over the past 7 days.The ORCHARDS Household Study Form  is used 2.12.3Household members are instructed on specimen collection using a Copan FLOQSwabs™ flocked midturbinate swab  or an anterior nasal Puritan® Sterile Foam Tipped Applicator . Each household participant collects the specimen without staff on Day 0 and Day 7. Upon retrieval, research coordinators review forms for completion and ensure specimens were collected and correspond with the appropriate labels.2.12.4Households completing the substudy receive a $50 gift card to local businesses.2.132.13.1We use the Quidel® Sofia® Influenza A + B fluorescent immunoassay2.13.2All specimens from students and household members (Day 0 and Day 7) are tested at WSLH for influenza A and B virus and Human RNase P (RP) using the in vitro diagnostic (IVD) Food and Drug Administration (FDA)‐approved CDC Human Influenza Virus Real‐time RT‐PCR Diagnostic Panel (Cat. # FluIVD03).2.13.3Chlamydia pneumoniae and Mycoplasma pneumoniae, using the multiplexed PCR respiratory pathogen panel (RPP: Luminex NxTAG Respiratory Pathogen Panel).All specimens from ORCHARDS students are tested for influenza A, influenza B, rhinovirus/enterovirus, adenovirus, parainfluenza virus,2.13.4For a subset of influenza‐positive subjects, we conduct next‐generation genome sequencing at the WSLH using the Illumina MiSeq™ platform. Sequence reads are trimmed using Trimmomatic with a 4‐bp sliding window quality score cutoff of Q30.2.13.5Aliquots of all residual specimens are archived at −70°C at WSLH.2.13.6We validate influenza immunization status for all ORCHARDS students using the Wisconsin Immunization Registry (WIR).2.13.7All identifying information on subjects is secured using a separate password‐protected security REDCap (Research Electronic Data Capture) database.2.13.8www.fammed.wisc.edu/orchards/) and Facebook page (www.facebook.com/orchardstudy/).ORCHARDS recruits using a reminder within the absenteeism reporting system. Information is provided at OSD registration each August and through e‐mails to OSD families. We employ flyers at community sites, presentations at community events, postcards, and the study website   and the University of Wisconsin Health Sciences IRB . The study is compliance with the Health Insurance Portability and Accountability Act of 1996 (HIPAA) and FERPA. The US Office of Management and Budget has approved all forms used in this study.2.13.10An independent surveillance system assesses medically attended influenza (MAI) at five family practice clinics within or adjoining the OSD. W‐IISP, sponsored by CDC and organized by the ORCHARDS team, has conducted active surveillance in medically attended patients with ARI/ILI since October 2009.n = 4069) individual patient visits.NP/OP swabs are collected from a subset of ambulatory patients with ARI/ILI. Specimens are tested at the WSLH for influenza A and B using the rRT‐PCR3The entire school district, including all schools, have participated since the initiation of ORCHARDS. Absenteeism data have been captured for all students, excluding pre‐kindergarten (for whom absenteeism is not recorded), since September 2014. The number of enrolled K‐12 students per year has increased from 3588 (2014/2015) to 3867 (2018/2019). Over the first 5 years of ORCHARDS, from September 2014 through June 2019, we evaluated 3,260,461 student days. Total absenteeism accounted for 301,427 (9.2%), a‐I for 58,126 (1.8%), and a‐ILI for 6634 (0.2%) of potential student days. The daily counts for each type of absenteeism, showing the variability, are depicted in Figure rs = 0.20; p < 0.001), a‐I , and a‐ILI . Most children (79%) reported school absenteeism due to the current illness episode, and about half (853/1728: 49%) were fully vaccinated against influenza at the time of the home visit.We completed 1728 home visits for children with ARI. Children ranged in age from 4 to 18 years (mean ± std. dev. = 9.9 ± 2.5 years). There were more male (57%) than female (43%) participants. Home visits occurred, on average, 56.3 ± 46.5 h after onset of symptoms. The number of home visits per day was positively correlated with a‐TOT (Pathogens were detected in 1115 (65%) specimens; the majority (99%) of these were viral. Codetections of viruses were noted in 66 students . Influenza was the most commonly detected virus, noted in 402/1728 23%) students, followed by rhinovirus/enterovirus. The numbers of viruses detected are provided in Table % student4In contrast to routine surveillance relying on MAI, ORCHARDS uses a community‐based design. Most cases of influenza do not present for medical attendanceA number of studies evaluating absenteeism and influenza predate ORCHARDSORCHARDS takes advantage of a long‐standing and highly effective influenza surveillance system as a “gold standard” for daily comparability. This parallel system is based on MAI surveillance at five family medicine clinics overlapping with the study catchment area and using very similar data instruments and identical laboratory methods. The community involvement, longitudinal nature, and external comparability make ORCHARDS a unique study platform to evaluate the role of school‐aged children on influenza transmission and the utility of cause‐specific absenteeism monitoring for identifying influenza outbreaks.Jonathan Temte: Conceptualization; data curation; funding acquisition; investigation; methodology; supervision. Shari Barlow: Conceptualization; funding acquisition; methodology; project administration; resources; supervision. Maureen Goss: Conceptualization; data curation; investigation; methodology; project administration; validation. Emily Temte: Conceptualization; data curation; investigation; methodology; project administration; validation. Cristalyne Bell: Data curation; project administration; validation. Cecilia He: Data curation; project administration; validation. Caroline Hamer: Data curation; project administration. Amber Schemmel: Data curation; project administration. Brad Maerz: Data curation; project administration. Lily Comp: Data curation; project administration; validation. Mitchell Arnold: Data curation; project administration; validation. Kimberly Breunig: Data curation; project administration; validation. Sarah Clifford: Data curation; project administration. Erik Reisdorf: Conceptualization; resources. Peter Shult: Conceptualization; resources. Mary Wedig: Conceptualization; resources. Thomas Haupt: Conceptualization; resources. James Conway: Conceptualization; methodology. Ronald Gangnon: Formal analysis; visualization. Ashley Fowlkes: Conceptualization; methodology. Amra Uzicanin: Conceptualization; methodology.https://publons.com/publon/10.1111/irv.12920.The peer review history for this article is available at Data S1. Supporting InformationClick here for additional data file.Data S2. Supporting InformationClick here for additional data file.


COVID‐19 pandemic, caused by severe acute respiratory syndrome coronavirus 2, has resulted in global social and economic disruption, putting the world economy to the largest global recession since the Great Depression. To control the spread of COVID‐19, cutting off the transmission route is a critical step. In this work, the efficient inactivation of human coronavirus with photodynamic therapy (PDT) by employing photosensitizers with aggregation‐induced emission characteristics (DTTPB) is reported. DTTPB is designed to bear a hydrophilic head and two hydrophobic tails, mimicking the structure of phospholipids on biological membranes. DTTPB demonstrates a broad absorption band covering the whole visible light range and high molar absorptivity, as well as excellent reactive oxygen species sensitizing ability, making it an excellent candidate for PDT. Besides, DTTPB can target membrane structure, and bind to the envelope of human coronaviruses. Upon light irradiation, DTTPB demonstrates highly effective antiviral behavior: human coronavirus treated with DTTPB and white‐light irradiation can be efficiently inactivated with complete loss of infectivity, as revealed by the significant decrease of virus RNA and proteins in host cells. Thus, DTTPB sensitized PDT can efficiently prevent the infection and the spread of human coronavirus, which provides a new avenue for photodynamic combating of COVID‐19. In this work, we report the application of a novel photosensitizer, DTTPB, with aggregation‐induced emission characteristics for photodynamic inactivation of human coronaviruses. With high molar absorbance coefficient, broad absorption band covering the whole white light region, and high membrane targeting and ROS sensitizing efficiency, DTTPB could effectively inactivate human coronaviruses and may contribute to preventing the spread of COVID‐19 pandemic. The COVID‐19 pandemic reminds us that human beings are still not well prepared in face of virus outbreaks, despite the simple structure of the virus as compared with mammalian cells. This could be partially ascribed to the high frequency of virus mutation. The mutation rate in RNA virus, such as human coronavirus, is even higher than DNA virus, which makes the development of vaccines and treatments very challenging.In 2020, we have witnessed the waging of COVID‐19, caused by the widespread of severe acute respiratory syndrome coronavirus 2 (SARS‐CoV‐2). Taking COVID‐19 pandemic with more than 147 million reported cases (data was from Johns Hopkins University till 26/04/2021) as an example, its widespread, to a considerable extent, was facilitated by SARS‐CoV‐2 contaminated high‐touch surfaces. Great efforts were spent to prevent virus transmission by killing and/or reducing the attachment of microbes. Though disinfectants, such as ethanol and hypochlorite, were frequently sprayed onto these high‐touch surfaces for inactivating virus, their frequency should be maintained at a high level, due to the rapid evaporation or deactivation of these disinfectants. Therefore, developing efficient surfaces and coatings materials to minimize the presence of active viral pathogens and prevent the spread of infectious pathogens in a variety of public places, such as hospitals, public transportations, and schools, are highly desirable.Cutting off the virus spreading route is an effective approach for thwarting virus outbreaks. In PDT, photosensitizers (PSs) and light irradiation were employed to sensitize the generation of reactive oxygen species (ROS), which can cause oxidative damages to nucleic acids, proteins, or lipids. Consequently, it induces irreversible microbe deaths with the superiority of minimal invasiveness, limited antibiotic resistance, low systemic toxicity, and minimal side effects. Photodynamic inactivation (PDI) of microbes has received considerable attention from scientists since the beginning of the 20th century. Recently, a resurgent on employing PDT for combating bacteria and fungi was observed. Besides, photodynamic antiviral studies also start to gain increasing attention, since Dr. Nicholas Kipshidze proposed “photodynamic therapy for COVID‐19” in Nature Photonics in 2020.As a prominent therapeutic strategy for contemporary and cost‐effective precision medicine, photodynamic therapy (PDT) has attracted more and more attention in antibiosis and tumor therapy. Conventional PSs, such as phenothiazinium salts, porphyrins, phthalocyanines, diketopyrrolopyrroles, and cyanines, have been widely employed for photodynamic anticancer and antibacterial studies. However, these PSs are usually featured with coplanar structures with large π conjugations, and tend to experience strong π‐π interactions in the aggregated state or at high concentrations, which could decay excited‐state energy through non‐radiative pathways, and result in a drastic decrease in both fluorescence (FL) intensity and ROS sensitizing efficiency. The low ROS sensitizing efficiency in the aggregated states is detrimental for applications in high‐touch surface disinfections, as well as fabrication into particles for PDT. Though the issue could be solved by introducing bulky groups to prevent π‐π interactions, it usually significantly increases the synthetic difficulties. PSs with aggregation‐induced emission (AIE) properties have recently become powerful tools for PDT of bacteria, fungi, and tumor. These PSs are usually featured with propeller‐shaped molecular conformations, and show not only enhanced FL but also elevated ROS sensitizing efficiency in the aggregated or solid‐state, as compared to that in the discrete molecular state. Despite the successful applications of AIEgen for PDT of bacteria, fungi, and tumor, research on PDI of the virus has been seldom reported.The outcome of PDT is closely related to the PS employed: effective PSs with high target specificity and ROS sensitizing efficiency are highly favorable for PDT applications. Since virus surface proteins vary among different human coronavirus species and mutants, lipid envelope may serve as a conservative target for PSs. Thus, we designed and synthesized an AIE‐active membrane‐targeting PS with near‐infrared FL, DTTPB , under white‐light irradiation, and were employed for highly efficient PDI of the virus. In the presence of 0.8 µm DTTPB, more than 99.9% human coronaviruses (HCoV‐229E) could be inactivated after irradiation with 9 mW cm−2 white light for 20 min.Human coronaviruses (including COVID‐19) are enveloped viruses with lipid bilayer covering their capsid and genetic materials, which could provide additional protection to the virus outside the host cell.22.1 Such structure feature could lower the energy gap between singlet excited state and triplet excited triplet, which was highly beneficial for facilitating intersystem crossing and could increase ROS sensitizing efficiency. In order to target the envelope of the virus, amphiphilic structure was preferred, mimicking the structure of phospholipid of the membrane. Based on these considerations, the molecular structure of DTTPB was adopted  structure feature was adopted.d Figure , in whic4 L mol−1 cm−1 . As toluene was a bad solvent for DTTPB, increasing fT resulted in the reduction of solvating power of the solvent mixture and finally led to the precipitation of DTTPB. As shown in Figure fT. When fT was raised to over 80%, an obvious redshift in the maximum absorption band was observed, indicative of the intermolecular interactions of DTTPB in the aggregated states. The DMSO solution of DTTPB fluoresced weakly with a red emission band peaked 606 nm , was used to investigate the interaction between DTTPB and phospholipid. DTTPB showed almost no FL in PBS solution Figure  and the p Figure . Compare We then examined the subcellular localization of DTTPB in living mammalian cell lines. Human cervical carcinoma cells (HeLa), human liver carcinoma cells (HepG2), and human embryonic kidney cells (HEK‐293T) were incubated with 5 µm DTTPB for 60 min, followed by the collection of FL images by using a confocal laser scanning microscopy. In HeLa cells, DTTPB specifically targeted cell surface regions and endowed HeLa cell membrane with red FL. The FL signal from DTTPB overlapped with that from the commercial plasma membrane probe, CellMask Green , HCoV‐OC43, HCoV‐229E, and foot‐and‐mouth disease virus (FMDV), and the FL spectra of these solutions were collected. Besides, the FL spectra of DTTPB in PBS and MEM, and MEM alone were also recorded for comparison. As illustrated in Figure 2.3m. As illustrated in Figure m DTTPB for 24 h, which demonstrated the excellent biocompatibility of DTTPB.For a PS to be employed for PDI of virus on a high‐touch surface, it should exert a minimum effect on humans. We thus evaluated the cytotoxicity of DTTPB by employing the cell counting kit‐8 (CCK‐8) method. Mammalian cell line, mouse embryonic fibroblast cells (NIH‐3T3), and two host cell lines, baby hamster kidney fibroblast cells (BHK‐21 for FMDV) and human fetal lung fibroblast cells (MRC‐5 for HCoV‐OC43 and HCoV‐229E), were incubated with different concentrations of DTTPB, ranging from 1.0 to 15.0 µ2.4FigureThe membrane targeting of DTTPB, and strong and broad absorption of DTTPB covering the whole visible light region are advantageous for anchoring to and PDI of coronavirus. As illustrated in −2) for up to 160 s, suggesting no or low ROS produced. In contrast, in the presence of both DTTPB and H2DCF‐DA, the FL intensity at 534 nm increased gradually with prolonged exposure time under white‐light irradiation (20 mW cm−2). The FL intensity increased by 220‐fold after 160 s irradiation, obviously proving the high ROS sensitizing efficiency of DTTPB.A key factor determining PDI efficiency was its ability to sensitize ROS generation. In our design of the PS, D‐π‐A structure feature was included to increase its ROS sensitizing efficiency through facilitating intersystem crossing. The ROS generation efficiency of DTTPB was first evaluated by the use of 2′,7′‐dichlorodihydrofluorescein diacetate (H2DCF‐DA) as an indicator. H2DCF‐DA emitted faintly, and reaction with ROS would turn on its FL at around 534 nm. Thus, by recording the FL of H2DCF‐DA, the efficiency of ROS production could be determined. As depicted in Figure 1O2 was considered as the first ROS generated during PDT, we thus evaluated the 1O2 sensitizing ability of DTTPB by measuring absorption changes of the 1O2 indicator, 9,10‐anthracenediyl‐bis(methylene)dimalonic acid (ABDA), under white‐light irradiation (20 mW cm−2). For comparison, the photosensitizing ability of a widely used PS, Rose Bengal (RB), was also evaluated under the same experimental conditions. As illustrated in Figure m) at 378 nm in the solution containing DTTPB (5 µm) gradually decreased to 20.2% of its original level upon white‐light irradiation for 5 min. In the control group without DTTPB, the absorption profile of ABDA solution was not changed under the white‐light irradiation. Under the same experimental conditions, the absorbance of ABDA at 378 nm in a solution containing 5 µm RB only decreased to 46.1% of its original value. These results clearly demonstrated that DTTPB could efficiently sensitize the generation of 1O2 for PDI applications. Encouragingly, the photosensitizing efficiency of DTTPB under white‐light irradiation was superior to RB, and the decomposition rate constants of ABDA in the presence of DTTPB were 2.06‐folds of that for RB . As illustrated in Figure m, the viral RNA expression levels for FMDV, HCoV‐OC43, and HCoV‐229E in corresponding host cells, were 91.7, 57.1, and 63.4%, respectively. In contrast, white‐light treatment could effectively reduce RNA copies in infected cells, suggesting the effective PDI of the virus. As shown in Figure m DTTPB and white‐light irradiation for 20 min. For HCoV‐OC43 and HCoV‐229E, with white‐light irradiation, their RNA copies could not be detected by qPCR at DTTPB concentrations higher than 7.5  secondary antibody, followed by imaging with an FL microscope. The virus antigen levels in each group were analyzed by immunofluorescence analysis with Image J. As shown in 2.850 assay was used to evaluate the number of infectious virus particles. In TCID50 assay, the viruses were incubated with different concentrations of DTTPB and then exposed to white light for 20 min, followed by incubation with corresponding host cells for 3 days and quantification of the plaques formed. As shown in Figure m of DTTPB and light treatment, the log10TCID50 of FMDV, HCoV‐OC43, and HCoV‐229E were significantly decreased by nearly 89.5 . By increasing the concentration of DTTPB from 0.01 to 10 µm, the CPE decreased gradually. It is noteworthy that all the BHK‐21 cells displayed almost no CPE after 24 h post infection by FMDV treated with 10 µm DTTPB and white‐light irradiation, which indicated that DTTPB could effectively inactivate FMDV and thus provide strong protection for BHK‐21 cells.We also investigated the morphology changes of BHK‐21 cell by using an inverted microscope (Figure 2.9Figurem, the relative RNA expression levels were decreased to 33.5, 21.0, and 3.9% for FMDV (Figure m, the relative RNA expression levels were decreased to 24, 0.4, and 0.9%, respectively, which ambiguously demonstrated that DTTPB could effectively inactivate viruses in environment and proved its great potential in photodynamic combat of SARS‐CoV‐2. The PDI performance of DTTPB on simulated high‐touch surface was better than that in culture media. This may be ascribed to the further restriction of intramolecular motions by absorbing onto high‐touch surfaces, which could enhance ROS sensitizing efficiency of DTTPB. The results demonstrated the potential of employing DTTPB to prevent the spread of infectious pathogens in public places.We further tested the feasibility of applying DTTPB in PDI of virus on high‐touch surfaces. In this experiment, filter paper was employed for simulating the high‐touch surface and the experimental procedures are illustrated in V Figure , HCoV‐OCV Figure , and HCoV Figure , respect31O2 sensitizing efficiency than the widely used PS, RB. DTTPB could selectively bind to and light up the cell membrane of mammalian cells, suggestive of its targeting to virus envelope. It exhibited good performance in the PDI of all the three tested viruses, as evaluated by the decreased expression of virus RNA copies after incubating with host cells. Specially, it exhibited highly efficient antiviral performance for the human coronavirus, HCoV‐229E: at a low concentration of 0.8 µm DTTPB and with white‐light irradiation, almost no HCoV‐229E RNA copies could be detected in host cells after incubation for 24 h. Similar conclusions could be drawn from western blotting analysis and immunofluorescence studies. The DTTPB‐mediated PDI of virus was also supported by viral tittering and examination of CPE, and the results clearly proved that DTTPB could efficiently prevent the infection and the spread of virus, and therefore directly provide strong protection for host cells. Furthermore, DTTPB also exhibited excellent performance in simulated high‐tough surfaces, demonstrative of its promising practical applications. As far as we are concerned, this contribution represents the first report of employing membrane‐targeting AIE‐type PS for efficient photodynamic killing of human coronaviruses. This work is expected to inspire new preventive strategies for combating with COVID‐19 and controlling the pandemic situation.To sum up, we've developed a near‐infrared membrane‐targeting PS, DTTPB, for photodynamic combat of virus. DTTPB showed typical AIE property with broad absorption band and high molar absorbance covering the whole visible light region. Besides, it also demonstrated excellent ROS sensitizing ability, with higher The authors declare no conflict of interest.Supporting InformationClick here for additional data file.


Gastrointestinal endoscopy (GIE) is useful for the early detection and treatment of many diseases; however, GIE is considered a high‐risk procedure in the coronavirus disease 2019 (COVID‐19) pandemic era. This study aimed to explore the rate of severe acute respiratory syndrome coronavirus 2 (SARS‐CoV‐2) positivity in saliva and gastrointestinal fluids to which endoscopy medical staff are exposed.The study was a single‐center cross‐sectional study. From June 1 to July 31, 2020, all patients who underwent GIE at Yokohama City University Hospital were registered. All patients provided 3 mL of saliva. For upper GIE, 10 mL of gastric fluid was collected through the endoscope. For lower GIE, 10 mL of intestinal fluid was collected through the endoscope. The primary outcome was the positive rate of SARS‐CoV‐2 in saliva and gastrointestinal fluids. We also analyzed serum‐specific antibodies for SARS‐CoV‐2 and patients’ background information.A total of 783 samples (560 upper GIE and 223 lower GIE samples) were analyzed. Polymerase chain reaction (PCR) on saliva samples did not show any positive results in either upper or lower GIE samples. However, 2.0% (16/783) of gastrointestinal fluid samples tested positive for SARS‐CoV‐2. No significant differences in age, sex, purpose of endoscopy, medication, or rate of antibody test positivity were found between PCR positive and PCR negative cases.Asymptomatic patients, even those with no detectable virus in their saliva, had SARS‐CoV‐2 in their gastrointestinal tract. Endoscopy medical staff should be aware of infection when performing procedures. The study was registered as UMIN000040587. The coronavirus disease 2019 (COVID‐19) pandemic is currently one of the greatest human challenges facing healthcare and economic systems worldwide.Gastrointestinal endoscopy (GIE) is useful for the early detection and treatment of gastrointestinal (GI) cancer, exclusion of organic diseases, and emergency cases, such as GI bleeding. However, upper GIE (esophagogastroduodenoscopy [EGD]) can induce coughing in patients; thus, there is a risk of aerosol transmission to healthcare workers.In Japan, COVID‐19 has affected 67,595 individuals and caused 1295 deaths as of September 1, 2020.This study aimed to explore the rate of SARS‐CoV‐2 positivity in saliva, gastric, and intestinal fluids to which endoscopy providers may be exposed.The study was a single‐center cross‐sectional study to evaluate the prevalence of SARS‐CoV‐2 positivity in saliva and gastric and intestinal fluids, in patients deemed to be at a low risk of COVID‐19 and who were scheduled to undergo endoscopy at YCU Hospital. From June 1 to July 31, 2020, all patients who underwent GIE at the YCU Hospital were registered.The institutional review board at YCU Hospital approved this prospective study on May 17, 2020 . Written consent for participation in the study was obtained from all patients. The study was registered in the University Hospital Medical Information Network Clinical Trials Registry as UMIN000040587.To reduce the risk of infection to healthcare providers, patients who underwent GIE were subject to a brief medical check and required to complete a questionnaire. Patients were classified as either COVID‐19 high‐risk or low‐risk patients. Patients who had (i) confirmed COVID‐19; (ii) a history of concentrated contact with a patient or suspected patient with COVID‐19 within 2 weeks; (iii) COVID‐19 related symptoms, such as fever (>37.5°C), dyspnea, or severe malaise; (iv) abnormalities of taste and smell with no obvious triggers; and (v) GI symptoms, such as diarrhea, lasting 4–5 days without obvious triggers, were classed as high‐risk patients. The remaining patients were classified as low‐risk patients.The inclusion criteria were as follows: (i) low‐risk patients; (ii) non‐emergency GIE cases; (iii) outpatients; (iv) patients who provided written informed consent for study participation.The exclusion criteria were as follows: (i) high‐risk patients ; (ii) emergency cases ; (iii) in patients ; and (iv) patients not willing to participate in the study.We analyzed baseline characteristics, including age, sex, purpose of endoscopy, and medications.In cases of upper GIE, including EGD and EUS, patients were required to fast for at least 12 h before the procedure. Before upper GIE, patients supplied 3 mL of saliva and received adequate sedation without oral dimethicone for antifoam. After insertion into the esophagus, the endoscope was immediately inserted into the stomach and used to vacuum 10 mL of gastric fluid. Then, endoscopists performed usual procedures. In cases of lower GIE, such as CS, bowel preparation was initiated 1 day before the procedure. Each patient was instructed to consume a low‐residue diet and to take 5 mg of oral sodium picosulfate on the evening before the procedure. On the day of the procedure, each patient was administered 1500 mL of polyethylene glycol (PEG). Before the procedure, patients also supplied 3 mL of saliva and received adequate sedation. After insertion into the cecum, the endoscope was used to vacuum 10 mL of intestinal fluid. Then, endoscopists performed usual procedures. Collected saliva and gastric and intestinal fluids were stored at −80°C until analysis. Patients who agreed to undergo additional blood sampling had 5 mL of blood drawn. Collected blood was separated from serum and stored at −80°C until analysis.The primary outcome was the positive rate of SARS‐CoV‐2 in saliva and gastric and intestinal fluids. Iwasaki et al. reported that the concordance rate of virus detection between saliva and nasopharyngeal samples was as high as 97.4%.Detection of SARS‐CoV‐2 genomic RNA was performed according to the Manual for the Detection of Pathogen 2019‐nCoV Ver.2.6 provided by the National Institute of Infectious Diseases in Japan.U‐test or the student’s t‐test was used to compare continuous data. P‐values < 0.05 were considered statistically significant. All statistical analyses were performed using JMP 15 software .Results are presented as the mean for quantitative data and frequency (percentage) for categorical data. Categorical data were assessed using the Chi‐squared test. The Mann–Whitney During the study period , the number of newly diagnosed patients with COVID‐19 was 1,116 in Kanagawa Prefecture. The prevalence of infected people was 0.46  in the last week of July.The clinical characteristics of patients are presented in Table The RT‐PCR testing for SARS‐CoV‐2 in saliva did not show any positive results in patients who underwent EGD, EUS, and CS. However, surprisingly 2.3% of upper GI samples (13/560) were positive, and limited EGD cases, 2.5% (13/511) were positive Table .During the study period, 55.2% of patients 432/783) agreed to have additional blood tests performed, including 271 patients who underwent GS, 45 patients who underwent EUS, and 116 patients who underwent CS. A total of 17 patients (3.9%) across all procedures showed a positive result with serum‐specific antibody testing, including 12 patients who underwent GS (4.4%) and five patients who underwent CS (4.3%) . Of the patients who tested positive for SARS‐CoV‐2 in GI fluid by RT‐PCR, only one patient tested positive for serum antibodies, which did not differ from the percentage of patients in the RT‐PCR‐negative group who tested positive for antibodies.We compared patients who were one or more of the samples positive with those who were all negative Table . No signn = 22), nurses (n = 7), receptionists (n = 3), and cleaning staff (n = 6), developed fever or other health concerns.Throughout the study period, no medical staff at the endoscopy center, including endoscopists  and specificity is 98.9% (95%CI 98.0–99.4%).We conclude that approximately 2.0% of asymptomatic patients, even patients with no detectable virus in their saliva, tested positive for SARS‐CoV‐2 in their GI tract. Endoscopy staff should continue to be aware of the risk of infection when performing procedures. Furthermore, even if people are asymptomatic, people should be careful when handling feces and when using the restroom during the COVID‐19 era. Further studies are needed to clarify the relationship between COVID‐19 and the presence of SARS‐CoV‐2 in the GI tract.Authors declare no conflicts of interest for this article.Grants for this research from the Japanese Foundation for Research and Promotion of Endoscopy (JFE) and Daiwa Securities Health Foundation were awarded to TH. We declare that the funding body has no role in the design, data collection, analysis, interpretation and writing of the study.Text S1 Detection of SARS‐Co‐V‐2 genomic RNA and serological tests for SARS‐CoV‐2 antibodies.Table S1 Sequences of the primers.Table S2 CT values in RT‐PCR positive cases.Table S3 Serological antibody test results in positive cases.Click here for additional data file.


Biocatalysis is an established chemical synthesis technology that has by no means been restricted to research laboratories. The use of enzymes for organic synthesis has evolved greatly from early development to proof‐of‐concept – from small batch production to industrial scale. Different enzyme immobilization strategies contributed to this success story. Recently, the use of hydrogel materials for the immobilization of enzymes has been attracting great interest. Within this review, we pay special attention to recent developments in this key emerging field of research. Firstly, we will briefly introduce the concepts of both biocatalysis and hydrogel worlds. Then, we list recent interesting publications that link both concepts. Finally, we provide an outlook and comment on future perspectives of further exploration of enzyme immobilization strategies in hydrogels. HEMA2‐hydroxyethyl methacrylateHRPhorseradish peroxidaseNAD[P]Hnicotinamide adenine dinucleotide [phosphate]PEGpolyethylene glycolPEGDApolyethylene glycol diacrylatepILspolymerized ionic liquidsSTYspace time yield.1For more than 100 years, enzymatic catalysis has made its way into modern organic chemistry. Nowadays, biocatalysis is a powerful tool for selective organic synthesis , 3, 4, 5product⋅volumereaction–1⋅timereaction–1) at technical scales can be possible with process engineering. Hence, researchers began to include and integrate medium engineering‐ [To overcome these challenges, modern molecular biological tools have been effectively used to optimize enzymes’ operational window for broader pH and temperature ranges as well as to increase their stability in organic media . For theneering‐ , 28, 29 neering‐ , 31 intoneering‐ , 33, orgneering‐ , 35, 36,neering‐ , 38, 39,neering‐ , 44, 45.neering‐ , improvineering‐ , 48, or neering‐ . Researcneering‐ , 51. Herneering‐ .Different forms of immobilization were described in the literature, and overall, three types of enzyme immobilization are generally known: (i) Binding the enzyme to a (porous) support, (ii) crosslinking of the enzyme, and (iii) encapsulation/entrapment of the biocatalyst into a matrix , 54. DivBesides the aforementioned methods for enzyme immobilization, the use of hydrogels for heterogenization of enzymes has attracted great attention in the last few years. An unbeatable argument compared to other immobilization materials is the fact that hydrogels, per se, contain water: Hydrogels are referred to as polymeric materials that are capable of absorbing a large amount of water. For most of the enzymes in non‐aqueous media, hydrogels a prerequisite that can provide the desired microenvironment for enzymes which is highly advantageous. Here, it is worth mentioning that enzyme immobilization in gels has been pioneered by Manfred Reetz with his great contribution since end of 90s , 57, 58.Hydrogels, as we understand the term within this publication, consist of a crosslinked polymer network. Generally, these polymers can absorb immense amounts of water, enlarging their volume and their whole mass while retaining their three‐dimensional shape. The mass fraction of the hydrogels does not change. Often, upon swelling, hydrogels retain their stiffness and therefore, their durability . The synzymes) [Although the technique of enzyme immobilization has long been known, as well as the knowledge of natural and synthetic hydrogels, we are not aware of any review article that explicitly reports on the immobilization of enzymes in hydrogels. It should be noted, however, that often a different terminology is used. For example, we would like to mention the recently published review article by Nöth et al. dealing with the synthesis, concepts, and emerging applications of biocatalytic microgels (μ‐Gelzymes) . Additiozymes) .In this review, we aim to present developments in the field of enzyme immobilization in hydrogels published in the last twenty years. In Section 2.1, we focus on the nature of hydrogel materials used for enzyme immobilization in general. In Section 2.2, we address the literature from the last five years with respect to the applications of hydrogel‐based immobilized enzymes.2At the beginning of every immobilization procedure there is one big question: Which immobilization carrier and which technique is the most suitable for the biocatalyst and the desired reaction? To answer this question, there are further criteria to be considered; like biocompatibility, product separation, high enzyme loading, biodegradability as well as mechanical and chemical stability . For ind2.12+, Mn2+, or Ba2+. Although sodium alginate is commercially available in a wide range of molecular weights  at low prices, an alginate solution with higher molecular weight suffers from extremely high viscosity  or proteins  , 78, 79.35 g⋅L–1 , comparee enzyme , 82. Thie enzyme , 85, 86.e enzyme , 88. Othe enzyme . To overe enzyme . Hydrophe enzyme . Anothere enzyme .ø = observed reaction rate/mass transfer rate) [ø ≤ 0.3) [To improve further characteristics of the immobilization matrix, additional monomers and crosslinkers can be introduced. A frequently applied method circumventing mass transport limitations is the covalent surface immobilization of enzymes on polymers by using spacers , 93. Ayher rate) . In geneø ≤ 0.3)  mean sufInteresting new features in the field of enzyme immobilization are polymerized ionic liquids (pILs) and polyelectrolytes and some of these can be referred to as hydrogels as well. Nakashima et al. reported a horseradish peroxidase (HRP) encapsulated in pILs‐microparticles being easily recovered by centrifugation of the reaction mixture. The pILs‐microparticles were obtained by emulsion polymerization of 1‐vinyl‐3‐ethylimidazolium bromide (VEtImBr) with the crosslinker N,N’‐methylene bisacrylamide (MBAA). The HRP was modified with a comb‐shaped PEG, allowing multivalent carboxylic anhydride groups to react with the amino groups of the enzyme. By combining the pILs‐microparticles and HRP‐PEG via free‐radical polymerization, the enzyme could be immobilized and possess higher activity compared to conventional polyacrylamide microparticles. The authors state, that this effect was generated by the flexible spacer arm groups. This allows the polymer surface to conform to the structure of the enzyme, thereby preserving and stabilizing the native catalytic activity to a greater extent .To summarize this section, enzymes can be immobilized in/on a large variety of polymeric carriers/matrices. The degree of swelling in aqueous media has an enormous impact on the effectiveness of the immobilization. For most of the enzymes, a high water content is associated with a desired high enzyme activity. However, it also often leads to increased leaching of the enzyme from the polymer matrix. To find the optimum of immobilization yield and activity, co‐polymerizations with other hydrophobic monomers or other immobilization methods such as covalent bonding can be used.2.2In this section, we will describe recent developments in the field of biocatalyst immobilization in hydrogels for enzymatic synthesis and devote detailed focus on their implementation in special applications.Trametes versicolor served as biocatalyst. The enzyme‐immobilized hydrogels were synthesized in different (co‐)polymer/crosslinker compositions and used as granules as well as coatings on porous aluminum  and 2‐((2‐(ethoxycarbonyl)prop‐2‐en‐1‐yl)oxy)‐ethyl phosphonic acid (ECPPA), to immobilize a laccase for wastewater treatment . N,N’‐dim Figure . DiffereR)‐selective alcohol dehydrogenase from Lactobacillus brevis (LbADH) and a NADPH‐regenerating glucose 1‐dehydrogenase GDH from Bacillus subtilis yielding a hydrogel whose mass consists of 77% enzymes. The SpyTag peptide/SpyCatcher protein (ST/SC) system together with a hexahistidin (His) tag were used to promote the enzyme gelation via crosslinking of the two enzyme building blocks  were transformed to their (R)‐configured alcohols with high conversions, proving the applicability of the system. In another publication, the authors compared different immobilization strategies and observed high space time yields (STYs) of > 450 g·L−1·h−1 and a continuous production for more than six days [Peschke et al. reported on self‐assembling all‐enzyme hydrogels . The auts Figure , left. Ts Figure , right, six days . Additiosix days .Streptomyces sp. was used for the reduction of 3,4‐dihydroisoquinoline to the corresponding amine in an all‐enzyme hydrogel loaded microreactor  .The auth. Figure . For theSaccharomyces cerevisiae cells in hydrogels and used it as a hydrogel film in a continuous flow reactor [l‐malic acid  from mesophilic organism Lactococcus lactis in agarose. Printed ADH discs and KIVD_mutant discs were combined in a flow setup and an enzymatic cascade was performed in continuous flow to produce isobutanol from ketoisovalerate  and rose bengal  together in alginate hydrogel capsules [R)‐2‐methylcyclohexanone with high enantioselectivities (> 99%) and a maximal conversion of 70.4%  of chemical denaturants like dimethylformamide, dimethyl sulfoxide, ethanol, or 1‐propanol.Yoon et al. co‐immobilized an ene‐reductase from capsules . With th% Figure . The autAspergillus oryzae  in PEGDA hydrogel materials [w/w and a pneumatic extrusion‐based 3D printer was used to print defined 3D lattice structures. UV‐light was used to harden the hydrogels with the help of the initiator 2‐hydroxy‐4′‐(2‐hydroxyethoxy)‐2‐methylpropiophenone. A standard β‐galactosidase activity assay was performed  of more than 20 with an effective diffusivity within the hydrogel of about 3·10–12 m2·s–1. In another study, the concept was broadened to the enzymes benzoylformate decarboxylase from Pseudomonas putida (BFD) and LbADH [Schmieg et al. studied the immobilization of β‐galactosidase from aterials . The connd LbADH . Again, Aspergillus oryzae , glucose oxidase from Aspergillus niger (GOx) and HRP were used whereas for the second cascade, phospholipase D from Streptomyces chromofuscus (PLD), choline oxidase from Alcaligenes sp. (ChOx), and HRP were used. Each cascade produces 2,2′‐azino‐bis(3‐ethylbenzothiazoline‐6‐sulfonic acid) ethyl methacrylate, and HEMA . Two sepWith respect to hydrogel/enzyme dots, the work of Thiele and co‐workers should also briefly be highlighted in this review. The authors use synthetic and natural monomers and modified hyaluronic acid microgels for enzymatic synthesis , 115.–1para‐nitrophenyl acetate for 1 h.Junshi Moriyama et al. entrapped bovine carbonic anhydrase (BCA) into calcium alginate hydrogel beads with the help of liposomes (BCALs) . Using tCandida antarctica  in pILs through encapsulation [rac‐1‐phenylethanol with vinyl acetate was studied . The easy upscaling procedure by numbering up the all‐enzyme hydrogel microreactors and the high space time yields being achieved are promising for future developments in this field. Furthermore, we reviewed the easy immobilization of whole permeabilized cells in hydrogels for the use in continuous flow reactors. Eventually, the use of hydrogels enables the creation of new enzyme‐containing microreactors and/or random column packing materials by 3D printing improving the handling of enzymes, their reusability, and robustness. Nevertheless, mass transport limitations may reduce the overall enzyme's efficiency and must be considered when immobilizing enzymes in hydrogels.3Within this review, our main aim was to cover recent developments in design and development of hydrogel‐based immobilized enzymes and their applications for chemical synthesis. The applications highlighted included both batch and continuous operations. We outlined the drivers behind the rise of this key emerging field: (i) Sustainable and biodegradable materials – considering natural building blocks, (ii) the biocompatibility of many hydrogel materials, (iii) their inherent high water absorption capacity, and (iv) the precision in the control of the production of the hydrogel materials especially thanks to 3D printing technology. Although a few exceptions might exist, mass transport limitations remain challenging and detailed systematic analyses are still missing in the field to a major extent.We think that future research will most likely concentrate on further developments of novel hydrogel materials for enzyme immobilization and their biocatalytic applications. Particularly, we expect a strong increase in the field of 3D‐printed enzymes being immobilized in hydrogel materials. This might also boost the research field in flow biocatalysis , for sinWe also predict that researchers will not only use ionic liquids (ILs) and deep eutectic solvents (DESs) as non‐conventional media for biocatalysis , but alsThe authors have declared no conflict of interest.


Cisplatin (CP) still is a novel choice for solid tumor therapy, but it is accompanied with the side effect of nephrotoxicity. Hydration may reduce the risk of CP-induced nephrotoxicity, while the issue is still challenging. In this study, five types of hydration protocols including saline, mannitol, dextrose saline, saline plus furosemide, and saline plus mannitol were examined in both sexes of rats during CP therapy.  Seventy-six male and female Wistar rats in 14 groups of experiments were subjected to CP therapy, and five types of hydration protocols were implemented, and the induced nephrotoxicity was evaluated via biochemical markers, kidney function parameters, and pathology investigation. P < 0.05) in female rats hydrated with saline plus furosemide or mannitol plus saline-treated groups. The worsened condition in male rats is observed in the mannitol hydration group with a significant decrease of ClCr and significant increase of serum BUN and Cr and ENa% (P < 0.05). The higher kidney tissue damage score (KTDS) in the mentioned groups verified the findings.  Male and female rats had different responses to hydration protocol types. The higher mortality rate was seen in female rats that received mannitol or dextrose hydration types. In addition, the serum levels of blood urea nitrogen (BUN) and creatinine (Cr) and sodium excretion fraction (ENa%) increased and the clearance of Cr (ClCr) decreased significantly ( Hydration with mannitol or dextrose promotes the risk of nephrotoxicity during CP therapy with more intensity on the female. Nowadays, cisplatin (CP) is recognized as a choice treatment for a wide range of solid tumors in clinic. In spite of its great effect on improvement of survival, it causes many complications such as nephrotoxicity. The renal manifestations of CP therapy may accompany with acute and chronic renal failure, tubular acidosis, or renal salt wasting . NephrotSeveral studies have been done in order to assess the protective effect of different synthetic and herbal antioxidants supplements against CP-induced nephrotoxicity –11. In aSeventy-six female and male (209 ± 1.7 g) Wistar rats  were used in this study. Animals were housed in the controlled conditions . This study approved by Isfahan University of Medical Sciences Ethics Committee .The animals were divided randomly into 14 groups of experiments .n = 6) and 8  are named NS + CP + NS. The animals in these groups initially received normal saline , and 1 hour later, CP  was administrated. Then, after 1 hour post-CP injection, the second injection of NS  was done.(1)  Groups 1  and 9  are named M + CP + M, and groups 3  and 10  are named DS + CP + DS. The animals in these groups had the same regimen of group 1, but the M + CP + M groups received mannitol  and the DS + CP + DS received dextrose saline  instead of NS.(2) Groups 2  and 11  are named NS + CP + NS, F. The rats initially received NS , and 1 hour later, CP  was administrated. Then, after 1 hour post-CP injection, the combination of NS  and furosemide  was administrated.(3) Groups 4  and 12  are named M, NS + CP + NS. The animals of these groups initially received combination of NS  and M , and 1 hour later, CP  was injected. Then, after 1 hour post-CP injection, the NS  was administrated.(4) Groups 5  and 13  are named CP. The rats in these groups received CP  alone without any hydration.(5) Groups 6  and 14  are named NS. These rats had the regimen-like group 6 or 13, but they received NS  instead of CP.(6) Groups 7 . The left kidney was used for histopathology investigations via hematoxylin and eosin (H&E) staining. Based on existence of vacuolization, dilatation, hyaline cast, debris, or degeneration, the renal damage was assigned as the kidney tissue damage score (KTDS), and it was scored from 1 to 4, while zero score was recorded for normal tissue , 18. Thet-Student test. Comparison of the KTDS between the groups was assessed using the Mann–Whitney test. P < 0.05 was considered as significant.Data are expressed as mean ± standard error of the mean. The levels of BUN, Cr, MDA, nitrite, UF, ClCr, ENa%, KW, and bodyweight (BW) change were compared between each two groups using the The survival time (in days) for each group of male and female rats is given in According to survival time table , the numIn order to determine the effect of CP alone, the data obtained in NS and CP groups  were compared statistically. The result is tabulated in P < 0.05) in the M + CP + M group, while these increased insignificantly in the DS + CP + DS group when compared with the CP-alone treated group. In addition, the ClCr also was decreased statistically in the M + CP + M group, as shown in P < 0.05). Also, a lower UF and a higher KTDS were detected insignificantly in M + CP + M groups, as shown in In male rats, the serum levels of BUN and Cr and ENa% were increased significantly (P < 0.05) in M, NS + CP + NS and NS + CP + NS, F groups when compared with the CP alone treated group, as shown in P < 0.05). The increase of KW and more BW lost were observed in the NS + CP + NS group from the CP group (P < 0.05), and a lower UF was collected significantly in NS + CP + NS and NS + CP + NS,F groups compared to the CP alone treated group (P < 0.05).In female rats, the serum levels of BUN and Cr and ENa% were increased significantly  and 3 male rats (50%) survived at the end of the experiment. In addition, the mortality rate in the female dextrose-treated group (group 10) was 66%.CP filters via the glomerulus, and it excretes through the renal tubules . ClCr isReviews of past studies illustrate controversial results about mannitol. Some of them reported that mannitol has a protective effect on the kidneys by its diuretic mechanism and reducing CP concentration , 25. On The nephroprotective effect of mannitol and furosemide have also been compared. Santoso et al. concluded that furosemide-saline has more nephroprotective effect compared with mannitol , while oAn increase in the glucose level to perform a suitable diuresis is another issue for challenging. Diabetes has been considered as one of the risk factors for various cancers such as colon, chest, pancreas, and liver, but it plays a protective role in reducing the toxic effects of CP. Intensity and duration of diabetes mellitus have a direct relationship with the severity of kidney toxicity, and the protective effects are also reduced by controlling diabetes . TherefoFinally, in the current study, we reported the data for male and female rats separately, and some differences were seen without any analysis. However, it is well documented that CP-induced nephrotoxicity is gender related . PezeshkAlthough mannitol and dextrose in clinical protocols for the reduction of CP-induced nephrotoxicity may be suggested, however, this study not only shows that these two hydration protocols do not protect the kidney but also they are harmful and the use of these substances need to be reconsidered seriously in male and female genders. It seems that hydration with saline alone is more safe than other protocols investigated in this study.


The ongoing SARS‐CoV‐2 pandemic has proven to be challenging, in both clinical and pathological terms. The viral tropism for angiotensin‐converting enzyme 2 (ACE‐2) receptor‐expressing cells is paralleled in the clinical setting by the development of a spectrum of manifestations, most notably acute respiratory distress syndrome (ARDS).A number of post‐mortem neuropathological studies have reported vascular, thrombotic, and ischaemic alterations in COVID‐19 cases.Our group aimed to describe the neuropathological findings in SARS‐CoV‐2 infected patients who died during the COVID‐19 pandemic and, by using real‐time (RT) PCR and immunohistochemistry (IHC), to define and quantify the presence of virus in selected areas showing pathological signs, as well as in areas of interest that might define a route of spread for the virus in the CNS.A total of 15 consecutive autopsies performed in patients with demonstrated or suspected SARS‐CoV‐2 infection  in L. Sacco Hospital, Milan, Italy, for which the brain was obtained were included in our study. Among them, 13/15 cases had positive SARS‐CoV‐2 RT‐PCR throat swab and the remaining 2/15 demonstrated viral infection by PCR analysis on post‐mortem specimens from the brain or other organs.The entire brain was fixed in neutral‐buffered formalin for a minimum of 21 days (range 21–42 days). Macroscopic examination and sampling were performed by cutting the brain in the coronal plane at level of the mammillary bodies and then sectioning at 1 cm intervals in order to comprehensively evaluate brain parenchyma and related structures. An extensive sampling of the brain was performed, which included areas from cervical spinal cord, brain stem , cerebellar hemispheres (cortex and dentate nuclei), cerebral hemispheres , and olfactory bulbs. Other organs, that is, lungs, heart, liver, kidneys, spleen, and bowel, were extensively sampled for diagnostic purposes and were routinely examined.For histopathological examination, 3 μm thick sections from each brain paraffin block were stained with haematoxylin and eosin. IHC was performed on the most representative areas in cases with signs of inflammation, with antibodies to glial fibrillary acidic protein (GFAP‐clone EP672Y), macrophages (CD68‐clone KP1), T‐lymphocytes (CD3‐clone 2GV6), B‐lymphocytes (CD20‐clone L26), and granulocytes . All antibodies were ready‐to‐use  and staining was done with the BenchMark Ultra IHC/ISH System  in accordance with the standard protocols supplied by the manufacturer. Additional IHC and special stains were used as appropriate.Neuropathological parameters assessed included gliosis, microglial activation, perivascular lymphocytic cuffing, red neurons, thrombi, and haemorrhage. Gliosis was assessed semi‐quantitatively as mild, moderate, and severe based on astrocytic morphology and GFAP expression.Selected areas with vascular and inflammatory alterations were tested via RT‐PCR to assess the presence of SARS‐CoV‐2, as well as the olfactory bulbs, olfactory tubercles with medial and lateral olfactory tracts, medulla oblongata, pons, amygdalae, hippocampi with entorhinal cortices, and hypothalamus. RNA was extracted from three 5 μm paraffin sections using Quick‐RNA FFPE Miniprep (Zymo research). Two target genes, RNA‐dependent RNA polymerase  and envelope , were amplified and tested in all selected samples. All olfactory bulbs and olfactory tubercle/medial olfactory tract/lateral olfactory tract samples, as well as all other areas positive for E‐gene and/or RdRp gene, underwent additional quantitative testing with one‐step reverse transcription and qPCR of two Nucleocapsid viral regions (N1 and N2), using the GoTaq® 1‐Step RT‐qPCR System  and the 2019‐nCoV CDC Diagnostic Panel  according to manufacturer's instructions. The N1 and N2 viral regions were detected in 10‐μl volume reactions using 0.2 μg of purified RNA. Reactions were performed on a CFX96  according to the following thermal profile: initial denaturation  followed by 45 cycles of 15 s at 95°C (denaturation) and 1 min at 60°C . A melting curve analysis was assessed for amplicon characterisation. Viral copy quantification was assessed by creating a standard curve from the quantified 2019‐nCoV N positive Plasmid Control . Cycle threshold values less than 35 were considered as positive test results. Human RNase P was used as an internal control to confirm that RNA was adequately conserved and extractedAreas positive with RT‐PCR testing underwent IHC for SARS‐CoV‐2 spike glycoprotein ,Our results are summarised in Table The neuropathological spectrum in our series comprises predominantly ischaemic and/or haemorrhagic alterations and a degree of reactive change, with no evidence of viral encephalitis  or viral inclusions. Parenchymal haemorrhages were observed in 9 (66.6%) cases and subarachnoid haemorrhage in 2 (12.7%) cases. Signs of acute neuronal ischaemic injury  were present in 7/15 (46.7%) cases. Areas of mild–moderate gliosis were found in 5/15 cases (33.3%). Microglial proliferation was demonstrated in 4/15 (26.7%) cases . A single case showed focal perivascular lymphocytic cuffing, without signs of gliosis or microglial proliferation. Two cases (12.7%) had thrombi in the middle cerebral arteries. One case demonstrated a thrombus in a leptomeningeal vein. Another single case demonstrated diffuse neutrophil infiltration of brain parenchyma and microhaemorrhages, with microglial proliferation and moderate gliosis. The most representative neuropathological findings are illustrated in Figure Using four different RT‐PCR assays , the olfactory bulbs demonstrated the presence of SARS‐CoV‐2 in 8/15 (53.3%) cases: four samples (26.7%) were positive for the E‐gene, five (33.3%) for the RdRp gene, five (33.3%) for the N1 gene, and seven (46.7%) for the N2 gene. This finding was not paralleled in the next area of connection in the olfactory pathway , which tested negative in all cases. The midbrain of cases no. 6 and no. 11 tested positive for E‐gene only. All other selected samples of all cases, including olfactory‐related areas and selected areas with signs of inflammation and/or haemorrhages, tested negative using molecular assays. Using quantitative RT‐PCR, viral copies in the olfactory bulbs ranged from 1 to 508 copies/μl (mean 123.4) with N1 gene and from 1 to 1798 copies/μl (mean 312.7) with N2 gene.Histopathological examination of the eight RT‐PCR positive olfactory bulbs was unremarkable. IHC for SARS‐CoV‐2 spike glycoprotein demonstrated a paranuclear localisation of the virus in endothelial cells of 1/8 olfactory bulb samples  showed positivity for SARS‐CoV‐2 in their olfactory bulbs, with high viral concentrations . Among these, only one case showed positivity for SARS‐CoV‐2 IHC. Notably, all areas with gliosis, microglial activation, microhaemorrhages, and other pathological changes tested negative using RT‐PCR.The olfactory bulb positivity in a subset of samples seems to indicate that SARS‐CoV‐2 can spread through the olfactory nerve fibres from the nasal cavity. However, the absence of virus within the neural and glial compartments in olfactory bulb samples, as well as in olfactory tubercles/lateral olfactory tract/medial olfactory tract, along with the endothelial localisation of the virus in such samples, seem to indicate that the virus spreads through a haematogenous route, which could be related to the common arterial supply of the olfactory fibres and olfactory bulb.The midbrain of 2 (12.7%) cases showed positivity for E‐gene using RT‐PCR. Both cases showed scant neuropathological features  showed satisfactory results. The discrepancy may be due to the long fixation of the CNS specimens (range 21–42 days), but this explanation is not completely convincing since our positive controls also underwent long fixation periods, as well as brain specimens in other studies which used FFPE samples.Our findings raise concerns regarding the equivalence of different genes of SARS‐CoV‐2 in PCR testing for the virus in the CNS. Further studies are needed to compare the performance of the genes E, RdRP, N1, and N2 in this setting, and clinicians and researchers should be aware that a negative result for a single gene does not preclude a positive result using another, and vice versa. The exact criteria to define the overall positivity for SARS‐CoV‐2 in this setting remains to be established.The authors declare no conflict of interests.The Ethics Committee of Luigi Sacco Hospital approved the use of patient data for scientific research related to the disease. This study followed the Italian general rules used for scientific research purposes (regulation no. 72–26/03/2012).GL was responsible for the original draft preparation, data collection and curation, and final draft preparation; CT, GO, EL, MB, and GC were responsible for the data collection and curation; LC, AP, and RSR performed autopsies; PZ performed autopsies and was responsible for the data collection and curation; MN performed autopsies and was responsible for the supervision, data collection and curation, and final draft preparation. All authors approved the final version of the manuscript.Figure S1. Gross and microscopic findings in central nervous system tissues of SARS‐CoV‐2 infected patients.Panel A. Case n.8 demonstrated grossly intraparenchymal bilateral multifocal haemorrhages, one of which extended to the cerebral ventricles.Panel B. Gross examination of case n. 10 revealed multiple areas of white matter discoloration (arrows).Panel C. Histology of case n.5 demonstrated a thrombus in a leptomeningeal vein. This was the only thrombosis‐related finding in our series .Panel D and E. Microscopically, areas of white matter discoloration of case n. 10 contained microhaemorrhages  and acute inflammatory cells parenchymal infiltrates ; Panel F and G. CD68‐KP1 immunostaining demonstrated microglial proliferation , and immunohistochemistry for myeloperoxidase  highlighted the neutrophilic infiltrate in brain parenchyma.Click here for additional data file.


Coronary artery lesions (CALs) are the most severe complication of Kawasaki disease (KD). Approximately 9–20% of the patients with KD develop CAL despite receiving regular treatment (intravenous immunoglobulin [IVIG] and aspirin). Some patients develop coronary aneurysms, leading to coronary artery stenosis or thrombosis, resulting in ischaemic heart disease and significantly affect the patients’ lives. The purpose of this study was to investigate the factors associated with the duration of CAL in patients with KD.The data of 464 patients with KD and CAL admitted to the Children’s Heart Centre, The Second Affiliated Hospital and Yuying Children’s Hospital from 2010 to 2018 were retrospectively analysed. Demographic and clinical information and echocardiographic follow-up data were collected. Kaplan–Meier curves were used to estimate the overall CAL duration, and the log-rank test was used to compare statistical differences. Univariate and multivariate Cox regression models were used to identify variables related to the CAL duration.p = 0.016), rash symptoms (p = 0.035), delayed IVIG treatment (p = 0.022), CAL type (p < 0.001), degree of CAL (p < 0.001), white blood cell count before IVIG treatment (p = 0.019), and platelet count after IVIG treatment (p = 0.003) were statistically significant factors associated with the overall CAL duration. Multivariable Cox regression showed that delayed IVIG treatment (p = 0.020), multiple dilatations (p < 0.001), a greater degree of dilatation (p < 0.001), and higher platelet count after IVIG treatment (p = 0.007) were positively related to CAL duration.The median CAL duration was 46 days . CALs were observed in 61.5, 41.5, 33.3, 22.3, 10.3, and 7.7% of the patients at 1 month, 2 months, 3 months, 6 months, 1 year, and 2 years after the onset of KD, respectively. Univariate Cox regression model showed that sex (CAL duration was affected by delayed IVIG treatment, type of CAL, degree of CAL, and platelet count after IVIG treatment. These factors should be monitored carefully during the follow-up and management of patients with KD and CAL. Kawasaki disease (KD) is an acute febrile vasculitis of unknown aetiology that predominantly affects children aged < 5 years. Its pathological features include systemic inflammation of medium-sized blood vessels and various tissues and organs. The coronary arteries are typically the most affected vessels , 2. AlthPersistent CALs may lead to thrombosis, stenosis, and obstruction, causing major adverse cardiac events, such as unstable angina pectoris and myocardial infarction, and even death , 8. One Recently, paediatricians have focused on patients with KD and CAA –16. HoweWe collected the demographic and clinical information of 511 patients with KD and CAL admitted to the Children’s Heart Centre of The Second Affiliated Hospital and Yuying Children’s Hospital from 1 January 2010 to 31 December 2018. KD diagnosis was based on the 2017 American Heart Association (AHA) guidelines and CAL diagnosis was based on the expert consensus in this region. Due to missing clinical information, 47 patients were excluded; therefore, 464 patients were included in the final analysis. Patients were divided into two groups based on the degree of CAL (patients with multiple CALs were grouped according CAL severity): the dilation group (absolute diameter of the coronary artery < 4 mm) and the aneurysm group (absolute diameter of the coronary artery ≥4 mm) ; haematocrit (%); erythrocyte sedimentation rate (mm/h); and haemoglobin (g/L), C-reactive protein (mg/L), alanine aminotransferase and aspartate aminotransferase (U/L), albumin (g/L), sodium (mmol/L), potassium (mmol/L), chloride (mmol/L), and brain natriuretic peptide (pg/mL) levels were recorded before and after IVIG treatment.The patients’ medical data and follow-up records, including the patients’ age, weight, sex, KD type, clinical symptoms, IVIG resistance, delayed IVIG treatment, CAL type, and degree of CAL, were collected from the electronic medical record system of the hospital. Laboratory data including the white blood cell, neutrophil, and platelet counts , left anterior descending artery (LAD), left circumflex (LCX), right coronary artery (RCA), and posterior descending coronary artery (PDCA) were recorded each time. This study’s primary endpoint was the overall duration of CAL, defined as the duration between the first date of CAL detection on echocardiography and the date of its disappearance on echocardiography. The disappearance of CAL in echocardiography was defined as normalization of absolute inner diameter, which means the absolute inner diameter is smaller than the diagnostic criteria.According to the 2017 AHA guidelines, the clinical criteria for the diagnosis of KD include a prolonged, unexplained fever lasting for ≥5 days and the presence of four or more of the five major clinical symptoms, namely bilateral conjunctival congestion, polymorphous rash, oral changes, acute non-purulent cervical lymphadenopathy, and extremity changes [A patient with unexplained fever for > 5 days, < 4 of the major clinical symptoms, and supportive laboratory findings or coronary artery abnormalities was considered to have incomplete  KD .IVIG resistance was defined as a body temperature of > 38.5 °C at 48 h after the end of IVIG infusion . DelayedCALs were classified according to their size as follows: absolute coronary artery diameter > 2.5 mm for children aged < 3 years, > 3 mm in children aged 3–9 years, and > 3.5 mm in children aged > 9 years. CAAs were diagnosed if the absolute diameter of the coronary artery was ≥4 mm. The range of small, medium, and giant CAAs was defined by a diameter < 5, 5–8, and > 8 mm, respectively . Single t- or the Mann–Whitney’s U tests (as appropriate) and the Pearson’s Chi-square or the Fisher’s exact tests (as appropriate), respectively. The overall duration of CAL was estimated using the Kaplan–Meier curve method, and the log-rank test was used to compare the variables. A univariate Cox analysis was conducted to explore the variables associated with the overall duration of CAL. Statistically significant variables in the univariate Cox regression analysis were included in the multivariate Cox model. Statistical significance was set at p < 0.05. All statistical analyses were performed using the R software . The Cox regression analyses were performed using the ‘survival’ package and the Kaplan–Meier curves were created using the R package ‘ggplot2’.Continuous variables are expressed as means ± standard deviation or interquartile range, and categorical variables are summarised as numbers and percentages. Continuous and categorical variables were compared among the groups using the p < 0.001) and heavier (p = 0.002) than those in the dilation group (Table p = 0.007) and oral symptoms (p = 0.025) in the aneurysm group were lower than those in the dilation group. A higher proportion of patients in the aneurysm group received delayed IVIG treatment (p = 0.018). In addition, more patients in the aneurysm group had multiple CALs (p < 0.001) than the dilation group. There were no significant differences in the sex, KD type, other clinical symptoms, or IVIG resistance between the two groups , and the aspartate aminotransferase level was lower in the aneurysm group (p = 0.029) than in the dilation group before IVIG treatment. There were no significant differences in the laboratory data after IVIG treatment between the two groups. The detailed demographic and clinical characteristics are presented in Tables The neutrophil count was higher p = 0.07, and thThe median CAL duration was 46 days . CALs were observed in 61.5, 41.5, 33.3, 22.3, 10.3, and 7.7% of the patients at 1 month, 2 months, 3 months, 6 months, 1 year, and 2 years after disease onset, respectively , sex (p = 0.016), and rash symptoms (p = 0.035) were correlated with the CAL duration , and platelet count after IVIG therapy (p = 0.003) were also associated with the CAL duration (Table p = 0.020), multiple dilations , greater dilation , and increased platelet count after IVIG  were found to be independently associated with the CAL duration  (p<0.001), delayed IVIG treatment p = 0.00, sex  are more likely to develop CAA and not respond to IVIG than patients with normal platelet counts (150–450 × 109/L) [Paediatricians often monitor the platelet count in patients with KD. In Japan, the platelet count is used to predict IVIG-resistant coronary aneurysms , 24. The× 109/L) . In our According to several guidelines, the optimal time for administering IVIG is 5–10 days after the onset of illness . The latWe did not find a relationship between IVIG resistance and the duration of CALs. This may be due to repeated IVIG treatments or additional corticosteroid treatments in patients who did not initially respond to the treatment, resulting in a suppressed immune response and inflammation. Therefore, no difference in the immune function was observed between patients who received multiple rounds of IVIG or additional corticosteroid treatment and patients who received only one round of IVIG. We did not find any other significant associations between other clinical symptoms or laboratory data and the duration of CALs.This study is not without limitations. First, because CAL was detected by echocardiography, both the occurrence and disappearance of CAL might show earlier than the check time point. According to previous research, the occurrence and the disappearance of CAL mostly happen in the subacute phase. Therefore, the follow-up strategy was made because the subacute-phase follow-up examination was more intensive than the chronic phase. We tried to make the duration of CAL obtained from the echocardiography closer to the real-time. Second, as this study began in 2010, we did not use the z-score to determine the degree of CALs, which has recently been reported as more appropriate , 29. InsIn this retrospective study, the duration of CAL was longer in patients with KD with a greater degree of CAL, multiple CALs, higher platelet counts after IVIG, and delayed IVIG treatment. Therefore, these factors should be monitored carefully by paediatricians. Cardiac coronary artery examinations, including angiography and percutaneous coronary interventions, and anticoagulant therapies should be used in patients with KD.


We present a case of atypical recurrent optic neuritis. A man in his 50s presented with right optic neuritis and profound visual loss, associated with elevated inflammatory markers. Lymph‐node biopsy was consistent with sarcoidosis. Aquaporin‐4 antibodies were also present. Three months following corticosteroid treatment, his right optic neuritis relapsed, again with raised inflammatory markers. He was started on azathioprine and prednisolone with good effect. A dual diagnosis of sarcoidosis and neuromyelitis optica with aquaporin‐4 antibodies is very rare. Long‐term immunosuppression is required. The case highlights the importance of identifying the features and cause of atypical optic neuritis. A man in his late 50s presented with a 5‐day history of rapid right eye visual loss associated with pain on eye movement and colour desaturation. Four weeks previously, he had experienced a flu‐like illness with significant weight loss. Past history was significant for factor V Leiden mutation with previous deep vein thrombosis and pulmonary embolism, as well as asthma and chronic obstructive pulmonary disease.On initial examination, his right eye had no perception of light and his left eye vision was 6/5 on Snellen chart. There was a right relative afferent pupillary defect with a hyperaemic and slightly swollen right disc. Systemic examination was unremarkable.Erythrocyte sedimentation rate (ESR) was elevated (93 mm/hr). Empirical treatment for giant cell arteritis was started. He was given 500 mg oral methylprednisolone daily for 5 days, followed by 80 mg prednisolone daily. A right temporal biopsy 4 days later was negative.Anti‐nuclear antibody (ANA) staining was positive at a titre of 1:1280 (homogenous). Peripheral anti‐neutrophil cytoplasmic antibody (pANCA) staining was positive, but myeloperoxidase and proteinase three antibodies were negative. Serum angiotensin‐converting enzyme was normal (32 U/L).CT head identified asymmetrical enlargement of the optic nerve sheath complex within the right orbit Fig. , with miNo improvement in vision was seen after 10 days of corticosteroids. A subsequent MRI scan of the orbits was normal and did not show pathological enhancement after contrast, although it was performed after 18 days of corticosteroids. MRI brain confirmed the CT findings in the deep grey matter and cerebellum. He was given 1 g of IV methylprednisolone daily for 3 days, starting on the same day as the MRI.Visual evoked potentials 3 weeks after presentation revealed severe right optic nerve conduction delay. An 18F‐fluorodeoxyglucose positron‐emission tomography (FDG‐PET) scan of the whole body demonstrated hypermetabolic mediastinal and hilar lymphadenopathy Fig. . HistoloIn addition, a live cell‐based assay for aquaporin‐4 (AQP4) antibodies later returned strongly positive. This was confirmed on repeat testing. Live cell‐based assay for myelin oligodendrocyte glycoprotein antibody was negative.There was a good clinical response to prolonged corticosteroids: at 3 months from onset, ESR had dropped to 18 mm/hr and corrected right‐eye vision had recovered (6/6‐2).However, 3 months later, whilst weaning corticosteroids, he experienced a relapse of his right optic neuritis (ON) with an elevated ESR (62 mm/hr). Optical coherence tomography showed supero‐temporal thinning of the peripapillary retinal nerve fibre layer in the right eye Fig. . He was He has been maintained on azathioprine and low‐dose prednisolone with no further clinical deterioration. Right‐eye vision has remained 6/9 with some colour‐vision deficits and infero‐temporal field depression. He has not had other neurological symptoms.The possibility of a deposition disorder (as suggested by his brain imaging) was thoroughly investigated, but no clear cause was found. This was felt to be an incidental finding not connected to his presentation.This is a case of atypical ON associated with both histologically proven sarcoidosis and AQP4 antibodies. To date, there is only one case of dual sarcoidosis and neuromyelitis optica spectrum disorder (NMOSD) reported in the literature, presenting with transverse myelitis and later bilateral ON, with both diagnoses made at the second presentation.It is difficult to conclusively establish whether sarcoidosis or NMOSD was responsible for the recurrent ON. The cell‐based assay for AQP4 antibodies has a specificity of 99%.For long‐term management, given the recurrent ON and the presence of AQP4 antibodies, azathioprine was selected due to its efficacy in both conditions.This case highlights the importance of identifying atypical features in ON, such as, in our case, age of onset and marked visual loss.Michael Foster is supported by a grant from the MRC (MR/S026088/1). Sara Collorone is supported by the Rosetrees Trust (MS632) and she was awarded a MAGNIMS‐ECTRIMS fellowship in 2016. Jacqueline Palace is partly funded by highly specialised services to run a national congenital myasthenia service and a neuromyelitis service. She has received support for scientific meetings and honorariums for advisory work from Merck Serono, Biogen Idec, Novartis, Teva, Chugai Pharma, Bayer Schering, Alexion, Roche, Genzyme, MedImmune, EuroImmun, MedDay, Abide ARGENX, UCB and Viela Bio and grants from Merck Serono, Novartis, Biogen Idec, Teva, Abide, MedImmune, Bayer Schering, Genzyme, Chugai and Alexion. She has received grants from the Multiple Sclerosis Society UK, Guthy Jackson Foundation, National Institute of Health Research, Oxford Health Services Research Committee, Medical Research Council, GMSI (Grant for MS Innovation), John Fell, Myaware and AMPLO for research studies. James Acheson has no competing interests to declare. Ahmed Toosy has received speaker honoraria from Biomedia, Sereno Symposia International Foundation, Bayer and meeting expenses from Biogen Idec and Novartis. He was the UK PI for two clinical trials sponsored by MEDDAY pharmaceutical company (MD1003 in optic neuropathy [MS‐ON] and progressive MS [MS‐SPI2]). He is supported by grants from the MRC (MR/S026088/1), NIHR BRC (541/CAP/OC/818837) and RoseTrees Trust (A1332).Michael Foster drafted and revised the manuscript and performed literature review. Sara Collorone supported in drafting the manuscript and performing literature review. Jacqueline Palace reviewed the manuscript and provided specialist scientific content. James Acheson reviewed the manuscript and provided specialist scientific content. Ahmed Toosy conceived of the report, supervised the project, reviewed the manuscript and provided specialist scientific content.Figure S1. Fundus photographs taken at first presentation, demonstrating hyperaemia and swelling of the right optic disc.Click here for additional data file.


To the Editor:The safety of NSAIDs, particularly ibuprofen, in COVID‐19 has been openly questioned.We investigated the effects of ibuprofen on lung levels of ACE2 and other major compounds of the lung RAS, both in healthy adult rats and in a well‐known rat model of metabolic syndrome . It is known that patients with MetS are more vulnerable to severe COVID‐19. In vitro, we used human alveolar type‐II pneumocyte cells to study the effects of ibuprofen on changes induced by viral spike protein on ACE2 levels, on levels of spike protein internalization, ADAM17 and TMPRSS2 activities, and on the release of major COVID‐19‐related cytokines. Details on methods are provided as Supporting Information.In healthy adult rats, treatment with ibuprofen (40 mg/kg) significantly increased lung ACE2 expression and enzymatic activity, and increased the expression of anti‐inflammatory RAS axis receptors ; furthermore, ibuprofen induced a significant decrease in proinflammatory AT1 receptor expression Figure . MetS raTreatment of cultures of human alveolar type‐II pneumocytes with ibuprofen also upregulated ACE2 expression 24 and 48 h after treatment Figure . TreatmeEffects of ibuprofen on spike protein internalization were studied using a laser confocal microscope. We initially observed that pneumocytes expressed ACE2 in our cultures and that spike protein was internalized by ACE2‐expressing cells Figure . Then, tAs several of the above‐mentioned observations, particularly ibuprofen‐induced decrease in spike protein internalization, cannot be explained by the ibuprofen‐induced increase in ACE2 expression, we investigated possible additional effects of ibuprofen on other major mechanisms involved in SARS‐CoV/spike protein internalization. First, we analyzed possible changes in ADAM17 activity because we observed that in the culture medium, pretreatment with ibuprofen inhibited the increase in levels of soluble (105 kDa) ACE2 induced by spike protein (see above). Consistent with this, we observed that ADAM17 activity was significantly upregulated in pneumocytes treated with spike protein, as compared with untreated control pneumocytes. However, this upregulation was significantly reduced by pretreatment of cultures with ibuprofen Figure . Second,The major mechanism of action of ibuprofen is inhibition of COX‐2, and interactions between COX‐2 and RAS have previously been observed in other tissues.In conclusion, in the present models, ibuprofen upregulated ACE2 expression and anti‐inflammatory RAS activity in the lung. It is known that this inhibits inflammatory, fibrotic, and thrombotic responses. In human lung cell cultures, ibuprofen‐induced upregulation of the SARS‐COV‐2 main receptor (ACE2) was counteracted by ibuprofen‐induced mechanisms that reduce SARS‐COV‐2 spike protein internalization, particularly by inhibition of ADAM17 and TMPRSS2 activities The authors declare that there is no conflict of interest.Animals were handled in accordance with the Directive 2010/63/EU, European Council Directive 86/609/EEC, and the Spanish legislation (RD53/2013). The corresponding committee at the University of Santiago de Compostela (15005/15/002) approved all animal experiments, which were performed in the Experimental Biomedicine Center .Jose L. Labandeira‐Garcia, Ana I. Rodriguez‐Perez, and Rita Valenzuela designed research and experiments. Ana I. Rodriguez‐Perez, Pablo Garrido‐Gil, and Maria A. Pedrosa performed in vivo experiments. Rita Valenzuela, Maria A. Pedrosa, Rafael Franco, and Gemma Navarro performed in vitro experiments. Ana I. Rodriguez‐Perez, Rita Valenzuela, Carmen M. Labandeira, Rafael Franco, and Jose L. Labandeira‐Garcia contributed to writing, review, and critique. All the authors edited the manuscript.Data are available from the corresponding author upon reasonable request.Supporting InformationClick here for additional data file.Figures S1‐S2Click here for additional data file.


A 29-year-old woman presented to the emergency department with the acute onset of palpitations, shortness of breath, and haemoptysis. She reported having an abortion (56 days of pregnancy) 1 week before admission because of hyperthyroidism diagnosis during pregnancy. The first diagnoses considered were cardiomyopathy associated with hyperthyroidism, acute left ventricular failure, and hyperthyroidism crisis. The young woman’s cardiocirculatory system collapsed within several hours. Hence, venoarterial extracorporeal membrane oxygenation (VA ECMO) was performed for this patient. Over the next 3 days after ECMO was established, repeat transthoracic echocardiography showed gradual improvements in biventricular function, and later the patient recovered almost completely. The patient’s blood pressure increased to 230/130 mm Hg when the ECMO catheter was removed, and then the diagnosis of phaeochromocytoma was suspected. Computed tomography showed a left suprarenal tumour. The tumour size was 5.8 cm × 5.7 cm with central necrosis. The vanillylmandelic acid concentration was 63.15 mg/24 h. Post-operation, pathology confirmed phaeochromocytoma. To our knowledge, this is the first case report of a patient with cardiogenic shock induced by phaeochromocytoma crisis mimicking hyperthyroidism which was successfully resuscitated by VA ECMO. All kinds of acute diseases, such as acute coronary syndrome, cardiogenic shock, septic shock , preecla2A 29-year-old woman presented to the emergency department with the acute onset of palpitations, shortness of breath, and haemoptysis. She reported having an abortion (56 days of pregnancy) 1 week prior to admission because of a hyperthyroidism diagnosis during pregnancy; the thyroid function increased significantly . The patient denied using any drugs or tobacco, and there was no history of allergies. Her family history was normal. She was admitted to the endocrinology department under the consideration of cardiomyopathy associated with hyperthyroidism, acute left ventricular failure, and hyperthyroidism crisis as potential diagnoses.2) by pulse oximetry was 93% with an oxygen delivery rate of 15 L/min by hydrogen mass, and her heart rate was 156 beats/min with hypotension of 85/43 mm Hg with a dopamine dose of 8 µg/kg min. This dose was increased slowly. Her shell temperature was 36.3°C. Abdominal examination revealed a soft abdomen with no organ enlargement or mass. She had auscultation of the lungs with wheezes or crackles. Laboratory studies showed hyperglycaemia (glucose: 9.43 mmol/L), myocardial damage (troponin I: 29.02 ng/mL and creatine kinase-MB: 64 U/mL), and mild renal insufficiency (creatinine: 146.4 mmol/L and anuria lasting for 3 h); her PCT level was 24 ng/mL and her CRP level was 2.0 mg/L. Transthoracic echocardiography (TTE) showed the normal left ventricular size and significantly reduced left ventricular systolic function. The left ventricular ejection fraction (LVEF) was 15%. Chest X-ray revealed severe interstitial and alveolar oedema  remained unstable despite high dose of norepinephrine (2.0 µg/kg/min), adrenaline (1.0 µg/kg/min), and dopamine (20 µg/kg/min) with ventilator support (FIO2: 100% and PEEP: 15 cm H2O). Blood gas analysis showed severe hyperlacticaemia and metabolic acidosis . Echocardiography revealed that the EF and global ventricular wall motion were much worse than before. PICCO showed severely low cardiac output and hyperpyrexia . She was considered to be having hyperthyroidism crisis-induced cardiogenic shock. Mechanical ventilation was started, and the haemofiltration solution temperature was decreased to room temperature. Two hours later, the patient was still hyperpyrexic (40.3°C) and abruptly developed severe bradycardia; intermittent intravenous epinephrine was administered to maintain her heart rate.On admission, she was conscious but displayed acrocyanosis, orthopnoea, excessive perspiration, moist cold skin, cough with pink bubbly expectorate, and respiratory distress. Her respiratory rate was 40 breaths/min, her oxygen saturation  with continuous ECMO and inotropic agent and vasopressor support; her urine output was approximately 200 cc/h. Over the next 3 days after ECMO was established, repeat TTE showed gradual improvement in biventricular function. Norepinephrine and adrenaline were withdrawn on the third day  and venous (21 F) cannulation using the Seldinger technique VA ECMO . The pathird day . The patThe patient’s blood pressure increased to 230/130 mm Hg when the catheter was removed; despite treatment with propofol and sufentanil at 200 mg/h and 50 µg separately, her blood pressure did improve and fluctuated from 130/60 mm Hg to 230/130 mm Hg in a short time. Intermittent sodium nitroprusside was required to control episodic hypertension; then, the diagnosis of phaeochromocytoma was suspected. Computed tomography revealed a left suprarenal tumour of 5.8 cm × 5.7 cm in size with central necrosis . The vanInformed consent: Informed consent has been obtained from all the individuals included in this study.Ethical approval: The research related to human use has been complied with all the relevant national regulations, institutional policies, and in accordance with the tenets of the Helsinki Declaration, and has been approved by the Medical Ethics Committee of The First Affiliated Hospital of Wannan Medical College .3This phaeochromocytoma case demonstrates fulminant cardiomyopathy; however, the patient’s clinical manifestation gave no evidence of phaeochromocytoma diagnosis. Suspicion was first raised when the blood pressure fluctuated after the patient was weaned from ECMO and it was then confirmed by imaging and laboratory test. PCC has been defined as the release of large amounts of catecholamines resulting in severe organ dysfunction, especially haemodynamic instability and myocardial damage. PCC has been reported to mimic cardiomyopathy, myocardial infarction , pulmonaThe rate of PCC misdiagnosis is very high. In a French multicentre cohort study  of PCC, An acute catecholamine crisis may be fatal before it is diagnosed . Previou4PCC mimics many conditions and is thus a masked killer. The misdiagnosis ratio was very high. For patients with unexplained cardiovascular impairment, especially in younger patients, we should consider the PCC.