Patients with coronavirus disease 2019 (COVID-19) occasionally develop ocular complications. We report a case of acute retinal necrosis (ARN) caused by Epstein–Barr Virus (EBV) that developed in a patient who had severe acute respiratory syndrome due to SARS-CoV-2 infection.A 68-year-old woman complained of floaters and blurred vision in her right eye as she was receiving systemic prednisolone for COVID-19 pneumonia under isolation in our hospital. The patient visited an ophthalmologist following her discharge from the hospital and after the 2 weeks of isolation had ended. At the initial examination, her best-corrected visual acuity (BCVA) was 20/100 in the right eye, and the eye showed moderate anterior segment inflammation and vitreous opacities. Treatment was initiated with topical 0.1% betamethasone and 1.5% levofloxacin. After 1 month, the inflammation in the right eye decreased and her BCVA improved to 20/40. However, on day 48 from her initial visit, the inflammation in her right eye worsened and her BCVA decreased to 20/2000 by day 80. Pars plana vitrectomy with silicone oil tamponade was performed to remove the vitreous opacities, and expanded white exudates peripherally and retinal vessels with white sheathing suggestive of acute retinal necrosis (ARN) were seen intraoperatively. Analysis of the vitreous sample revealed EBV positivity on polymerase chain reaction. The patient was diagnosed with EBV-associated ARN and treated with systemic steroids and valaciclovir. The ocular inflammation gradually decreased, and she was discharged from the hospital. However, a week later, the inflammation in the right eye markedly worsened. Despite another course of steroids, the inflammation worsened, resulting in total retinal detachment and absolute glaucoma. Because of the severe pain, the right eye was enucleated.Clinicians should be aware that COVID-19 and immunosuppressive treatment can reactivate EBV in the eye.The online version contains supplementary material available at 10.1186/s12886-022-02692-5. The pandemic due to severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has had health implications of unprecedented magnitude. The disease can affect every organ of the body.Patients with coronavirus disease 2019 (COVID-19) occasionally develop ocular complications, and various manifestations in the eye directly or indirectly associated with the virus have been reported, i.e, conjunctivitis, retinal hemorrhage, disc edema, uveitis, vitritis, and retinitis [Ophthalmologists should be aware of the possible association of ocular disease in patients affected by COVID-19 because ophthalmic manifestations may be the presenting feature of COVID-19 or they may develop several weeks after recovery.Isolation of COVID-19-affected patients may delay opportunities for eye examination and worsen eye inflammation, resulting in a delay in treatment and difficulties in diagnosis.We report a case of acute retinal necrosis (ARN) in a patient on immunosuppressive treatment for COVID-19 pneumonia in whom Epstein–Barr virus (EBV) was detected from the vitreous.A 68-year-old woman presented with floaters in her right eye. Her review of systems showed that she had cough, fever, and shortness of breath that had started 2 weeks earlier, and her symptoms had worsened after a week. She visited the hospital and was diagnosed with COVID-19 pneumonia by general examination and polymerase chain reaction (PCR). She had not been vaccinated against COVID-19. She had undergone surgery for breast cancer 2 years prior and the cancer treatment had ended. She was not on any medication before she acquired COVID-19. She received systemic antibiotics and remdesivir for COVID-19 pneumonia and was in isolation at our hospital.Systemic prednisolone treatment was also started from 0.6 mg/kg/day and tapered gradually for 14 days. The patient was under systemic treatment in isolation when she presented with floaters in her right eye.Five days after finishing prednisolone treatment, she was discharged from the hospital. She complained of worsening floaters and blurred vision but she was required to self-isolate for 2 weeks, thus she visited an ophthalmologist 3 weeks after the primary ocular symptoms.At the initial visit, she reported an increase in the floaters and blurred vision in the right eye. Her best-corrected visual acuity (BCVA) was 20/100 in the right eye and 20/16 in the left eye. The intraocular pressure was 16 mmHg in the right eye and 17 mmHg in the left eye. No significant findings were noted in the left eye. Slit-lamp examination of the right eye revealed grade 2+ cells in the anterior chamber and posterior synechia. Fundus examination showed vitreous opacities and retinal exudates  was re-administered, the inflammation progressed, leading to the development of total retinal detachment and absolute glaucoma Fig. . The rigCandida infection was ruled out. Based on these findings, the patient was eventually diagnosed with EBV-related ARN.EBV, also known as human herpesvirus 4, is a member of the herpesvirus family, and most individuals are infected by EBV during childhood. It is a well-known cause of infectious mononucleosis . EBV is Few cases of ARN caused by EBV have been reported where EBV was detected in vitreous samples by PCR –11. It hCOVID-19 can induce a state of hyperimmunity –15. In oCandida albicans.There is no established treatment for EBV uveitis. Therefore, our patient was treated with steroids and valaciclovir as reported . This reIn conclusion, we have presented a rare case of EBV-related ARN. The intensive treatment resulted in a temporary resolution of the inflammation. However, systemic steroids may lead to further immunosuppression, resulting in additional infections. Clinicians should be aware that COVID-19 and treatment can reactivate EBV in the eye.Additional file 1.


Human breast milk (HBM) is the ideal source of nutrients for infants and is rich in microRNA (miRNA). In recent years, expressed breast milk feeding rather than direct breastfeeding has become increasingly prevalent for various reasons. Expressed HBM requires storage and processing, which can cause various changes in the ingredients. We investigated how the miRNAs in HBM change due to processes often used in real life. HBM samples collected from 10 participants were each divided into seven groups according to the storage temperature, thawing method, and storage period. In addition, we analyzed the miRNA changes in each group. The number of microRNAs that showed significant expression was not large compared to the thousands of miRNAs contained in breast milk. Therefore, it is difficult to suggest that the various storage and thawing processes have a great influence on the overall expression of miRNA. However, a short-term refrigeration storage method revealed little change in nutrients compared to other storage and thawing methods. Taking all factors into consideration, short-term refrigeration is recommended to minimize changes in the composition or function of breast milk. Human breast milk (HBM) contains complex proteins, lipids, and carbohydrates, and is the ideal source of nutrients for infants. Additionally, it contains antibodies involved in the human immune response and oligosaccharides involved in shaping the intestinal microbiota and supporting health, among numerous other biologically active compounds ,2. BreasBreast milk is particularly diverse and rich in microRNA (miRNA). Accordingly, there is much interest in the role of miRNA in breast milk, particularly how miRNAs contained in breast milk are nutritionally absorbed through the infant’s digestive tract and benefit the infant, as well as their role as epigenetic regulators of gene expression in infants ,10. miRNTo perform their functions, miRNAs must remain stable. Despite the rise in expressed breast milk feeding, there has been little analysis of changes in miRNA in expressed breast milk. Our research seeks to determine how the miRNA in expressed breast milk is affected by various storage and handling conditions often used in real life. Based on these studies, optimal breastfeeding management guidelines can be presented, and an environment can be provided to enhance the convenience of nursing mothers and promote the best health conditions for infants.Human breast milk was collected at the Chung-Ang University Breastfeeding Research Institute  for current lactating mothers who agreed to participate in the study. There was no particular restriction on the research participants because our focus was to compare only the change according to storage and thawing with initially collected HBM and confirm the stability of miRNAs. Breast milk was collected in a fresh state within a day of milking and was collected after refrigerating for less than 24 h at home except when delivered immediately after the milking. The donated breast milk of 40 to 50 cc was divided into seven aliquots (samples) immediately after collection, and each sample was treated, stored, and thawed as follows : Sample The seven samples were compared for their changes in miRNAs according to microwave treatment , storage condition , thawing method , and duration of frozen storage .g, 4 °C, 10 min) twice, and fat, cells, and debris were removed. The skim milk samples were centrifuged  and sequentially filtered through 0.8, 0.45, and 0.22 μm syringe filters  to remove residual fat and cell debris. miRNAs of skim milk were extracted by acid-phenol/chloroform separation combined with column-based filtration using the mirVana miRNA Isolation Kit . The concentration and purity (260:280 ratio) of miRNAs were analyzed using a NanoDrop™ 1000 spectrophotometer . Aliquots (>20 ng RNA) of the miRNA samples were used for small RNA sequencing (RNA-seq).miRNA analysis of each sample was performed according to the breast milk collection date and storage period. To prepare the nonfat skim milk fraction, human breast milk samples were centrifuged  according to the manufacturer’s instructions. Briefly, for library construction, total RNA from each sample was used to ligate the adaptors, and cDNA was then synthesized using reverse-transcriptase with adaptor-specific primers. PCR was performed for library amplification, and the libraries were cleaned up using a QIAquick PCR Purification Kit  and polyacrylamide gel electrophoresis gel. The yield and size distribution of the small RNA libraries were assessed with an Agilent High Sensitivity DNA Assay on an Agilent 2100 Bioanalyzer instrument . High-throughput sequences were produced by a NextSeq 500 system using 75 bp single-end sequencing .p-value of 0.05 in the t-test.Sequence reads were mapped by the Bowtie2 software tool to obtain a bam file. Mature miRNA sequences were used as a reference for mapping. Read counts mapped to mature miRNA sequences were extracted from the alignment file using BEDtools v2.25.0 and a bioconductor using the R statistical programming language. Read counts were used for determining the expression level of miRNAs. The counts per million-trimmed mean of M-values (CPM-TMM) normalization method was used to compare samples. For the miRNA target study, miRWalk 2.0 was performed. Functional gene classification was performed by DIANA. In addition, differentially expressed gene analysis was used to identify miRNAs with a significant expression. We found miRNA with a normalized RC (log2) value of 4 or higher, with the expression of miRNA increasing or decreasing by more than twofold on a subgroup basis and the Breast milk was collected 10 times from nine lactating mothers . ParticiWhen comparing the expression level of miRNAs between the control (Sample 1) and microwave treatment (Sample 2), two miRNAs showed significant changes in all participants (hsa-miR-24-3p and hsa-miR-27a-3p). However, there was no tendency for increased and decreased gene expression, according to the participant .When we compared the expression level of miRNAs between the control (Sample 1) and one week of storage at room temperature (Sample 3), we saw that four miRNAs showed significant changes in all participants . The expression level of hsa-miR-378a-3p was significantly decreased in all participants. Furthermore, hsa-miR-365a-3p and hsa-miR-365b-3p were significantly decreased in all participants except for Participant 6 .When we compared the expression level of miRNAs between the control (Sample 1) and one week of refrigerated storage (Sample 4), we saw that no miRNAs showed significant changes in all participants .When we compared the expression level of miRNAs between the control (Sample 1) and bottle warmer thawing after one week of frozen storage (Sample 5), we saw that two miRNAs showed significant changes in all participants (hsa-miR-205-5p and hsa-miR-3182). hsa-miR-3182 was significantly decreased in all participants except for Participant 8 .To explore the change in miRNA of breast milk according to the thawing method, breast milk was exposed to different thawing methods (bottle warmer and microwave treatment) after one week of frozen storage. Again, when we compared the expression level of miRNAs between the control (Sample 1) and bottle warmer thawing after one week of frozen storage (Sample 5), we saw that two miRNAs showed significant changes in all participants (hsa-miR-205-5p and hsa-miR-3182). hsa-miR-3182 was significantly decreased in all participants except for Participant 8 .When we compared the expression level of miRNAs between the control (Sample 1) and microwave thawing after one week of frozen storage (Sample 6), six miRNAs we saw significant changes in all participants . hsa-miR-3960 was significantly increased in all participants except for Participant 4, and hsa-miR-24-3p was significantly decreased in all participants except for Participant 4 .To assess the changes in miRNA of breast milk according to the duration of frozen storage, all samples were thawed by the same method in a bottle warmer after frozen storage at −20 °C for one and four weeks. When we compared the expression level of miRNAs between the control (Sample 1) and bottle warmer thawing after one week of frozen storage (Sample 5), we saw that two miRNAs showed significant changes in all participants (hsa-miR-205-5p and hsa-miR-3182). Moreover, hsa-miR-3182 was significantly decreased in all participants except for Participant 8 .When we compared the expression level of miRNAs between the control (Sample 1) and bottle warmer thawing after four weeks of frozen storage (Sample 7), we saw that four miRNAs showed significant changes in all participants . hsa-miR-103a-3p and hsa-miR-103b were significantly decreased in all participants except for Participant 9. hsa-miR-193b-5p was significantly increased in all participants except for Participant 10 .Direct breastfeeding is the ideal method, but due to various social or medical issues, expressed breast milk feeding is often performed, necessitating the collection, processing, and storage of breast milk ,15. BreaIn previous studies, Zhao et al. confirmed a decrease in the amount of miRNA and extracellular vesicles in cow milk according to a microwave treatment (305 W for 32 s) . RecentlIn our findings, there were up to six miRNAs with significant differences in expression in all participants, which were less significant than the thousands of miRNAs contained in breast milk. Therefore, it is difficult to suggest that the various storage and thawing processes previously performed have a great influence on the overall expression of miRNA. However, there was no significant change in the amount of miRNA expressed in breast milk subjected to refrigerated storage for one week. This means that refrigerated storage has little effect on miRNA among the storage methods. Many preterm infants are unable to breastfeed; hence, refrigeration storage of breast milk is a common practice in neonatal intensive care units. In previous studies, breast milk refrigerated at 4 °C for up to 96 h showed no significant difference in pH, osmolality, white blood cell counts, bacterial colony counts, and the concentrations of secretory IgA, protein, total fat, and free fatty acids ,22. TakiTwo other common storage methods for breast milk other than refrigeration are frozen storage and room temperature storage. When comparing breast milk stored at room temperature for one week (Sample 3) with breast milk thawed in a bottle warmer after one week of frozen storage (Sample 5), four and two miRNAs, respectively, had a significant expression level difference in all participants. After one week of storage at room temperature (Sample 3), the expression level of hsa-miR-378a-3p decreased in all participants, and hsa-miR-365a-3p and hsa-miR-365b-3p decreased in all participants except for Participant 6. hsa-miR-378a-3p has been found to function as a negative regulator of cytokine production involved in interleukin (IL)-33 production and the inflammatory response, and hsa-miR-365a-3p and hsa-miR-365b-3p have been found to function as negative regulators of interleukin-6 production ,24. AccoWhen we compared thawing by bottle warming (Sample 5) and microwave (Sample 6) after one week of frozen storage, we saw that the difference in miRNA expression was greater for microwave thawing. The number of miRNAs showing a significant difference in expression was higher when breast milk was frozen for four weeks (Sample 7) than one week (Sample 5). It can be inferred that the longer the frozen storage, the more the denaturation of miRNA. Additionally, a study by García-Lara et al. confirmed that the fat concentration and energy content decreased with the increase in the freezing time of breast milk . When a The composition and expression of miRNAs in HBM can differ due to various maternal, infant, or other factors. However, in this study, we investigated the change in, and stability of, miRNAs according to various storage and thawing conditions rather than the differences between each miRNA according to such factors. Although not in HBM, previous studies have confirmed the change in miRNAs, similar to our research. In a study by Kupec et al., serum miRNA was not affected by food intake or sample collection times. However, there was a significant difference in expression according to the storage temperature (−80 vs. 4 °C) . Glinge Our research has some limitations. First, only 10 nursing mothers participated in the study; therefore, the total number of participants was insufficient. Second, in this study, seven storage/treatment processes were compared, but if the storage period and processing time had been further subdivided, the results would have been more detailed. Third, more research is needed on how miRNAs are absorbed through the gastrointestinal tract and their functions after being ingested by infants. A study by Luo et al. found that the concentration of corn miRNAs in corn-fed animals remained stable in serum for seven days, indicating that dietary miRNAs are absorbed and distributed in the circulation . Zhang eIn conclusion, it is difficult to suggest that the various storage and thawing processes have a great influence on the overall expression of miRNA. However, in the case of refrigeration storage, miRNA changes were minimal compared to other storage and treatment methods. It was possible to suggest that refrigerated storage may be an ideal method to maintain a state of miRNA that is almost similar to that in direct lactation by minimizing changes in miRNAs compared to other storage methods.


BNT162b2 vaccine, developed by BioNTech and Pfizer ha recently approved for use in children aged 5 to 11 years. Recent data show evidence of safety on the administration and serious adverse events have been rarely reported. However, allergic systemic reactions could occur. In some cases, a correct allergic evaluation allows identifying patients at risk of developing an anaphylactic reaction. Risk assessment of allergic reactions to COVID-19 vaccines is useful to limit contraindications to vaccination and help to safely vaccinate people supposed to be at risk of allergic reactions. The Committee for Medicinal Products for Human Use of the European Medicines Agency  and AIFALow risk (Green zone)This zone includes patients with allergic rhinoconjunctivitis, well-controlled asthma, and non-anaphylactic allergic reactions to foods, insects, and latex.Action: Proceed with vaccination as usual , according to local guidelines. In children treated with allergen immunotherapy, COVID-19 vaccines should be administered at the interval of 7 days from subcutaneous immunotherapy Likewise, sublingual daily dose should be stopped 3 days before COVID-19 vaccine administration and restarted 7 days after .Medium risk (Yellow zone)History of anaphylactic reactions to foods, insects, latex.This zone includes the following clinical pictures.The following definition of anaphylaxis was considered in these cases: “Anaphylaxis is a serious systemic hypersensitivity reaction that is usually rapid in onset and may cause death. Severe anaphylaxis is characterized by potentially life-threatening compromise in breathing and/or the circulation, and may occur without typical skin features or cardio-circulatory shock being present” .Idiopathic anaphylaxis and exercise-induced anaphylaxis.Action: If the cause of anaphylaxis is confirmed, COVID-19 vaccination can be performed. Most international documents suggest , 20 to p..MastocytosisCOVID-19 vaccination is generally recommended in patients with mastocytosis. Safety measures, including pharmacological pre-medication and post-vaccination observation, should be considered in all patients with mastocytosis, depending on the individual risk and general conditions of every single case , 25.Action: In most cases, proceed with routine vaccination in an outpatient setting with emergency awareness and emergency medication available (24). Pre-medication with sedating or non-sedating H1 antihistamine and supervision for 60 minutes after vaccination should be considered.For the high-risk population : see red zone.Uncontrolled asthma.According to GINA document , asthma Action: Try to reach the best possible asthma control and then proceed to vaccination. If asthma is severe or only partially controlled, proceed with vaccination in a hospital setting with an observation of at least 60 minutes , 27. COVLarge local reaction to previous COVID-19 vaccination.Action: some documents , 14, 28 Action: Proceed with the vaccination  allergy is required , 16, 29.Generally, allergy assessment or teleconsulting are indicated in all above cases, if requested by pediatricians or vaccination centers. According to the allergy assessment or teleconsulting, vaccination should be performed in settings where symptoms of anaphylaxis can be recognized as early as possible and promptly treated with epinephrine and further emergency treatment or in hospital settings where it is possible to continue monitoring/treating of the patient in case of need , 17, 27.3)High risk (Red zone)Positive skin tests to excipientsPrevious allergic reaction to the specific vaccinePrevious severe allergic reaction to a vaccine component or drugs, including PEG or trometamolAction: perform allergy risk assessment; consider that sensitivity and specificity for a skin test to excipients is unknown, and the diagnostic utility of PEG and polysorbate allergy testing is uncertain . AccordiThis zone includes patients with the following:On the basis of international documents –14, recoEven if the risk of anaphylaxis to COVID-19 mRNA vaccines, particularly to BNT162b2 vaccine, in children aged 5–11 years seems very low , 29, nevPrimary and acquired immunodeficiency syndromes encompass several heterogeneous conditions ranging from mild oligosymptomatic defects to severe diseases, with the phenotype of overwhelming opportunistic infections, autoinflammation, or immune dysregulation. Therefore, it is challenging to provide a “one-fits-all” recommendation for all children and adolescents affected by immunodeficiency syndromes. However, in general, no absolute contraindication exists to vaccination with approved mRNA or viral vector inactivated vaccines in individuals affected by primary or secondary immune defects , 31.A consistent number of patients affected by inborn errors of immunity (IEI), including antibody deficiency syndromes under immunoglobulin replacement therapy, may mount an effective immune response against SARS-CoV-2 vaccines. Indeed, the type and strength of immune response could be very different accordingly to the type of IEI and the main immune compartment involved  but also the patient’s therapy regimen under which the COVID-19 vaccine is administrated –34. At tVery few data also exist about the risk/benefit ratio of anti-SARS-CoV-2 vaccination in children 5 to 11-years-old with previous MIS-C/PIMS-TS, although in theory even in these patients the benefits of COVID-19 vaccination outweigh the potential risks of triggering a relapse in the disease , 35. HowA teleconsulting visit with the referral pediatric immunologist is advisable in patients affected by IEI, acquired immunodeficiencies, and previous MIS-C/PIMS-TS.


Gastric cancer occupies the fourth highest morbidity rate of cancers worldwide. A higher incidence of gastric cancer had been found in East Asia compared to the other regions. Gastrectomy with radical lymph node dissection is the cornerstone of curative treatment for Stage III gastric cancer, and postoperative systemic chemotherapy with docetaxel, S-1 improved patients’ disease-free survival rates. However, advances in immunotherapy bring innovations in the management of patients with gastric cancer. The objective of this study was to explore the efficacy and safety of camrelizumab in combination with docetaxel + S-1, sequenced by camrelizumab + S-1 in stage III gastric cancer patients who are EBV positive, with defective mismatch repair and CPS ≥5.This prospective, open-label, single-arm trial was performed at Renji Hospital. In this study, a total of 70 adult patients aged 18–80 years with Stage III (PD-1+/MSI-H/EBV+/dMMR) gastric cancer confirmed by post-operative pathology will be enrolled after screening. Participants will receive the specific chemotherapy regimen until 1 year after the operation or until tumor recurrence or metastasis. The primary outcome is the 3-year disease-free survival rate measured by the Clopper-Pearson method and 95% confidence intervals. The secondary outcomes include overall survival, incidence and severity of adverse effects, and laboratory abnormalities. The data will be analyzed by the Kaplan-Meier method and log-rank test. The patients will be followed up every 3 months with imaging investigation until clinical remission.All participants will provide informed consent. The protocol has been approved by the Shanghai Jiaotong University School of Medicine, Renji Hospital Ethics Committee (KY2019-191). The results will be disseminated through peer-reviewed manuscripts, reports and presentations.ClinicalTrials.gov, identifier: ChiCTR1900027123. Registration date November 2019; first enrolment December 2019; expected end date December 2021; trial status: Ongoing.A clinical trial for Stage III (PD-1+/MSI-H/EBV+/dMMR) gastric cancer patients who accepted anti-PD-1 therapy combined with docetaxel + S-1 as the first-line treatment and explored improvements in three-year disease-free survival rate. The trial was in compliance with the applicable regulations and the Helsinki Declaration. Furthermore, the protocol has been approved by the Shanghai Jiaotong University School of Medicine, Renji Hospital Ethics Committee (KY2019-191) and registered in the Chinese Clinical Trial Registry (ChiCTR1900027123).The written informed consent will be provided before starting the trial and then the participants will be screened by a gastrointestinal surgeon to establish whether they meet the eligibility criteria. The qualified patients will be admitted within 4 weeks after surgery and begin treatment. During the follow-up period, the patients will be scheduled for a return visit every 3 weeks and an imaging evaluation every 4 weeks until the end of the treatment. They will be assessed for the primary and secondary outcomes after the intervention and during the 3-year follow-up. The end of the trial is defined as the last follow-up date for the last patient. Complete inclusion/exclusion criteria are detailed in Camrelizumab (200 mg) was administered intravenously for 30 min  every 3 weeks. Repeated every 21 days and 3 weeks for a course. The drug was discontinued 1 year after the operation or with tumor recurrence or metastasis, determined by imaging.Camrelizumab dosing adjustment will not be allowed. However, if the patient develops severe complications, the researchers can suspend the medication.Tegafur 80–120 mg (adjusted dose by the researcher according to the patient’s tolerance) d1-d14 taken orally followed by resting for 7 days. The cycle will be repeated every 21 days. And the course of treatment is 3 weeks and the drug will be discontinued 1 year after the operation or on tumor recurrence or metastasis, determined by imaging.According to the patient’s condition, the researcher will decide to adjust the drug dosage. Each dose was increased or decreased in the order of 40 mg, 50 mg, 60 mg and 75 mg. If the 40 mg dose is not tolerated, the drug administration will be discontinued.Docetaxel 40 mg/m2 (the dose is adjusted by the researcher according to the patient’s tolerance level) is given intravenously for 1 h every 3 weeks from the second cycle to the seventh cycle. The drug was discontinued 1 year after the operation or on tumor recurrence or metastasis, determined by imaging.3. If severe neutropenia occurs (<500/mm3 for 7 days or more), a reduction in dosage is recommended in the next course of treatment. If there is no improvement, further dose reduction or discontinuation will be recommended.The white blood cell count should be monitored regularly during the treatment. Docetaxel will only be given to patients with a neutrophil count greater than 1500/mmThe treatment protocol described above will be given continuously for 1 year after the operation or until tumor recurrence or metastasis, determined by imaging. Safety visits and follow-ups will be performed after treatment. Cancer progression will also be followed up after the end of treatment for subjects who have non-disease progression and have completed the treatment.To observe and evaluate the 3-year disease-free survival (DFS) rate in patients with stage III gastric cancer (PD-L1 + / MSI-H / EBV +/dMMR).1.To observe and evaluate the overall survival (OS) in patients with stage III gastric cancer (PD-L1 + / MSI-H / EBV +/dMMR).2.To observe and evaluate the safety and adverse events in patients with stage III gastric cancer (PD-L1 + / MSI-H / EBV +/dMMR).The sample size calculation will be based on the 3-year disease-free survival (DFS) rate of the included patients. The study was designed as a prospective single-arm trial, focusing on patients with stage III gastric cancer (PD-L1+/MSI-H/EBV+/dMMR). The sample size was estimated based on the following assumptions. According to previous literature, standard treatment has a 3-year disease-free survival rate of 67% . We estiThe statistical analysis will be based on intention-to-treat (ITT) data or safety analysis set (SAT). The demographic characteristics of each group will be compared using the independent t-test and the chi-squared test for continuous variables and dichotomous variables. Measurement data will be expressed as mean, standard deviation, median, maximum, minimum. Enumeration data and ranked data will be expressed as a constituent ratio, rate, confidence interval. All statistical data will be analyzed by SPSS. The disease-free survival (DFS) rate will be estimated by Clopper-Pearson and the 95% confidence interval will be calculated. The overall survival (OS) rate will be analyzed by the Kaplan-Meier method and log-rank test. TTR is described by mean, standard deviation, median, maximum and minimum. Tumor markers associated with anti-PD-1 antibodies, such as levels of PD-L1 or other biomarkers in tumor tissue, will also be analyzed using descriptive statistics.Data collection will include patient demographics and disease subclassification based on the Montreal Classification, such as clinical phenotype, disease location and surgical history. The collected patient demographic data will include the identity card number, sex, date of birth, age at diagnosis, duration, nationality, height and weight. Laboratory tests  should be completed within 14 days prior to the first treatment cycle Tumor imaging  and echocardiography will be completed within 28 days. The participants and specific follow-up staff will screen for infectious diseases such as HIV, hepatitis B virus and hepatitis C virus.Follow-up will be conducted by specific staff for all the participants enrolled in this study. The follow-up program will be established after the surgery and will be performed annually thereafter. In principle, all the examinations were suggested to be carried out in Renji Hospital. If the patients are reexamined in other hospitals, grade III, first-class hospitals are recommended and the follow-up staff will track and record the results. The tests included: (1) Adverse reactions; (2) Physical examination: superficial lymph nodes, abdomen, metastatic signs and so on; (3) Peripheral blood routine examination: HB, RBC, WBC, LYM, NEU, NEU%, PLT; (4) Blood biochemistry: albumin, prealbumin, total bilirubin, AST, ALT, creatinine, urea nitrogen, fasting blood-glucose; (5) Serum tumor markers: CEA, CA19-9, CA72-4; (6) Abdominal and pelvic CT.All participants should be evaluated for safety and adverse events within 90 days of the last administration, and the patients’ condition should be recorded. The following examinations should be performed on the 90th day of follow-up after treatment. (1) ECOG score standard; (2) Blood routine examination: hemoglobin, red blood cell, white blood cell, neutrophil count, and so on; (3) Routine urine test; (4) Blood biochemistry; (5) Evaluation for adverse events.Combined with these results, the researchers assessed and recorded the postoperative survival status of all patients to determine tumor recurrence or metastasis. If patients refuse to be followed up according to our protocol, they will be recorded as missing cases and analyzed with those patients who met the study criteria at the end of the study.After completion of the 90-day follow-up after discontinuation, the patients will enter the survival follow-up period. The participants, their family members, or local physicians are interviewed by telephone at least every three months. Follow-up staff will collect information on survival (date of death and cause of death) and post-treatment until either the end of death or the subject were lost to follow-up or the study was terminated by the researchers .(1)The original medical condition/disease is aggravated prior to entry into the clinical trial;(2)Any new adverse medical condition ;(3)Abnormal laboratory results with clinical significance.The researchers will keep detailed records of any AE in the subjects.An adverse event (AE) is any undesirable clinical event that occurs to a study participant during the study but does not necessarily have a causal relationship with the treatment. AEs may include any unexpected adverse symptom, sign, laboratory abnormality, or disease. For example:Severe adverse event (SAE) refers to a medical event occurring during a clinical trial that requires hospitalization or prolonging the current hospitalization, causes persistent or significant disability, affects working capacity, endangers life, or results in congenital malformation/birth defect and other medical events. Hospitalization events exclude rehabilitation facilities, nursing homes, admission to a routine emergency room, day surgery (outpatient/ambulatory) and social reasons. Furthermore, if hospitalization or prolonged stay is unrelated to the exacerbation of AE, these events should not be included under SAE, such as annual physical examination, admission for pre-existing disease and so on. First, the subjects should sign an informed consent effective until 90 days after the last drug administration. The researchers should keep detailed records of symptoms, severity, association with the experimental drug, time of occurrence and treatment, measures that have been taken, time and manner of follow-up, and outcome. Any severe adverse event should immediately be reported by completing the SAE Report Form provided by the CFDA. Then the researcher should report the condition to the provincial, autonomous regional and municipal medical products administration, CFDA, and health administrative departments within 24 h and timely report to the ethics committee. SAEs occurring after 90 days following the last drug administration are generally not reported unless suspected to be related to the trial.All AE/SAE should be followed up until disappearance, remission to baseline level, reaching a stable state, or a reasonable explanation .The study has been approved by Shanghai Jiaotong University School of Medicine, Renji Hospital Ethics Committee (KY2019-191) and registered in the Chinese Clinical Trial Registry (ChiCTR1900027123). The trial will be conducted according to the principles of the Declaration of Helsinki and in accordance with Good Clinical Practice (GCP) standards. Informed consent will be obtained by the coordinators or researchers associated with this protocol. It details the drug use, the course of the study, and the risks of the study are fully explained. Written informed consent must be obtained before proceeding with the study. Throughout the course of the clinical study, participants could withdraw their consent at any time. All the personal information about the subjects will be kept strictly confidential.Results will be published in the open-access peer-reviewed medical literature as well as submitted for presentation at national and international meetings.For decades, chemotherapy has remained the mainstay of effective treatment for advanced GC . For morP < .001).Docetaxel, a cytotoxic anti-cancer agent, is a notable treatment option available for patients with advanced gastric cancer . DocetaxAlthough the DS regimen assessment results reported significant benefits for the patients, the advances in immunotherapy bring considerable changes. The “FORCE1” study  suggestsBased on the results of these clinical studies, although guidelines recommend camrelizumab as a third-line treatment for gastric cancer, we would like to explore its efficacy and safety as a first-line treatment for patients with specific phenotypes of gastric cancer. To our knowledge, these strategies have not been compared in a clinical trial to date. Therefore, this study aims to investigate if camrelizumab, in combination with docetaxel + S-1, sequenced by camrelizumab + S-1treatment, can improve the outcome of patients who are EBV positive with defective mismatch repair and CPS ≥5.However, this research has some shortcomings. Firstly, the number of eligible patients is relatively small. It may take long time and much money to enroll all patients. Secondly, the study we conducted is exploratory and subsequent large-scale clinical studies are still needed to provide higher level of evidence. Finally, the trial is open label so subjects and researchers can know what procedure was undertaken to treat.The final review paper will be submitted to a peer-reviewed journal for publication and presented at relevant conferences .


Gastroenteropancreatic neuroendocrine carcinoma (GEP-NEC) is a group of rare but highly aggressive malignancies. The standard chemotherapy regimens composed of etoposide and cisplatin/carboplatin (EP/EC) are of limited efficacy. This prospective, multicenter, phase II study is conducted to explore the effectiveness and safety of first-line anti-PD-1 antibody (camrelizumab) combined with chemotherapy in advanced GEP-NEC patients.2, administered intravenously on days 1–3; cisplatin 75 mg/m2, administered intravenously on day 1 or carboplatin area under the curve 5 mg/ml per min, administered intravenously on day 1). All patients were naïve to systemic therapy in the advanced setting. The primary endpoint is a 6-month progression-free survival (PFS) rate. The secondary endpoints are objective response rate, PFS, overall survival and adverse reactions.Patients with unresectable or metastatic GEP-NEC will receive camrelizumab combined with standard first-line chemotherapy every 3 weeks (camrelizumab 200 mg, administered intravenously on day 1; etoposide 100 mg/mThis is the first study to investigate the therapeutic potential of camrelizumab plus chemotherapy for advanced GEP-NEC. It is expected that this trial will propose a new and effective treatment strategy for GEP-NEC in the first-line setting.http://www.chictr.org.cn, identifier ChiCTR2100047314.This trial is registered at the Chinese Clinical Trial Registry June 12, 2021. In recent decades, the incidence of GEP-NEC has been reported to have increased rapidly  are classified into well-differentiated neuroendocrine tumors (NETs), poorly differentiated neuroendocrine carcinomas (NECs), and mixed neuroendocrine–non-neuroendocrine neoplasms  have shown remarkable responses in various solid malignancies, especially in tumors with a high mutational rate, deficient mismatch repair (dMMR) status, or positive PD-L1 expression. Preclinical studies have shown a high tumor mutational rate (TMB) and high frequency of positive PD-L1 expression in neuroendocrine carcinomas, which indicates immunogenic and promising responsiveness to ICIs –11. In sInspired by preclinical work and immunotherapy in small cell lung cancer, we conducted this study to explore the benefit of an anti-PD-1 antibody, camrelizumab, combined with chemotherapy as first-line treatment in advanced GEP-NEC patients.This prospective, multicenter, single-arm, phase II study is designed to investigate the efficacy and safety of first-line camrelizumab combined with standard chemotherapy in patients diagnosed with advanced GEP-NEC. The study protocol is formulated in accordance with the SPIRIT  statement . The triThe main inclusion criteria are pathologically confirmed local advanced or metastatic GEP-NEC according to the 2019 WHO Classification of Tumors of Digestive System; primary tumor located in the gastrointestinal tract, pancreas, or bile duct; without previous first-line systemic antitumor therapy since advanced GEP-NEC diagnosis; at least a 6-month interval between cancer recurrence or metastasis and the end of adjuvant chemotherapy for patients who received prior radical surgery and adjuvant chemotherapy; aged 18 to 75 years; life expectancy of over 3 months; ECOG performance status 0 or 1; at least one measurable lesion according to the Response Evaluation Criteria in Solid Tumors (RECIST) version 1.1 . The total dose of cisplatin can be administered in 3 days. Treatment continues until disease progression, intolerable toxicity, patient refusal, or investigator decision. Patients will receive maintenance treatment of camrelizumab  if disease stable or remission after 6 cycles of combination therapy. The treatment duration of camrelizumab is at most 24 months. The study design is shown in Patients eligible for participation and having signed informed consents will receive camrelizumab and EP/EC chemotherapy every 3 weeks  based on RECIST version 1.1. Patients who get radiological disease progression (PD) could be permitted to continue treatment if investigators judge the clinical benefit from the continued treatment . Moreover, these patients should receive imaging examination again to determine whether they get confirmed PD after 4 weeks according to immune-related RECIST (irRECIST) . PFS is Serum and tissue samples will be collected before treatment start for cytokines, TMB, microsatellite instability, and PD-L1 analyses. All adverse events will be monitored during the treatment period, which will be assessed according to the Common Terminology Criteria for Adverse Events (CTCAE) version 5.0. The quality of life will be evaluated at baseline and prior to every imaging evaluation, using the validated European Organization for Research and Treatment of Cancer Quality of Life Core Questionnaire 30 (EORTC QLQ-C30) .The primary endpoint of this study is the 6-month PFS rate. Previous convincing reference data are very limited. Due to the rarity of GEP-NEC, there is no published large-scale phase III study about first-line treatment. Due to similarities in clinical, pathological, and prognostic characteristics between GEP-NEC and small cell lung cancer (SCLC), the first-line chemotherapy for GEP-NEC is recommended based on guidelines of SCLC and some small sample size and retrospective studies of GEP-NEC. Based on phase III results of SCLC and retrospective data of GEP-NEC, the reported 6-month PFS rate is about 22% for reference. In our study, the expected 6-month PFS rate is set at 40%. To achieve 80% power with a two-sided alpha level of 0.05, we need to enroll 44 patients. Considering a dropout rate of 10%, a total of 49 patients are required. SPSS Statistics version 22.0  will be used to perform all the statistical analyses.GEP-NECs are characterized by rapid deterioration and poor prognosis. The current recommended first-line platinum-based chemotherapy is of limited efficacy. A new treatment strategy is in urgent need for GEP-NECs.As aforementioned, studies have revealed that NECs have features of high TMB and PD-L1-positive expression, indicating immunogenic and promising responsiveness to immune checkpoint inhibitors –11. In aAt present, there are a few studies investigating the value of immunotherapy in refractory advanced gastrointestinal NECs , 16. TheThere are several limitations in our study, including lack of a control group, a small sample size, and uncertain predictive biomarkers of therapeutic effect. Besides, as this is an investigator-initiated trial with limited budgets, the histological and radiological evaluations are performed in local centers without centralized assessment.The original contributions presented in the study are included in the article/Supplementary Material. Further inquiries can be directed to the corresponding author.This study was reviewed and approved by Chinese Ethics Committee of Registering Clinical Trials. The patients/participants provided their written informed consent to participate in this study.DC and XL are responsible for the trial design. XL is responsible for recruitment and patient information. QM and CC are responsible for patient follow-up. QM and HL are responsible for data collection. XL and CC are responsible for statistical analysis. XL, QM, and DC drafted and revised the manuscript. All authors contributed to the article and approved the submitted version.This study is supported by the Sichuan Province Science and Technology Support Program (2021YFS0047). The main role of the funding is providing financial support for this study.We gratefully acknowledge the understanding and cooperation of enrolled patients.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.


Bleaching agents can cause certain surface alterations on the enamel, such as depressions, surface porosity, and surface irregularities; this makes the dentin more susceptible to post-operative tooth sensitivity (PoS). In addition, the presence of flawed or leaky restorations, gingival recession, or defects in the cementoenamel junction can also cause severe tooth sensitivity post tooth bleaching.Hence, the current study aimed to perform a systematic review to determine the efficacy of various desensitizing agents (DA) in managing post-operative tooth sensitivity and color alteration when applied before in-office bleaching procedures. Randomized clinical trials were searched to conduct an SR where the post-operative tooth sensitivity was evaluated after in-office bleaching with various desensitizing agents used before the procedure. Post-operative pain assessment was measured using the Visual Analogue Scale (VAS). Outcomes were evaluated up to an hour and 24 hours post the procedure. Out of 163 articles, only 13 titles were selected that met the eligibility criteria. Eight hundred and forty-one adult patients with vital pulp status were included. The participants receiving prior desensitizing agent applications reported significantly lower pain scores in the VAS reports. The most significant reduction of post-operative sensitivity was observed in the immediate (up to an hour) and 24 hours after the in-office bleaching. The popular desensitizing agent that could manage post-operative tooth sensitivity (TS) is 5% potassium nitrate and 2% sodium fluoride used before the in-office bleaching procedure.  A person's smile is one of the most striking features of their appearance; hence people want their smiles to look perfect. The hue of the teeth has a significant cosmetic value when it comes to smiling. Dark, brownish discolorations can be caused by pigments from beverages such as coffee, tea, tar from smoking, or constant betel nut chewing. In addition, excessive consumption of oranges, carrots, or chocolates may lead to food-related stains . ExternaExternal tooth bleaching is a safe procedure that allows tooth whitening to be retained for several years. In-office bleaching or chair-side techniques are entirely dependent on the dental professional. Almost all the techniques involve applying a hydrogen peroxide gel of concentrations ranging from 25% to 40% [The presence of hypersensitivity is an indication of inflammatory changes in the dental pulp. Histologically, only a few pulps have demonstrated symptoms of inflammation post-in-office bleaching after the usage of hydrogen peroxide gels . Yet, toAt present, the mechanism resulting in tooth sensitivity followed by bleaching is unclear. The literature explains this is a result of hydrodynamic theory . AccordiAccording to various studies, it has been shown that bleaching agents can cause alterations on the dental surface, probably due to the low pH and the presence of chelating agents . BleachiFocused questionDo desensitizing agents in randomized clinical trials help manage post-operative tooth sensitivity and color alteration when used before the in-office bleaching procedures?Search strategyThe literature search was carried out for appropriate studies such as PubMed-MEDLINE, Scopus, and the Cochrane Central Register of Controlled Trials. Furthermore, sources like Google Scholar, and IndMed, along with the journal of web databases, were searched from the year 2009 up to April 30, 2021, lacking any limitations on language. In addition, ongoing clinical trials, including The National Institutes of Health Trials and Clinical Trial Registry India, were explored. Finally, the authors were contacted via mail for any grey literature. The search terms used by the authors were: “tooth bleaching agents,” “dentin desensitizing agents,” and “pain measurement/VAS.”Eligibility criteria(A) Randomized controlled clinical trials (RCTs) of vital in-office bleaching in a single or multi visits treatment using desensitizing agents. (B) Adult patients indicated for tooth whitening to improve the tooth shade were included. (C) Patients with fluorosis, tetracycline stains, caries, and restored or endodontically treated teeth were excluded. (D) A visual analogue scale (VAS) was used for pain measurement.Screening and selectionThe articles were screened by title and consequently by abstract by two reviewers (KK & AST) independently. The search did not include narrative or historical reviews, letters, or case reports. Full-text reading was conducted only on those articles that presented the keywords either in the title or abstract, or both. To screen the full text for the eligibility of the papers sans abstract but titles evincing of objectives which were related to the eligibility criteria were selected. After the primary selection of papers, the two reviewers thoroughly read the full-text articles (KK & AST). Post this, the articles that fulfilled all the eligibility criteria of this review were processed for further data extraction. Finally, both the reviewers (KK & AST) went through all the selected article’s references to search for additional pertinent articles. All disagreements were resolved through a discussion amongst the reviewers. Yet, any persisting disagreements were judged by the third reviewer and were considered conclusive. Quality assessmentTwo reviewers (KK & AST) analyzed the risk of bias in every trial using the Cochrane Collaboration’s tool for assessing the risk of bias , with thData extractionTwo authors (KK & AST) made use of the Microsoft Excel software  and specially designed data extraction forms to independently extract data. The authors maintained discussion among them to help resolve any disagreement. For each selected study, the following data were then excerpted from a standard form (when available). These included author and year of publication, study design, sample size, participant’s characteristics, desensitizing agent, comparator, hydrogen peroxide (%), attrition, analgesics, attrition, and author’s conclusion.OutcomesPost-operative sensitivity experienced by the participants during and after the in-office bleaching procedure was the primary outcome. We considered the postoperative tooth sensitivity after 1 hour and 24 hours. The varying responses regarding different desensitizing agents were recorded.The secondary outcome of this review was to assess the difference in tooth color when a desensitizing agent was used before the bleaching procedure. We considered clinical trials measuring the color change in delta shade guide units or color change in delta E (CIEL*a*b*) after bleaching.ResultsThe reporting is based on the Preferred Reporting Items for Systematic Reviews and Meta-Analyses (PRISMA) guidelines . FigurePrisma guidelineThe terms “tooth bleaching agents,” “dentin desensitizing agents,” and “pain measurement/VAS” were searched for in various databases. The PubMed-Medline, Scopus, Cochrane Library, and additional sources identified 163 search results, out of which 80 were duplicates. The remaining 83 unique studies were screened for the titles and abstracts, and 33 articles were selected for full-text screening. Thirteen articles -21 that Study design and subject characteristicsUsing the Cochrane Collaboration’s tool for assessing the risk of bias with the consensus resolving any arguments, two reviewers (KK & AST) analyzed the risk of bias in every trial.All selected studies were randomized clinical trials. Eleven studies ,17,18,20The review included 841 adult patients with vital pulp status who received an intervention of a DA before the in-office bleaching procedure. The in-office bleaching procedure was done in multiple sessions. The in-office bleaching procedure and concentration varied between the included studies. One study  used 28%In the study by RK Henry et al., sugar-free chewing gum containing a desensitizing agent was given to the participants to chew for seven days before the in-office bleaching procedure . CH ThieThe post-operative pain was evaluated as post-operative tooth sensitivity as risk and post-operative tooth sensitivity as intensity. The former evaluated the incidence of experiencing post-operative tooth sensitivity, whereas the latter evaluated the intensity of the tooth's sensitivity. The results extracted as tooth sensitivity experienced by the participants as the risk were included in the systematic review.Meta-analysis was not possible due to varied outcome units and heterogenous studies. Forest plots are not added since a meta-analysis was not done. A pooled estimate cannot be calculated with different outcome units and heterogenous studies.Pain scores immediately after in-office bleachingPain scores immediately after the in-office bleaching procedure were investigated in 11 studies ,13,15,16Pain scores at 24 hoursPain scores at 24 hours after the in-office bleaching procedure were investigated in nine studies ,16,18-21DiscussionThe present systematic review included 13 RCT’s examining the effectiveness of various desensitizing agents and placebo with preceding application to the in-office bleaching procedure -21. ThisImmediately after the in-office bleaching procedure, a significant reduction in the incidence of post-operative sensitivity was experienced with the use of 5% KNO3 with 2% NaF (Desensibile KF 2%) ,11,19-21Post-operative tooth sensitivity is a common problem experienced by patients after vital bleaching. Since the tooth bleaching procedure usually requires multiple sessions to achieve the required shade, sore tooth sensitivity experienced by the patients during the first visit could be a factor for patients to be discouraged from continuing the treatment -26. SeveTooth sensitivity should be distinguished from PoS experienced after the tooth bleaching procedure. Investigators have theorized that there could be some degree of pulpal inflammation associated with the vital bleaching procedure; otherwise, the PoS experience is thought to be related to the hydrodynamic theory ,26,30,31In a study by A Reis et al., the use of 5% KNO3 with 2% NaF did not produce any significant change immediately after the in-office bleaching procedure. Still, it was observed that during the first 24 hours, there was a remarkable reduction in the post-operative PoS intensity experienced in the experimental group in comparison to the placebo group . During The clinical significance of applying the desensitizing agent before the in-office bleaching procedure is to prepare the tooth surface to be resistant to TS in an action of the higher concentration of the bleaching agents, especially in cases of in-office bleaching procedure. A higher concentration of bleaching agent is applied for short periods, mostly three 15 minutes sessions for a total of 45 minutes. This increases a higher risk of the incidence of post-operative TS experienced by the patient ,26,30,31Regarding the color change, efficacy alteration due to the prior application of desensitizing agents was found to be void in the 14 studies assessed in this systematic review -16,18-21Bleaching agents can cause certain surface alterations on the enamel, such as depressions, surface porosity, and surface irregularities; this makes the dentin more susceptible to post-operative tooth sensitivity (PoS). In addition, the presence of flawed or leaky restorations, gingival recession, or defects in the cementoenamel junction can also cause severe tooth sensitivity post tooth bleaching. Hence, the current study aimed to perform a systematic review to determine the efficacy of various desensitizing agents (DA) in managing post-operative tooth sensitivity and color alteration when applied before in-office bleaching procedures. The most significant reduction of post-operative sensitivity was observed in the immediate (up to one hour) and 24 hours after the in-office bleaching. Therefore, more studies are required for the prior use of dentifrices to manage post-operative sensitivity after in-office bleaching. The popular desensitizing agent that could manage post-operative TS is 5% potassium nitrate and 2% sodium fluoride used before the in-office bleaching procedure. However, newer desensitizing agents must be researched with improved randomized clinical trials for a better assessment of their efficacy.


As the COVID-19 spread over the globe and new variants of COVID-19 keep occurring, reliable real-time forecasts of COVID-19 hospitalizations are critical for public health decisions on medical resources allocations. This paper aims to forecast future 2 weeks national and state-level COVID-19 new hospital admissions in the United States. Our method is inspired by the strong association between public search behavior and hospitalization admissions and is extended from a previously-proposed influenza tracking model, AutoRegression with GOogle search data (ARGO). Our LASSO-penalized linear regression method efficiently combines Google search information and COVID-19 related time series information with dynamic training and rolling window prediction. Compared to other publicly available models collected from COVID-19 forecast hub, our method achieves substantial error reduction in a retrospective out-of-sample evaluation from Jan 4, 2021, to Dec 27, 2021. Overall, we showed that our method is flexible, self-correcting, robust, accurate, and interpretable, making it a potentially powerful tool to assist healthcare officials and decision making for the current and future infectious disease outbreaks. During the continuous spread of COVID-19, many variants  of COVID-19 emerge, leading to drastic surges in hospital admissions and shortages in health care resources2. Therefore, an accurate hospital admissions forecasting model is crucial to assist hospitals and policymakers with the possibilities and the timings of rapid changes, so as to further respond to and prepare for future COVID-19 spread.COVID-19 (SARS-CoV-19), an acute respiratory syndrome disease caused by a coronavirus, has spread worldwide causing over 120,695,785 reported cases and 4,987,755 reported deaths3 has been collecting hospitalizations predictions from various research teams and making ensemble and baseline predictions since May 2020. According to the weekly COVID-19 forecasts submissions compiled by CDC3, machine learning5 and compartmental models7 are the most popular forecasting approaches5. For example, Rodríguez et al.4 use a neural network architecture incorporating COVID-19 time series, and mobility information as inputs, whereas Jin et al.5 utilize attention and transformer models to compare and combine past COVID-19 trends for future forecasts. On the other hand, Vespignani et al.6 and Kinsey el al.7 adapt SEIR  as the baseline structure and combine different exogenous variables including spatial-temporal and mobility information to build more sophisticated models to capture COVID-19 disease dynamics and forecast hospitalizations. Meanwhile, statistical and data-driven models, taking advantage of COVID-19 public search information for hospitalizations predictions, have not drawn much attention.The Centers for Disease Control and Prevention (CDC)13, HIV/AIDS14, dengue15, etc. For instance, Google Flu Trend (GFT)8, a digital disease detection system that uses the volume of selected Google search terms to estimate current influenza-like illnesses (ILI) activity, was the first among many studies to demonstrate how big data and public search behavior compliment traditional statistical predictive analysis. Later, many studies proposed different methodologies to improve upon GFT and provide more robust and accurate real-time forecast estimates of influenza activity in the US, including machine learning models10, statistical models9, ensemble models11. Online search information from other sources, such as Yahoo12 and Baidu13, have demonstrated their predictive power for influenza tracking as well.For the last decade, numerous studies have suggested online search data could be a valuable component to monitor and forecast infectious diseases, such as influenza16. VECM combines Google search data and healthcare chatbot scores and produces COVID-19 hospitalizations predictions in Greater Charlotte market area, where the Google search queries are selected based on existing literature, and filtered through hand-craft procedures. Yet, their selection approach highly depends on literature considered and healthcare chatbot scores can be limited in-access across different geographical areas. So far, none of the existing Internet-search-based methods provide robust weeks-ahead hospitalisation forecasts for different geographical areas in the United States that fully utilize COVID-19 related online search queries and account for their sparsity and noisiness.On the other hand, to best of our knowledge, the only hospitalizations prediction model that is based on the Internet search data is a vector error correction model (VECM) proposed by Turk et al.In this paper, we propose the ARGO inspired model (ARGO), which combines online search data and lagged COVID-19 related information in a L1-norm penalized linear regression model to produce real-time 1–2 weeks ahead national and state-level hospitalizations predictions over 12-month out-of-sample evaluation period in the United States. Our results show that ARGO, leveraging predictive information in the selected interpretable Google search queries, is able to largely outperform baseline models and all other public available models from CDC’s COVID-19 forecast hub, which implies potential in ARGO to help with urgent public health decisions on health resources allocations.We propose a LASSO-penalized linear regression model, efficiently combining Google search information and COVID-19 related time series information, for United States national and state-level 1 and 2-week-ahead COVID-19 hospitalization forecasts, with dynamic training and rolling window prediction. We identified 11 important query terms from the initial large pool of relevant Google search queries from previous studies, and calculated the optimal lag between search data and COVID-19 hospitalization. The important terms consist of symptom-related terms and general COVID-19 related terms, possessing strong representation of upcoming COVID-19 surges, which enhances the robustness and the interpretability of our forecasts. Finally the selected Google search data, vaccination rate, past COVID-19 cases and hospitalizations are all combined to produce both national and state-level forecasts. Our selection of search queries is systematic and comprehensive, and our method is intentionally straightforward and unified to prevent overfitting. Numerical comparisons show that our method performs competitively with other publicly available COVID-19 forecasts. This study further emphasizes the general applicability and the predictive power of online search data for various tasks in disease surveillance.17 , the mean absolute error (MAE) and the Pearson correlation coefficient (Cor) of one-week-ahead and two-week-ahead predictions (detailed in the Methods section). All comparisons are based on the original scale of the ground truth of new hospital admissions released by US Department of Health and Human Services (HHS)Figure Figure 3, the official CDC COVID-19 Forecasting page. COVID-19 Forecast hub collects predictions from all contributing teams that submit hospitalization forecasts since January 15th, 2020. The COVIDhub-baseline is a persistent method that uses latest daily observation as future daily predictions3. The COVIDhub-ensemble uses medians of hospitalizations predictions submitted to the COVID-19 forecast hub as its “ensemble” forecasts. We also consider the top 5 CDC published teams for prediction comparison, among over 100 teams submitted to CDC after filtering out missing values in reporting periods and/or states. Due to the space limit, we only include COVIDhub-baseline and COVIDhub-ensemble in the main text, leaving the full comparison of the top individual teams from CDC’s COVID-19 forecast hub in Supplementary Information .To further demonstrate the prediction accuracy and robustness of ARGO, we collected hospitalizations predictions of the two benchmark methods (COVIDhub-baseline and COVIDhub-ensemble) from COVID-19 forecast hub18). For fair comparisons, all models were trained on a 56-day rolling window basis. Retrospective out-of-sample predictions of daily national hospitalizations were made every week from January 4, 2021 to December 27, 2021 by the three models and were then aggregated into weekly new hospitalizations.National one-week-ahead and two-week-ahead predictions of new hospitalizations were generated using (i) ARGO inspired model, (ii) persistence (naive) model and (iii) AR7 model. The naive method simply uses past week’s hospitalizations as current week’s forecasts, without any modeling effort. AR7 is an autoregressive model of lag 7  during the same period of January 4, 2021 to December 27, 2021.Table To further analyze the importance of four features used in ARGO , we conduct sensitivity analysis by excluding one feature at a time in state-level predictions. Table S5 shows the comparison between ARGO (full model) and each sub-model (excluding one feature) in the three error metrics for 1 and 2 weeks-ahead state-level predictions. Our full model ARGO outperforms all other simplified models throughout the forecasting horizon. In particular, the model that excludes Google search terms has relatively higher MSE and MAE compared against other submodels, suggesting the importance of Google search terms in the method. Also, Google search information alone is not predictive enough, as all other submodels (with Google search information) cannot beat ARGO (full model) in any of the error metrics, indicating the importance of information combination.9, which reduces the chance of overfitting and again demonstrates ARGO’s robustness and flexibility.The ARGO inspired model combines autoregressive COVID-19 related information and online search data. It is able to produce accurate, reliable real-time hospitalizations predictions at both national and state-level for 1–2 weeks ahead predictions. The state-level real-time hospitalizations predictions made by ARGO could help local public health officials make timely allocation decisions of healthcare resources, such as ventilator, ICU beds, personal protective equipment, personnel, etc, as well as account and promptly prepare for future surges of COVID-19 pandemics caused by new virus variants. Moreover, ARGO allows us to transparently understand how Google search information and historical COVID-19 information complement one another. If any of the COVID-19-related information and Google search information is missing, the error of ARGO’s prediction will increase as shown in Table S5 in SI, indicating all features help improve predictions’ accuracy. Past week’s COVID-19 hospitalization contributes positively to the current national COVID-19 hospitalization trend predictions , while the effect of past week’s vaccination rate is negative, which indicates that the current trend is likely to follow from the past week’s growth/drop, and vaccination rates potentially lead to drops in hospitalization. Figure S2a and S2b indicate a time-varying relationship between COVID-19 hospitalization trend and people’s search behavior for COVID-19 related terms since coefficients of Google search queries are changing over time. Furthermore, our ARGO hospitalization prediction model is a straightforward adaptation from the original ARGO model for influenza19 and moving average smoothing applied to Google search data are able to minimize the risk caused by such noisiness, and help ARGO produce robust output. To further account for the instability in the state-level Google search queries, the query terms are identified using the national level data where the search frequencies are more representative with lower noise and higher stability. ARGO selects the most representative search queries according to their Pearson correlation coefficients with hospital admission. In addition, the national level search frequency is directly used as input features for state-level predictions. An optimal delay between the search data and the hospital admissions is also identified for each query term. All together, ARGO is able to overcome the sparsity issues of Google search queries and produce robust future estimations .All data used in this study are publicly available from respective data sources outlined here. For completeness, the datasets analysed during the current study and the code are also deposited in the Harvard dataverse repository, DOI: 10.7910/DVN/S7HOTD. All analyses, including the generation of all figures, were performed with the R statistical software21, percentage of fully vaccinated population from Centers for Disease Control and Prevention (CDC)22 which is averaged among percentages of fully vaccinated population in all states and COVID-19 confirmed new hospital admissions from HHS17. The data sets were collected from July 15, 2020 to January 15, 2021.We used reported COVID-19 confirmed incremental cases from JHU CSSE data23. We obtained online search data from Google Trends23 for the period from July 15, 2020 to January 15, 2021. To retrieve the time series search frequencies of a desired query, one needs to specify the query’s geographical information and time frame on Google Trends. The returned frequency from Google Trends is obtained by sampling all raw Google search frequencies that contain this query23. The detailed data collection procedure and subsequent data pre-processing (introduced in sections below) are illustrated in the flowchart . In step 1 , to curate the pool of potentially predictive queries, we first started with 129 influenza (flu) related queries based on prior studies25. Then, we changed “Influnza” and “Flu” keywords to “Coronavirus” and “COVID-19”, respectively. We also added in additional COVID-19 specific search terms from Google Trends Coronavirus Story page26. Lastly, for each query, we also included its top “related queries and topics” based on Google Trends website23. Finally, we end up with 256 COVID-19 related queries (Table S1). Next two sections will describe the subsequent data cleaning and pre-processing in detail, illustrated in step 2 and step 3 of Figure S1.Google Trends provides estimated Google search frequency for the specified query term23 are observed to be unstable and sparse19. Such instability and sparsity can negatively affect prediction performance of linear regression models which are sensitive to outliers. To deal with such outliers in Google search data, we used an IQR filter19 to remove and correct outliers on a rolling window basis. The search data that is beyond 3 standard deviation from the past 7-day average are examined and removed19, which is also shown in step 2 (orange highlighted box) first sub-step in Figure S1.The raw Google search frequencies obtained from Google Trends19 to filtered Google search frequencies to match the trends of national hospitalizations. For each query, a linear regression of COVID-19 new hospitalizations is fitted against lagged Google search frequency, considering a range of lags (from 4 to 35). The lag results in lowest mean square error is selected as the optimal lag for that query. The data used to find optimal lags are from August 1, 2020 to December 31, 2020, and this is shown in the second sub-step in step 2 .The trends of Google search frequencies are often a few days ahead of hospitalizations, indicating that the search data might contain predictive information about hospitalizations. Figure After applying optimal lags to the 256 COVID-19 related terms, we further selected the queries that have correlation coefficients with national COVID-19 hospitalizations larger than 0.5 for the period from August 1, 2020 to December 31, 2020. We applied 7-day moving average to further smooth out weekly fluctuations in the selected Google search queries. All three procedures above are shown in step 3 in Figure S1, serving as the final step of the entire data pre-processing procedure. Table r on day t; k on day t; t of region r; t of region r; t (i.e. t being Monday). Standing on day T, to predict l-day-ahead hospital admission of state r, r; k; l-day-ahead predictions of region r, Let T of region r, respectively.With the formulation above, on each Monday from January 4, 2021 to December 27, 2021, we iteratively trained our model and made national and state-level retrospective out-of-sample hospitalizations predictions up to 14 days into future. We then aggregated daily predictions into one-week-ahead and two-week-ahead predictions. For example, Root Mean Squared Error (RMSE) between a hospitalization estimate This study did not involve human participants, data, or tissue. It was conducted using only aggregated and anonymized data. Institutional review board approval was not required. All methods were carried out in accordance with relevant guidelines and regulations.Supplementary Information.


A systematic literature review was conducted up to 15th February 2022 to summarize long COVID evidence and to assess prevalence and clinical presentation in children and adolescents. Articles reporting long COVID prevalence and symptoms based on original data in the paediatric population were included. Case series quality was assessed through the JBI Critical Appraisal Checklist. For observational studies, adherence to STROBE checklist was evaluated. Twenty-two articles were included: 19 observational studies  and 3 case series. Nine studies provided a control group. We found a high variability in terms of prevalence (1.6–70%). The most frequently reported symptoms were fatigue (2–87%), headache (3.5–80%), arthro-myalgias (5.4–66%), chest tightness or pain (1.4–51%), and dyspnoea (2–57.1%). Five studies reported limitations in daily function due to long COVID. Alterations at brain imaging were described in one study, transient electrocardiographic abnormalities were described in a minority of children, while most authors did not evidence long-term pulmonary sequelae. Older age, female sex, and previous long-term pathological conditions were more frequently associated with persistent symptoms.Conclusion: Long COVID evidence in children is limited, heterogeneous, and based on low-quality studies. The lockdown consequences are difficult to distinguish from long COVID symptoms. High-quality studies are required: WHO definition of long COVID should be used, controlled clinical studies should be encouraged, and the impact of new variants on long COVID prevalence should be investigated to ensure an objective analysis of long COVID characteristics in children and a proper allocation of healthcare system resources.The online version contains supplementary material available at 10.1007/s00431-022-04600-x. The challenges of coronavirus-associated acute respiratory disease called coronavirus disease 19 (COVID-19) are now extending to its long-term sequelae. Since the beginning of the severe acute respiratory syndrome coronavirus type 2 (SARS-CoV-2) –5. RecenChildren rarely develop a severe respiratory disease in the acute phase of COVID-19, though a limited number of patients exhibits a well-defined multisystem inflammatory condition, that can lead to multiorgan failure and shock, known as paediatric inflammatory multisystem syndrome temporally associated with SARS-CoV-2 (PIMS-TS)  or multiSince October 2020, parents’ concerns about persistent symptoms in children months after the acute SARS-CoV-2 infection have been emerging . In NoveA systematic review of the literature was performed according to the Preferred Reporting Items for Systematic Reviews and Meta-analyses (PRISMA) guideline recommendations . The resThe research was restricted to English language. Articles reporting long COVID prevalence and symptoms based on original data in paediatric population were included independently from the study design. Review articles, commentaries, editorials, and letters to the author with no original data were excluded. Sample dimension was not an exclusion criterion. Studies concerning PIMS-TS were excluded, except where the number of patients with PIMS-TS was minimal , 14.Duplicate publications were removed, then two authors separately (RP and EC) checked the titles and abstracts and removed irrelevant studies according to the inclusion and exclusion criteria. Articles were categorized as cohort studies or case series and, according to the source of information, as based on surveys or questionnaires or on clinician-assessed data. From each study information about children population included, test used for SARS-CoV-2 infection diagnosis, follow-up time, long COVID definition, and clinical presentation were extracted. Studies including a minimal number of patients with PIMS-TS were included, and the prevalence of persistent symptoms was recalculated after excluding PIMS-TS cases for the sake of comparability.For observational studies, adherence to Strengthening the Reporting of Observational Studies in Epidemiology (STROBE) recommendations  was asseEthics approval was not required for the systematic review component of this study.Overall, 214 articles have been initially retrieved, and after screening and selection, 22 have been included in the review 4). The lThe clinical spectrum assessed across studies varied notably. The most frequently reported symptoms were the following: fatigue (2  to 87% ), headacEight of the studies included in our review provided a control group , 32, 33.Molteni et al. identified two classes of children based on illness duration, termed LC28 for duration over 28 days and LC56 over 56 days . The obsA Latvian study compared children with previous SARS-CoV-2 infection to children with other non-SARS-CoV-2 infections stating that symptoms persistence is more evident with COVID-19 than any different infection . On the Among controlled studies, the long COVID clinical spectrum is undefined. Stephenson et al. described tiredness (23% vs 14.2%) and headache 39% vs 24.2%) as more frequently reported within the case group, and no difference in the distribution of mental health and well-being scores was found between the two groups . Similar.2% as moThe persistence of long COVID symptoms has been associated with a hypometabolic pattern at positron emission tomography (PET) with 2-[18F]-fluorodeoxyglucose (FDG) of the brain, involving bilateral medial temporal lobes, brain-stream, cerebellum, and the right olfactory gyrus in 7 French children with long COVID .Data regarding possible cardiac involvement are contrasting. Erol and colleagues described a statistically significant difference in systolic blood pressure, left ventricular posterior wall diameter, relative wall thickness, and tricuspid annular plane systolic excursion values between children with a history of SARS-CoV-2 infection and controls . In an IA mild obstructive reversible pattern at lung function test was evidenced in nearly half the children in the Israelian cohort , whereasIn the CLoCK study cohort, in both positive and negative groups, those with multiple symptoms were more likely to be female, adolescent, and to have poorer baseline physical and mental health status . The samOlder age as a risk factor for persistent symptoms after SARS-CoV-2 infection has been reported in 9 studies , 29, 32.Overweight has been described as a long COVID risk factor in adults . Among sNo correlation between acute illness severity and duration of symptoms was noticed , 31, excThe need of rehabilitation plans for long COVID patients in adults has been claimed , whereasIn the present systematic review, 7 studies , 34, 35 Interestingly, persisting symptoms were described also in children with previous mild or asymptomatic COVID-19 and no correlation between the severity of acute illness and long COVID has been noted , 31.Furthermore, it is unclear whether persisting symptoms are related to viral infection itself or they express the effects of pandemic, lockdown, and school suspension on children. Lockdown and social limitation negatively impacted on children and adolescent mental health . This faWhen a control group was provided, patients with a history of SARS-CoV-2 infection were more prone to show higher prevalence of persistence of symptoms , 33, 37,The symptoms observed affect cardio-respiratory, gastrointestinal, and neurological systems, and rehabilitation and psychologist support are needed . TherefoThe mechanisms underlying post COVID condition are not clearly defined; however, several pathogenesis models have been put forward. One of the most supported hypotheses is based on the persistence of the virus or a virus component . SeveralOur review may have limitations, including that some articles might have been missed. Considering that the literature regarding long COVID is rapidly increasing, a continuous updating of evidence is mandatory. Methodological issues were frequent among the included studies: matched cohort studies were limited, rarely a comparison with other viral illness was provided, and most of the data were based on questionnaire-based studies. Symptoms prevalence mainly relies on self-reporting and online surveys; hence, recall and selection biases must be considered. Furthermore, most of the studies included in our review were published prior to the WHO post COVID-19 definition, resulting in a heterogeneous delineation of the condition among studies. Lastly, the exclusion of children with PIMS-TS, who typically complain more severe and persistent symptoms, could have an impact on the long COVID prevalence estimation.Evidence on long COVID in children is limited, heterogeneous, and based on low-quality studies. Given that an accurate prevalence of the condition remains undefined, it is difficult to distinguish between functional complaints of post-acute COVID syndrome and social restriction effects.Further high-quality studies are required to define the optimal management of this emergent condition and to establish which resources are needed to face long COVID syndrome and the overall lifelong negative effects of SARS-CoV-2 pandemic in children and adolescents. Since WHO provided a research definition of long COVID, its use should be promoted in future studies to harmonize data. Controlled clinical studies should be encouraged over questionnaire-based ones to ensure an objective analysis of the actual prevalence and long COVID characteristics in children. Moreover, the impact of new variants on long COVID prevalence needs to be investigated to ensure healthcare systems properly allocate their resources.Supplementary file1 (DOCX 30 KB)Below is the link to the electronic supplementary material.