Escherichia coli, followed by Clostridium perfringens and Citrobacter freundii. Antibiograms and the determination of resistance genes showed a reduced spread of bacteria carrying antibiotic resistance among wild animals in Sicily. However, since several wild animals are becoming increasingly close to residential areas, it is important to monitor their health status and to perform microbiological analyses following a One Health approach.Wild environments and wildlife can be reservoirs of pathogens and antibiotic resistance. Various studies have reported the presence of zoonotic bacteria, resistant strains, and genetic elements that determine antibiotic resistance in wild animals, especially near urban centers or agricultural and zootechnical activities. The purpose of this study was the analysis, by cultural and molecular methods, of bacteria isolated from wild animals in Sicily, Italy, regarding their susceptibility profile to antibiotics and the presence of antibiotic resistance genes. Bacteriological analyses were conducted on 368 wild animals, leading to the isolation of 222 bacterial strains identified by biochemical tests and 16S rRNA sequencing. The most isolated species was  Sus scrofa), wild rabbit (Oryctolagus cuniculus), fox (Vulpes vulpes), and porcupine (Hystrix cristata), and many common birds together with endangered species, such as the golden eagle (Aquila chrysaetos), the smaller Bonelli’s eagle (Aquila fasciata), and the griffon vulture (Gyps fulvus).Sicily is the largest island in the Mediterranean Sea and the biggest Italian region. Located in the middle of the Mediterranean area, Sicily has a strategically important position for bird migration because it represents a natural bridge between Europe and Africa. The Sicilian territory is characterized by a considerable extension of rural areas and natural parks and by the presence of urbanized and industrial areas. These regional parks, such as Parco delle Madonie and Parco dei Nebrodi, as well as several smaller regional reserves, host many wildlife mammal species, including wild boar  List A diseases and, particularly those subject to state public health measures and the zoonotic diseases . For theIt is well known and widely reported that wild animals play a role in the maintenance of pathogens, bacteria, and parasites in a territory, some of which are responsible for zoonoses . Cattle,Another critical concern regards the possibility that these animals could have a role in the diffusion of antimicrobial resistance (AMR) . AlthougThe acquisition of resistance genes is considered the main factor that contributes to the wide distribution and diffusion of antimicrobial resistance; indeed, through vertical or horizontal transfer, there is an exchange of elements, such as plasmids and transposons. Transported by plasmids or contained within transposons, there are integrons that provide bacteria with a gene capture system perfectly adapted to the challenges of multiple antibiotic treatment regimens and that have been considered to contribute to the release of “superbugs” . SeveralBased on these observations, the presence of pathogenic bacteria, including zoonotic agents, in animals  from different areas of Sicily, Italy, was evaluated to monitor the health status of wildlife populations. To better understand the spread of antimicrobial resistance in wild animals as part of a One Health approach, the susceptibility profile of some of the isolated bacterial strains was determined by microbiological assays, and the presence of genes coding for antimicrobial resistance was evaluated by molecular analysis.Between 2017 and 2019, samples from 193 wild birds, 119 mammals, 35 terrestrial, and 21 swamp tortoises, from occasionally found carcasses, hunting activities, and regional recovery centers, located mainly in the provinces of Messina and Palermo, were analyzed .The recovery centers collect wounded live animals that need care and carcasses from most of the Sicilian territory, although they are located near two of the largest Sicilian regional parks (Parco delle Madonie and Parco dei Nebrodi). Before sample collection, each animal was identified on a morphological basis to define the species. The 275 carcasses were subjected to necropsy, including gross examinations and tissue sample collections from the organs  for laboratory investigations.From the live animals, mainly birds and turtles, housed in recovery centers, samples, including 79 swabs  and 14 feces, were collected. These samples were collected by veterinary practitioners, who visited the rescued animals in the recovery centers, and were sent to the laboratory to facilitate the diagnosis and perform appropriate antibiotic treatments prior to their release into the wild. In addition, feces samples and rectal/cloacal swabs were screened for pathogenic bacteria that were potentially transmissible to other animals housed in the centers.Salmonella spp. species, a pre-enrichment in buffered peptone water (APT) and two subsequent enrichments in selenite cystine and Rappaport–Vassiliadis broths , incubated, respectively, at 37 °C and 42 °C for 24 h, were performed from the intestine, spleen, liver, feces, and rectal/cloacal swabs. The enrichment broths were then seeded in xylose lysine desoxycholate (XLD) and brilliant green agar (BGA) [Swabs and feces from live animals and tissue samples collected from carcasses were subjected to bacteriological procedures to isolate bacterial species. Bacteriological cultures were performed using selective and differential medium  to promote the growth of the main commensal and pathogenic species from the collected samples ,20. For ar (BGA) . The col® high-fidelity DNA polymerase kit  and a pair of universal primers (5′-CCAGCAGCCGGCGGGTAATACG-3′ as the forward primer and 5′-ATCGGYTACCTTGTTACGACTTC-3′ as the reverse primer) were used in a polymerase chain reaction (PCR) assay [For the most significant strains, amplification and nucleotide sequencing of the 16S rRNA genes was performed. Bacterial DNA was extracted using 100 µL of PrepMan™ ultra sample preparation reagent , according to the protocol indicated by the manufacturer. The Q5R) assay .An aliquot of extracted DNA (5 µL) was added to a PCR reaction mix prepared by mixing a final concentration of 1X of Q5 reaction buffer, 200 µM of dNTPs, 0.5 µM of each universal primer, 0.02 U/µL of Q5 Hot Start high fidelity DNA polymerase, and 1X of Q5 High GC enhancer, in a total volume of 50 µL. After 10 min of denaturation at 98 °C, the reaction mixture was run through 35 cycles of denaturation for 30 s at 98 °C, annealing for 1 min at 55 °C, and extension for 30 s at 72 °C, followed by a final extension of 2 min at 72 °C. Subsequently, 10 µL of the PCR product was used for electrophoresis on a 1% agarose gel to determine the size of the product. The polymerase chain reaction (PCR) products were purified and sequenced at BMR Genomics Srl . The nucleotide sequences were identified using the National Center for Biotechnology Information  nucleotide basic local alignment search tool (BLASTn) program.Unfortunately, only 61 strains of the 222 isolates at the first stage were successfully recovered in purity after storage. However, the analyzed strains were representative of the species present in the tested animals.The antibiotic susceptibility of 61 isolated strains was determined by the disk diffusion method on Mueller–Hinton agar  (Kirby–Bauer). A set of 12 antibiotics representative of the main classes used in human and veterinary medicine was chosen: amikacin (30 µg), gentamicin (10 µg), ampicillin (10 µg), amoxicillin/clavulanic acid (30 µg), ceftiofur (30 µg), ceftriaxone (30 µg), clindamycin (2 µg), chloramphenicol (30 µg), ciprofloxacin (5 µg), enrofloxacin (5 µg), sulfamethoxazole/trimethoprim (23.75 + 1.25 µg), and tetracycline (30 µg). The interpretation of the results was performed in accordance with the Clinical and Laboratory Standards Institute (CLSI) ranges . HoweverTEMbla, CTXMbla, tetA, qnrS, and sull II antibiotic resistance genes and of the mobile element int1 in 61 bacterial strains was determined using PCR. These genes were chosen for their contribution to resistance against the most commonly used antibiotics, especially in veterinary medicine, and to assess any correspondence between the phenotypic resistance found and the presence of these genes in the tested strains. A PCR reaction mix containing 5 µL of the DNA template and a final concentration of 1X DreamTaq buffer, 2 mM of dNTPs, 0.5 μM of the forward primer, 0.5 μM of the reverse primer, and 1.25 U of DreamTaq DNA polymerase  in a total volume of 50 µL was utilized. Subsequently, 10 µL of the PCR product was used for electrophoresis on a 1% agarose gel to determine the size of the product. The primers and the annealing temperatures used are reported in The presence of n = 193), followed by mammals (n = 119), and, with a minor number, reptiles (n = 56).During the three-year period 2017–2019, 275 animal carcasses, 79 fecal samples, and 14 swabs from 368 wild animals were analyzed. Details on the species and number of the animals tested are presented in All carcasses were subjected to autopsy, which revealed several anatomopathological lesions, many of which can be attributable to direct contact with humans. For the birds, indeed, the most frequent causa mortis was from gunshot wounds with fractured forelimbs; moreover, many of the subjects analyzed appeared emaciated and dehydrated. During the autopsy of mammals, subcutaneous hematomas, abdominal hemorrhagic effusions with a ruptured spleen, and fractures were observed, all injuries attributable to impact trauma. Thus, fecal samples and swabs  from live animals were submitted to the laboratory by veterinary practitioners of the recovery centers who suspected bacterial infections, which were then confirmed by laboratory analyses and subsequently treated during the hospitalization before the release of the animal in the wild.Escherichia, found in 114 animals (51.3%), followed by Clostridium (22.5%), Citrobacter (16.6%), and Aeromonas (14.4%) , Clostridium perfringens , and Citrobacter freundii . Unique bacteria species were isolated in 57.6% (128/222) of the subjects, whereas in 14.4% (32/222) and 24.7% (55/222) of the subjects, two and three bacteria species were isolated, respectively. In addition, the presence of four different bacterial species was found in six animals, while five were isolated in only one subject. The prevalence of the isolated bacterial genera varied among the host classes  of the subjects analyzed. The most isolated genus was  (14.4%) . The mos classes .Enterobacterales species, such as Citrobacter spp. and Klebsiella spp., whose resistance to this antibiotic is indicated as intrinsic by EUCAST. This resistance was present, in particular among strains isolated from mammals  and birds . The results obtained for the other antibiotics tested showed high percentages of susceptibility, particularly to chloramphenicol (93%), ceftriaxone (90%), sulfamethoxazole/trimethoprim (85%), and amoxicillin/clavulanic acid (84%).The susceptibility to the chosen set of antibiotics was determined for 61 strains. The results of the antibiograms performed with the Kirby–Bauer method are reported in E. coli from one owl (id. 13) and one tortoise (id. 10) resistant to penicillins, sulfonamides, and tetracyclines; E. coli from one kestrel (id. 28) resistant to penicillins, cephalosporins, and aminoglycosides; Campylobacter jejuni from a crow (id. 30) resistant to penicillins, cephalosporins, and sulfonamides; Pseudomonas aeruginosa from a peregrine falcon (id. 33) resistant to cephalosporins, lincosamides, and sulfonamides; Enterobacter cloacae from a crow (id. 25) resistant to penicillins, cephalosporins, sulfonamides, and tetracyclines; and Staphylococcus simulans from a peregrine falcon (id. 32) resistant to penicillins, phenols, fluoroquinolones, lincosamides, and tetracyclines.In addition, the results obtained showed the presence of seven multidrug-resistant strains (MDR), five of which were resistant to three antibiotic classes, one to four, and another to five. Specifically, the MDR strains were: two TEM, blaCTXM, tetA, sulII,bla and qnrS) and mobile element class-1 integron (int1) demonstrated a reduced incidence of these in the bacterial strains isolated. Indeed, of the 61 strains analyzed, only seven (11.47%) were positive for the presence of one or more genes among those investigated. These seven strains, all belonging to the Enterobacterales family, were three strains of E. coli  in which only the TEMbla gene was present, an E. coli (from golden eagle id. 5), which harbored two resistance genes, TEMbla and sulII, and E. cloacae (from rabbit id. 25), in which tetA and sulII were present.Molecular analysis conducted for the detection of resistance genes . The presence of CTXMbla and qnrS genes was not found in any of the strains tested. Finally, we found the presence of the mobile element int1 in three strains (5.2%), two E. coli, from golden eagle id. 5 and owl id. 13 and E. cloacae from rabbit id. 25. Three resistance genes, Staphylococcus aureus or P. aeruginosa, were the isolated bacteria responsible for the clinical signs of the lesions.This study reports the data regarding the presence of antimicrobial resistance in bacteria isolated in 2017–2019 from wildlife in Sicily. Most of the analyzed samples were collected from subjects with death due to anthropic activities (gunshot wounds or impacts with vehicles). Bacteriological analysis allowed the isolation of different bacterial species. Although some of the bacterial species isolated may have pathogenic and/or zoonotic potential, in most cases, they were bacterial species commensal with those animals, without pathogenic activity, and were not related to the anatomopathological findings observed during post-mortem inspection. Only in a few cases, for example, in live animals with wounds or skin lesions infected by Enterobacterales (76%) represent the species most prevalent in wildlife. This result is in accordance with those reported in previous studies on wild birds in Sicily, in which the most commonly reported species were E. coli, C. freundii, and Klebsiella oxytoca [C. perfringens, which, after E. coli, was the most isolated species from both birds and mammals, was indicated as a possible part of the microbiota in wild animals by some authors, although more studies on its role in the wild are needed [In fact, although wildlife could represent a risk to humans and domestic animals when it acts as a reservoir of disease, intermediate host, or biological amplifier, most interactions between microorganisms and wildlife are harmless and present relatively few risks . The ana oxytoca . In addie needed ,27.The phenotypic resistance was evaluated by testing antibiotics belonging to eight different classes: penicillin (ampicillin and amoxicillin/clavulanic acid), cephalosporins (ceftriaxone and ceftiofur), phenicols (chloramphenicol), fluoroquinolones (enrofloxacin and ciprofloxacin), aminoglycosides (gentamicin and amikacin), lincosamides (clindamycin), tetracyclines (tetracycline), and sulfonamides (sulfamethoxazole/trimethoprim). To avoid overestimating the incidence of resistance, the data obtained were interpreted, taking into account, in accordance with the bacterial species tested, the intrinsic resistance reported by EUCAST .Enterobacterales isolated from wild animals were also found in other Italian regions: strains resistant to cephalothin (94.3%), amoxicillin/clavulanic acid (86.9%), and tetracycline (44.6%) were found in wild boars in Tuscany, while, in strains from migratory Passeriformes transiting through the Metaponto territory , resistances to amoxicillin (64. 8%), ampicillin (63.1%), rifampicin (61.5%), and amoxicillin-clavulanic acid (54.1%) were found [The data we collected showed the percentage of resistance to the antibiotics tested as less than or equal to 16%, except for ampicillin, for which the detected resistance was 41.8%. However, in a previous study conducted on Gram-negative bacteria isolated from wild birds in Sicily in 2013, significant levels of resistance against sulfamethoxazole/trimethoprim and amoxicillin/clavulanic acid were reported in a population of live wild birds . High pere found ,29.E. coli isolated from wild boar and deer in Poland; Smith et al. in a study conducted on herring gulls and deer in Ireland found that all strains isolated were resistant to rifampicin, oxacillin, and penicillin; and a moderately high level of resistance to tetracycline, streptomycin, ampicillin, and sulfamethoxazole/trimethoprim (35%) was found in fecal samples obtained from wild animals in Portugal [In addition, in contrast to our data, a high incidence of resistance to several antibiotics was also reported in studies conducted on wild species from other European areas: Wasyl et al. detected resistance to 11 antibiotic molecules, including streptomycin, tetracycline, sulfamethoxazole/ trimethoprim, and colistin, in Portugal ,31,32.The results in the present study showed that, except for a multidrug-resistant strain from a terrestrial turtle, the other six MDR strains were isolated from wild birds , as wellTo confirm these data, further sampling, including more subjects from the different classes of animals, will be necessary. However, the data collected regarding the antibiotic susceptibility, determined using the phenotypic method, indicated that resistance at least for the 12 antibiotics tested in this study was not widespread among strains isolated from the wild animals in our study. In contrast to data related to companion animals, which showed a higher prevalence of resistant bacteria , the resEnterobacterales family, and mainly in E. coli strains (6/7). All these E. coli harbored the TEMbla genes, which, together with the CTXMbla absent in our strains, are the most widespread resistance genes. The TEMbla gene coding for β-lactamase TEM is the most common mechanism of resistance to ampicillin in E. coli.A low incidence of genes for antimicrobial resistance was also present in these strains from the wild. Although they were investigated in both Gram-positive and negative strains, these genes have only been found in a few strains  belonging to the E. coli strains tested were found to be phenotypically resistant to ampicillin, only for six of these strains, could the resistance be attributable to the presence of resistance genes. In fact, as reported by several authors, there are multiple complex mechanisms that lead a bacterium to be resistant to antimicrobials, and phenotypic expression does not always correspond to genotypic resistance and vice versa [E. coli strains id. 15, 33, and 10, which, although they harbored only the TEMbla gene, had different phenotypic resistance profiles. Indeed, while strain id. 15 was resistant only to ampicillin, the other two strains were also resistant to other antibiotics: strain id. 33 showed resistance also to amikacin and strain id 10, also to sulfamethoxazole/trimethoprim and tetracycline (Although 87% (20/23) of the ce versa ,34,35. Tacycline .sulII (responsible for resistance to sulfonamides) and tetA (to tetracyclines) were found in only four (three E. coli and one E. cloacae) and three strains (two E. coli and one E. cloacae), respectively, although not all these strains showed phenotypic resistance to these antibiotics.For the same reason, E. coli, and some Gram-positive species, such as Staphylococcus spp. and Corynebacterium spp. of clinical origins [E. coli, is not able to acquire or maintain class-1 integrons without antibiotic pressure [Three of the strains that harbored resistance genes were also found to contain class-1 integron, a mobile element carrying resistance cassettes that has been identified in Gram-negative species, mainly  origins ,37. With origins . In addi origins ,40. Howepressure  and thatThe natural microbiota of animals, as well as humans, are subject to numerous antimicrobial pressures due not only to the use and abuse of antibiotic drugs but also to their use in agriculture. In addition, antibiotics and resistant bacteria present in urban, agricultural, and livestock wastewater can also arrive in wild environments where factors, such as the huge number of bacterial species present and the high-density, can promote gene exchange between bacteria and the spread of antibiotic resistance.Although this study demonstrated a reduced prevalence of pathogenic and/or zoonotic bacteria, as well as a reduced prevalence of phenotypic resistance and genes determining resistance to the main antibiotic classes, in a One Health approach, it is particularly important to monitor wildlife. One Health is an ideal approach to achieve global health by recognizing that human, animal, and ecosystem health are inextricably linked. In fact, antimicrobial resistance is now considered a zoonotic health threat. Worldwide, antimicrobial resistance is on the rise, and, if current trends continue unabated, it has been estimated that this could be the cause of 10 million deaths by 2050 .Wild animals, especially species such as wild boars, foxes, and seagulls, which are increasingly closer to human environments, can be reservoirs and diffusers not only of pathogenic and/or zoonotic bacteria but also of resistant commensal bacteria or bacteria that possess potentially transferable antibiotic resistance genes. Even in aquatic environments, including remote ones, such as glaciers, the presence of antimicrobial-resistant bacteria has been detected . Thus, i


In this review these pathways are explored, including the impact of VO on the tumor microenvironment, and on cancer outcomes. The current evidence-based literature regarding the role of dietary, lifestyle, pharmacologic and surgical interventions to modulate the risk of EAC is explored.Esophageal adenocarcinoma (EAC) represents an exemplar of obesity-associated carcinogenesis, with a progressive increase in EAC risk with increased body mass index. In this context, there is increased focus on visceral adipose tissue and associated metabolic dysfunction, including hypertension, diabetes mellitus and hyperlipidemia, or combinations of these in the metabolic syndrome. Visceral obesity (VO) may promote EAC  The past four decades have seen a marked rise in the incidence of esophageal adenocarcinoma (EAC), with data from the United States demonstrating a continued rise in the overall incidence from ~0.4 per 100,000 in 1975 to 3.5 per 100,000 in 2015—an over 8-fold increase , non-alcoholic fatty liver disease (NAFLD), and cardiovascular disease  (Metabolic syndrome (MetS) defines a distinct clinical phenotype that is associated with VO, with added features of T2DM, hypertension and hyperlipidemia, and commonly NAFLD , 22. Patgus (BE) .Notwithstanding a clear epidemiologic association, the impact of VO and MetS on oncologic outcomes of EAC remains uncertain, with various data suggesting improved, worsened or an unchanged survival and treatment outcomes –27. WithThe present review aims to summarize key data outlining the roles of obesity, VO, and the MetS in the pathophysiology of EAC, with a focus on potential targets for prevention and treatment.2]; OR = 2.79 [95% CI: 1.89–4.12] for class II obesity [BMI: 35–39.9 kg/m2]; OR = 2.39 [95% CI: 1.86–3.06] for class I obesity [BMI: 30–34.9 kg/m2]; and OR = 1.54 [95% CI: 1.26–1.88] for overweight [BMI: 25–29.9 kg/m2], vs. BMI <25 kg/m2) , and over 10,000 control subjects. This reported a dose-response effect of BMI with the greatest risk among individuals with the most severe obesity  were associated with increased EAC risk on multivariable analysis . NotablyMetS defines VO in association with metabolic consequences such as T2DM and hyperlipidemia, and typically is associated with a pro-inflammatory systemic and local response, and end-organ consequences such as NAFLD. Studies have identified an association between MetS and both BE and EAC. A systematic review and meta-analysis of MetS and BE identified 12 publications including 355,311 subjects and reported a significant association . For EAC, with MetS defined using a standardized metabolic risk score, a significant association between the metabolic risk score and EAC was observed exclusively in male patients . Analysivia GERD, as long duration or severe GERD is associated with an up to 40-fold increase risk of EAC  and pro-inflammatory mediators, with potential consequences including NAFLD and insulin resistance  gene product in mice, and we now understand that leptin serves an important role as an adipostasis hormone. Leptin is secreted by white adipocytes, and exists in the circulation at levels proportionate to body fat mass , with interstitial fibrosis within the TME altering cytokine signaling, and epithelial morphology and differentiation (A current focus of much research in EAC is the impact of obesity on the tumor microenvironment (TME), the cellular environment in which a tumor exists. The TME includes the extracellular matrix, fibroblasts, immune and inflammatory cells, signaling molecules, and tumor-related blood vessels. A number of processes are dysregulated both in VAT and within the TME, highlighting potential links between obesity and cancer, including hypoxia, inflammation, angiogenesis, energy metabolism, and epithelial to mesenchymal transition (EMT) . Obesityntiation , 20, 73.ntiation . This montiation .There is compelling evidence that the gut microbiome plays a central role in the regulation of inflammation and metabolism, with recent data implicating dysbiosis in the pathophysiology of a number of inflammatory and metabolic conditions, such as inflammatory bowel disease, obesity and MetS. Interestingly, the impact of dietary factors with respect to the development of obesity and MetS may be mediated by host-microbiota interactions, as germ-free mice do not develop metabolic dysregulation in response to a high fat diet (HFD), however fecal transplant to HFD-fed germ-free mice from obese mice increases recipient adiposity . FurtherObesity, HFD, and GERD may alter the composition of the esophageal microenvironment, resulting in changes in the resident microbiota. Early data indicate that individuals with GERD and BE exhibit distinct patterns of microbiota , 121, wiIRS1) gene and confers improved insulin sensitivity, fat distribution and metabolism. Variants in IRS1 were recently assessed among participants in the Swedish Obese Subjects (SOS) study  study , a largeS) study –134. VarFOXF1, are associated with increased risk of BE, in addition to variants within the major histocompatibility complex locus on the short arm of chromosome 6. Furthermore, for 29 of 40 SNPs associated with obesity, the same allele increased risk of BE  chemotherapy or chemoradiotherapy in esophageal cancer, limited data suggest that obesity is associated with similar response rates following neoadjuvant therapy , 154. InInsulin resistance, IGF-1, leptin, and IL-6 may be increased among patients with MetS, and may negatively impact outcomes for cancer as for cardiovascular diseases , 159. MeMouse studies demonstrate that HFD is associated with the development of esophageal dysplasia and adenocarcinoma, associated with increased IL-8 and a shift in gut microbiota . DespiteBariatric/Metabolic surgery is a highly effective intervention for the treatment of obesity, resulting in sustained weight loss and improvements in aspects of the MetS such as insulin resistance and altered lipid metabolism . ApproxiReduced physical activity has been found to be associated with increased risk of EAC in multiple studies , 176. A A previous multicenter prospective cohort study demonstrated reduced risk of BE progression among patients treated with proton pump inhibitors (PPIs) over a median 5.2 year follow-up period . A systeGiven the role of systemic inflammation in the pathogenesis of BE and EAC, a key question is whether modulation of the inflammatory response may prevent BE or its progression . ObservaEpidemiologic data indicates that aspirin may confer a protective effect with respect to BE/EAC risk –186. AspMetformin has also been suggested as a possible therapeutic to prevent obesity-associated cancers, by attenuating hyperinsulinemia. A randomized controlled study of patients with BE assessed the impact of metformin on levels of phosphorylated S6 kinase (pS6K1), a marker of insulin pathway activation. The study found no significant change in pS6K1, and a trend toward reduced insulin levels and improved insulin resistance, and there was no significant difference in epithelial proliferation or apoptosis in esophageal biopsies. As such current data do not support the use of metformin as chemoprevention among patients with BE . Observa2 increase in BMI, suggesting a dose-response effect  recently reporting a progressive increase in relative risk for EAC for each 5 kg/me effect . VisceraJE and JR designed and conducted the research, provided essential materials, analyzed data, wrote the paper, and have primary responsibility for final content. All authors contributed to the article and approved the submitted version.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.


Ulcerative colitis (UC) is a chronic inflammatory bowel disease (IBD) characterized by a relapsing-remitting course owing to recurrent intestinal inflammation. UC often has symptoms such as intermittent rectal bleeding, diarrhea, and abdominal pain. As the precise etiology of UC has not completely clarified, UC has become a public health challenge worldwide. According to an epidemiological survey, there were about 350,000 new cases of IBD in China from 2005 to 2014. By 2025, the number of IBD patients in China will reach 1.5 million. Traditional Chinese medicine (TCM) has been widely used to treat UC in China, however, it is still challenging to systematically determine the efficacy of in UC. Therefore, this trial aims to evaluate the clinical efficacy and safety of CHM in the treatment of mild active UC patients.A multi-center, double-blinding, double-dummy, active-controlled, randomized trial will be established. A total of 240 patients in 6 centers with mild active UC (Mayo score is 3–5 points) and TCM syndrome of damp-heat stasis blocking and spleen-qi deficiency will be randomly allocated in the ratio of 1:1 to 2 groups: the experimental group and the control group. The experimental group will receive Hudi enteric-coated capsules (HEC) and enteric-coated mesalazine tablets placebo; the control group will receive enteric-coated mesalazine tablets and HEC placebo. Each group will be treated for 8 weeks. The primary therapeutic outcome: the rate of clinical efficacy and clinical remission at 8 weeks of treatment (last survey point) according to the modified Mayo score. The secondary outcomes: individual symptom score, TCM syndrome score, endoscopic response rate, mucosal healing rate, and quality of life scale score. Outcomes will be assessed at baseline and the end of the trial. Besides, intestinal mucosa, stools and blood biopsies from the mild active UC patients before and after treatment will be collected to reveal the underlying mechanisms.The results of this trial will provide compelling evidence of the efficacy and safety of HEC for treatment of mild active UC and preliminarily show the potential mechanism of how HEC acts. Finally, it will widen treatment options for patients with mild active UC. UC was mainly prevalent in developed countries of Europe and North America. Studies have reported that the annual incidence of UC varies from 8.8 to 23.14 per 100,000 person-years in North America, 1.7 to 57.9 per 100,000 person-years in Europe, and prevalence was 139.8 to 286.3 per 100,000, and 2.42 to 412.0 per 100,000, respectively. According to an epidemiological survey, there were about 350,000 new cases of IBD in China from 2005 to 2014, and by 2025, the amount of IBD patients in China will reach 1.5 million. As the precise etiology of UC has not completely clarified, UC has become a public health challenge worldwide, and because of the widespread of the mild active period, our trail will focus on this specific type.Ulcerative colitis (UC) is a kind of inflammatory bowel diseases (IBD) characterized by chronic nonspecific inflammatory colorectal disease with a yet-uncharacterized etiology. UC often has symptoms such as intermittent rectal bleeding, diarrhea, and abdominal pain. It mainly affects young and middle-aged people between 18 and 50 years old. But those medicine often result in different adverse effects, a low response rate and a high risk of infection, however, complementary and alternative medicine (CAM), like traditional Chinese medicine (TCM), with lower adverse effects were used seldom in UC. TCM is one of the most commonly used modality for UC patients, accounting for 19% to 54% of CAM users, that is been known as useful in improving symptoms, reducing medical expenses and clinical evaluation for UC patients, but it is still challenging to systematically determine the efficacy of TCM in treatment.UC is treated with 5-aminosalycilate acid (5-ASA), corticosteroids, immune suppressants, and biological agents currently, especially, 5-ASA is recommended as a first-line therapy for mild-to-moderate UC patients. To obtain more conclusive results on the risk-benefit ratio of TCM for UC, it is necessary to design and conduct a high-quality clinical trial with appropriate blinding method and a large number of patients. Hudi enteric-coated capsules (HEC) has finished a multi-center clinic randomized controlled trial in China, it confirmed that HEC played a pivotal role in treating mild to moderate UC by relieving symptoms effectively, but as the shortage of colonoscopy examination and treatment time, this trial was not rigorous enough. Besides, the mechanism of HEC in treating UC was not clear.Numerous studies based on UC mouse models and clinical trials, especially randomized double-blinding clinical trials, remains the gold standard for evaluating the efficacy of TCM.Therefore, the primary aim of this multi-center, double-blinding, double-dummy, randomized controlled trial is to evaluate the clinical efficacy and safety of HEC in the treatment of mild active UC patients. Meanwhile, we want to reveal the underlying mechanism of HEC how to treat mild active UC by molecular biology technology from the clinic specimens.22.1http://www.chictr.org.cn) in time. Each participating center has conducted ethical filing and has the ethical approval of the leader unit. All volunteers signed the informed consent form before the trial and details could be inquired in the registry web or the corresponding author.The study is conducted in accordance with the Declaration of Helsinki (Edinburgh 2000 version). The final protocol  of this trial has been approved by the team leader unit, Research Ethical Committee of Jiangsu Province Hospital of Chinese Medicine (Version number: 2019NL-039-02). In addition, this trial has been registered in the Chinese Clinical Trial Registry . Before the enrollment, all participants would sign written informed consents. If there will be any significant modification of the protocol, it should be reviewed by the research ethical committee and updated on the registry web  placebo, while HEC placebo and EMT were used in the control group. A total of 240 patients diagnosed with mild active ulcerative colitis (MAUC) and TCM syndrome of damp-heat stasis blocking and spleen-qi deficiency in 6 clinical trial centers are eligible for enrolment. We will implement randomization with a 1:1 allocation ratio in the experimental group (n = 120) or the control group (n = 120), and each patient will undergo an 8-week treatment. The study flowchart is showed in Figure This study is a multi-center, double-blind, double-dummy, randomized, parallel positive controlled trial. We adopted a noninferiority trial design and followed the Standardized Protocol Interventions: Recommendations for Interventional Trials (SPIRIT) 2013 Statement.2.3,13 and the average rate is about 68%. Our previous study found that the efficacy rates of HEC is 83.11% in treating MAUC. The sample size was calculated by the following formula: Number of Samples was determined based on the calculation of noninferiority trial design. The efficacy rates of EMT ranges from 65% to 71%,Uα, Uβ is the corresponding U value of α, β. When α equaling to 0.05, β equaling to 0.1, Uα (0.05) is 1.645, Uβ (0.2) is 0.842. P1 and P2 represent the efficacy rates of HEC and EMT. P equals to (P1 + P2) /2 × 100%. The certainty was set at 80%, considering 20% of sample lose. The sample number was set to 120 for each group, 240 in total, and 40 samples in each center.2.4A total of 240 MAUC patients were recruited by 6 clinical trial centers in China from June 2019 to December 2021. Six clinical trial centers were listed as follows: Jiangsu Provincial Hospital of Chinese Medicine, Peking Union Medical College Hospital of Chinese Academy of Medical Sciences, Guangdong Provincial Hospital of Chinese Medicine, Chongqing Traditional Chinese Medicine Hospital, The Second Affiliated Hospital of Fujian Traditional Chinese Medicine University, and Shaanxi Traditional Chinese Medicine Hospital. The inclusion of the case complies with the principle of geographical balance nationwide. Internal physicians will screen eligible patients from outpatient clinics. Before the enrollment, all patients will receive a comprehensive view of informed consent, including objective, interventions, scheduling, the benefits and risks of this trial. An informed consent form is mandatory for enrollment.2.5Diagnostic criteria:1.The western medicine diagnostic criteria of UC will refer to Chinese consensus on diagnosis and treatment of IBD .2.,16 Diagnosis of this syndrome including the main and minor symptoms as following:The TCM diagnostic criteria of the damp-heat stasis blocking and spleen-qi deficiency syndrome will refer to the Clinic terminology of traditional Chinese medical diagnosis and Consensus of traditional Chinese medical in diagnosis and treatment of UC.Main symptoms:1.Diarrhea, mucus and bloody stools;2.Abdominal pain;3.Tesmus;4.Tired and lazy;Minor symptoms:1.burning anus;2.short and dark-color urine;3.dry and bitter in the mouth;4.drowsiness of limbs;5.poor appetite.Tongue and pulse: red tongue, thick yellow fur with tooth marks on the sides, and smooth pulse.Confirmation of the syndrome type: 2 main symptoms (required for the first item) plus 2 minor symptoms, which can be diagnosed by referring to the tongue and pulse.Inclusion criteria:1.Diagnosed with active UC.2.Defined by modified Mayo score of 3 to 5 points (mild period).3.Differentiation of TCM syndrome is damp-heat stasis blocking accompanied with spleen-qi deficiency.4.Age >18 and <65 years.5.Voluntary to participate and providing written informed consents, as required by Good Clinical Practice.Exclusion criteria:1.Patients during remission period, moderate or severe period.2.Pregnancy, lactation, or have desire for pregnancy.3.Severe allergy or allergic to mesalazine or any of the ingredients in HEC.4.Have cardiovascular, pulmonary, liver, renal, endocrine, nerve, and hematology diseases. Blood creatinine level, blood alanine transaminase or aspartate aminotransferase level above the upper limit of normal.5.Have serious complications, such as intestinal stenosis, intestinal obstruction, intestinal perforation, multiple intestinal polyps, toxic megacolon or colorectal cancer, etc.6.Mental disorders or other causes which result in inability of proper expression of feelings.7.Have participated in other medical clinical studies in the last 3 months.8.In addition to mesalazine, receiving other treatments for UC.9.Serious and unstable condition requiring emergency treatment.10.Inappropriate to participate in this trial judged by clinical physicians.Termination and withdrawal criteria:1.The participant is not willing to continue the clinical trial.2.No medication or any follow-up records;3.Important deviations occur in the process of the clinical trial, such as poor compliance.4.Occurrence of allergic reactions or serious adverse events during the trial.5.Not alleviated or the symptoms are aggravated.2.6Institute of Clinical Pharmacology of Xiyuan Hospital, China Academy of Chinese Medical Sciences took the responsibility of generating the allocation sequence. Consecutive numbers were assigned to each clinical center according to a center-stratified random order generated by SAS9.4 , and an interactive voice-response or web-response system will be used to assign each patient a number and group. A researcher who will not participate in this study set up an emergency letter for each blinding number, and emergency letters will be sealed in opaque envelopes. In patients with serious adverse events, an emergency unblinding procedure will be initiated. Meanwhile, the whole process of drug coding and documentation is performed blinded. Participants, clinical investigators, and statisticians will all be blinded until completion of the whole trial.2.7Experienced physicians will be responsible for recruiting and evaluating the participates in this trial. Postgraduates in gastroenterology will be trained by the principal investigator before participating in this trial. They will dispatch drugs, fill in case report forms (CRFs), and record detailed reasons for the patient's withdrawal from the trial.2.8Six trained investigators and their postgraduates will perform this procedure together. A total 240 patients will be recruited from out-patient and ward of the 6 clinic centers mentioned above, from May 2019 until December 2021. Before the enrollment, all the patients will receive a comprehensive view of informed consent, including objective, therapeutic interventions, scheduling, trial benefits, and possible risks of this trial. Only after signing a written informed consent, patients could be enrolled in it. Meanwhile, to guarantee the completeness of this trial, we will handsel drugs to the patients who have participated it completely.2.9The patient's stool, blood and colorectal mucosa samples will be collected before and after intervention, and clearly marked according to the unified format. A designated investigator will centrifuge blood samples in the laboratory. Then, the samples were transferred in frozen pipe and stored at −80°C for preservation in the biological sample bank of School of Life Sciences, Beijing University of Chinese Medicine. Consents and other documents like the collection and usage of participant data and biological specimens could be inquired from the registry web or the corresponding author.33.1HEC , EMT ,and placebos prepared identical in size, color, shape, taste, smell, and consistency to the 2 above drugs, respectively . HEC is a combination of Polygonum cillinerve Ohwi (Zhushaqi), Polygonum Cuspidate (Huzhang), Oldenlandia diffusa (Baihuasheshecao), Sonchus Brachyotus DC (Beibai’jiang), Limonium bicolor (Er'sebuxuecao), Radix Sanguisorbae Preparata Nigra (Diyutan), Bletilla striata (Baiji) and Radix Glycyrrhizae (Gancao). In this trial, an administrator was assigned to manage the drugs, including drug storage, distribution, and recycling independently and kept detailed records.3.2Both 2 groups will be treated with drugs for 8 weeks continuously, and the administration method of drugs has been designed as described in Table 3.3Each patient was provided with a participant diary card to record diary signs and symptoms of diarrhea, mucilage or blood–pus stools, abdominal pain, and tenesmus. Patients will be interviewed every 2 weeks after enrollment in the trial, while they should return the remaining drugs and packages, and receive the drugs they need to take for the next 2 weeks. Assessment of study endpoints and duration of follow-up was shown in Figure 3.4Primary therapeutic outcome: the rate of clinical efficacy and clinical remission at 8 weeks of treatment (last survey point) according to the modified Mayo score Table . ClinicaMinor outcomes including individual symptom score, TCM syndrome score, endoscopic response rate, mucosal healing rate, and quality of life scale score.1. All symptoms were divided into four levels: none, mild, moderate, and severe. The main symptoms were scored as 0, 2, 4, and 6 points, and the minor symptoms are scored as 0, 1, 2, and 3 points. The tongue and pulse were divided into normal and abnormal grades, 0 and 2 points were recorded in the main symptom, and 0 and 1 points were recorded in the minor symptom.Individual symptom score: referred Guidance Principle of Clinical Study on New Drug of Traditional Chinese Medicine.2.TCM syndrome score: The score was graded based on the degree of individual symptom score. All indicators were determined by comparing the values obtained at 2, 4, and 8 weeks with baseline values.Clinical recovery: clinical symptoms disappeared or syndrome score decreased by ≥95% from the baseline;Marked efficacy: the syndrome score decreased by ≥70% and <95% compared with the baseline;Efficacy: the syndrome score was a decrease of at least 30% and less than 70% from the baseline;Invalid: the syndrome score was a decrease of less than 30% from the baseline;Worsen: the syndrome score exceeded the baseline after treatment.TCM syndrome efficacy rate =  cases /a total number of cases ×100%.Endoscopic response rate: was defined as endoscopic score decreased at least 1 point from the baseline in the modified Mayo score. Endoscopic response rate = endoscopic response cases / total number of the group ×100%.(2)Mucosal healing rate: was defined as absolute subscore for endoscopy of 0 or 1. Mucosal healing rate = Mucosal healing cases / total number of the group ×100%.(3) included the 32 most frequent and important items. Inflammatory Bowel Disease Questionnaire was divided into 4 modules: bowel symptoms (10 questions), systemic symptoms (5 questions), emotional function (12 questions), and social function (5 questions). Each question has graded responses from 1 to 7, and thus the total score is ranging from 32 to 224, with higher scores representing a better quality of life.Quality of life assessment: Patients were evaluated by Inflammatory Bowel Disease Questionnaire,3.5Adverse events (AEs) will be documented and assessed by the local principal investigators during this trial, including clinical signs, symptoms, and laboratory tests. All patients will undergo the following laboratory texts between the start and end of the trial. The safety assessment indices included blood pressure, respiration rate, heart rate, body temperature, and physical examination in addition to blood routine examination, urine routine test, stool routine examination and occult blood, liver function tests , kidney function tests  and electrocardiogram. Any AEs will be requested to record in electronic CRFs (eCRFs) regardless of their relationship to the intervention. Severe AEs will be stopped immediately and reported to the local drug administration authorities within 24 hours including a detailed description of the time, severity, possible relationship with the drug, and the treatment measurement will be recorded.3.6The quality assessment of the trial will be conducted in the following aspects: the CRFs, patient diary card, original laboratory examination, informed consent, and drug distribution and storage. The trial information of patient will be recorded in CRFs by a trained and qualified investigator, and the completed CRF will not be allowed to any corrections. The CRFs of each center will be reviewed regularly by clinical inspectors for data quality inspection. The investigators will input and manage the electronic CRFs (eCRFs) online through the Clinical data management system (eCDMS3.0) of Xiyuan Hospital, China Academy of Chinese Medical Sciences. Data input was repeated by 2 investigators to ensure the data accuracy. In addition, all paper documents will be preserved in the Jiangsu Provincial Hospital of Chinese Medicine for 5 years after trial completing and only can be viewed by the research team. Besides, all the datasets will be conducted and monitored by Guangzhou Hipower Pharmaceutical R&D Co, Ltd, which acted as Composition of data monitoring committee of this trial. It will audit the trial conduct and datasets every week.3.7t test. If the data does not conform to the normal distribution or the uniformity of variance, Wilcoxon rank sum test or Wilcoxon signed test will be used to analyze them to compare the two groups. A two-sided P-value <.05 will be set as the significant level.Data analysis will be performed by professional statisticians and the main researchers using SPSS 22.0. Statistical analysis included the actual number of subjects selected, cases of shedding and rejection, demography and other baseline characteristics, patient compliance, efficacy and safety analysis. We used frequency tables or percentages for categorical variables, and mean ± standard deviation for continuous variables to describe the characteristics of patients in both groups. Categorical data will be analyzed by chi-square test or Fisher exact test to compare the differences between the 2 groups, while quantitative data with normal distribution will be analyzed by 4 It is of great significance to find Chinese medicine preparations for the treatment of UC to supplement the selection of clinical drugs. UC is a chronic IBD characterized by a relapsing-remitting course owing to recurrent intestinal inflammation. If the disease can be effectively controlled during mild to moderate period, which will greatly reduce the patient's pain and anxiety. In a clinical randomized controlled trial of HEC in the treatment of active UC, we found the efficacy of TCM syndromes of HEC group was 91.09%, and the positive control group  was 84.62% after 6 weeks of treatment. HEC was effective in improving the symptoms of patients with mild to moderate UC. The shortcomings of the study were the short study time, lack of enteroscopy, and the Mayo score cannot be used as the primary therapeutic outcome. Therefore, this study extended the treatment time to 8 weeks, completed colonoscopy and intestinal mucosa biopsy, and evaluated the efficacy from the modified Mayo score before and after treatment. The results from this study may provide evidence-based clinical evidence on the effectiveness, safety, and reduce or substitute mesalazine of HEC.Compared with UC in Western countries, UC in China has some differences in clinical characteristics. And the prevalence and incidence rates of UC in China were increased rapidly.We are grateful to all the researchers and patients who have been involved in this trial.Conceptualization: Jun-Xiang LiProject administration: Jun Dong, Jun-Xiang LiInvestigation: Xiao Ke, Bei-Ping Zhang, Xiao-Jun Yang, Xin-Li Wen, Wei-Yang Zheng, Jun DongFormal analysis: Xiao Han, Pang-Hua DingMethodology: Zhi-Bin Wang, Rui Shi, Tang-You MaoWriting – original draft: Fu-Shun Kou, Lei ShiWriting – review & editing: Lei Shi, Fu-Shun Kou, Jun-Xiang Li


The uncontrolled spread of the coronavirus disease 2019 (COVID-19) has called for unprecedented measures, to the extent that the Italian government has imposed a quarantine on the entire country. Quarantine has a huge impact and can cause considerable psychological strain. The present study aims to establish the prevalence of psychiatric symptoms and identify risk and protective factors for psychological distress in the general population. An online survey was administered from 18–22 March 2020 to 2766 participants. Multivariate ordinal logistic regression models were constructed to examine the associations between sociodemographic variables; personality traits; depression, anxiety, and stress. Female gender, negative affect, and detachment were associated with higher levels of depression, anxiety, and stress. Having an acquaintance infected was associated with increased levels of both depression and stress, whereas a history of stressful situations and medical problems was associated with higher levels of depression and anxiety. Finally, those with a family member infected and young person who had to work outside their domicile presented higher levels of anxiety and stress, respectively. This epidemiological picture is an important benchmark for identifying persons at greater risk of suffering from psychological distress and the results are useful for tailoring psychological interventions targeting the post-traumatic nature of the distress. The exceptional spread of COVID-19 in Italy led the government to implement unprecedented quarantine measures, sealing off the northern regions, initially, followed by the entire country.The coronavirus disease 2019 (COVID-19) pandemic emerged in Wuhan, China, in late 2019; by the start of 2020, it had spread worldwide. At present, Italy is one of the most affected countries: official data  highlighted that, when comparing the psychological outcomes of quarantined versus non-quarantined persons, the former are more likely to show psychological distress ,3,4,5. AThe international literature has examined—for both general and specific  populations—the impact of sociodemographic factors on individuals’ responses to quarantine, levels of distress during health-related emergencies, and other variables associated with a higher likelihood of developing psychological problems. Recent studies ,7,8, addOverall, the aforementioned literature supports the view that imposed isolation has a huge impact on many aspects of people’s lives, causing considerable psychological strain and triggering a variety of psychological problems. Therefore, in the current context of Italy, the negative effects of the quarantine must be addressed alongside the virus outbreak itself. The present study was the first to administer and analyse a nationwide large-scale survey of psychological distress in the general population of Italy during the COVID-19 pandemic. A self-report questionnaire, integrated with the Italian version of the Depression, Anxiety and Stress Scale–21 items (DASS-21) , and twoThe results may assist government agencies and healthcare professionals in safeguarding the psychological wellbeing of the community in the wake of the COVID-19 outbreak in Italy and other affected countries.We assessed the public’s immediate psychological response to the COVID-19 epidemic in Italy using an anonymous online questionnaire that had been assembled by the research team, inspired by the literature. Data were collected over 5 days (18–22 March 2020). The questionnaire was administered cross-sectionally on an online survey platform, which participants accessed via a designated link. The link was disseminated through the main means of communication and social networks, in order to reach a large number of subjects. Expedited ethics approval was obtained from the Institutional Board of the Department of Human Neuroscience, Faculty of Medicine and Dentistry, “Sapienza” University of Rome (IRB-2020-6), in conformity with the principles embodied in the Declaration of Helsinki.A total of 2812 respondents participated in the survey. Inclusion criteria were: (a) 18 years and older, and (b) living in Italy. The online survey was closed on the sixth day following dissemination of the link. All participants voluntarily responded to the anonymous survey and indicated their informed consent within the survey. The procedures were clearly explained, and participants could interrupt or quit the survey at any point without explaining their reasons for doing so. Two respondents were excluded from the sample because they were younger than 18 years of age, and 44 participants were excluded because they were outside Italy during the outbreak.n = 1194, 43.2%) held a high school diploma and were unmarried , students , and childless . Furthermore, most participants reported following the advice of the government to stay at home . More descriptive statistics, including all characteristics considered, are presented in The final sample consisted of 2766 participants: 1982 (71.7%) females and 784 (28.3%) males. The mean age of the sample was 32.94  and most of the sample  sociodemographic data; (b) working life ; (c) acquaintances infected with COVID-19; (d) loved ones infected with COVID-19; (e) previous physical diseases ; (d) previous stressful situations ; and (h) the psychological impact of COVID-19 on depression, anxiety, and stress levels over the past 7 days.Sociodemographic data were collected on biological sex, age, education, marital and parental status, employment status, residential location during the COVID-19 outbreak, and history of stressful situations and medical problems. Moreover, further information related to COVID-19 was collected. Specifically, participants were asked if they were following the government advice to stay at home or if they were continuing to go to work; they were also asked if any acquaintances or loved ones were (or had been) infected with COVID-19.never) to 3 . DASS-21 outcome scores are classified into three ranges: average, high, and extremely high. The DASS-21 obtained high reliabilities in the Italian validation study, with Cronbach’s alphas of 0.74, 0.82, and 0.85 for the Anxiety, Depression, and Stress subscales, respectively; Cronbach’s alpha for the total scales was 0.90. In the Lovibond and Lovibond version of the DASS-21 [Mental health was measured using the Depression, Anxiety and Stress Scale–21 items (DASS-21) .The DASS DASS-21 , the subvery false or often false) to 3 (very true or often true). The overall measure generates scores in the range of 0–75, with higher scores indicating greater overall personality dysfunction. Each trait domain receives a score in the range of 0–5, with higher scores indicating greater dysfunction in that specific personality trait domain. In the present study, we evaluated the dimensions of negative affect and detachment. We selected these two dimensions because the literature indicates a relationship between these traits and internalizing psychopathology  [Personality functioning was investigated using the Personality Inventory for DSM-5–Brief Form–Adult (PID-5-BF) . The PIDanxiety) ,13,14. TDescriptive statistics of the DASS-21 and PID-5-BF subscales are reported in p < 0.05.Referring to the community sample descriptive statistics reported by Bottesi et al. , the DASAssociations between sociodemographic variables and DASS-21 depression, anxiety, and stress levels during the COVID-19 outbreak, are presented in A second multiple ordinal logistic regression analysis was performed, with depression, anxiety, and stress levels as the dependent variables. Negative affect and detachment were entered as covariates together with the sociodemographic variables that resulted as significant in the previous models, considered or covariates  or factors.n = 1859) of respondents had an average level, 17% (n = 470) were in the high range, and 434 (15.4%) were in the extremely high range. The prediction model for depression showed goodness of fit to our observed data (χ2 (22) = 202.781, p < 0.001). The test of parallel lines was not significant (χ2 (22) = 16.783, p = 0.775). Nagelkerke’s pseudo R2 of 0.086 indicated that the significant sociodemographic variables explained approximately 9% of the variability. Lower education levels, female gender, unemployment compared to employment, not having a child, having an acquaintance infected with COVID-19, and having a history of stressful situations and medical problems were associated with higher depression levels.In relation to depression, 67.3% (2 (12) = 721.328, p < 0.001). The test of parallel lines was not significant (χ2 (12) = 20.953, p = 0.051). Nagelkerke’s pseudo R2 of 0.280 indicated that the significant sociodemographic variables explained approximately 30% of the variability. Higher levels of negative affect and detachment were significantly associated with higher levels of depression. Education level and working position were excluded from the final model  of respondents had an average level, 7.2% (n = 198) were in the high range, and 318 (11.5%) were in the extremely high range. The prediction model for anxiety showed goodness of fit to our observed data (χ2 (22) = 166.487, p < 0.001). The test of parallel lines was not significant (χ2 (22) = 19.765, p = 0.598). Nagelkerke’s pseudo R2 of 0.083 indicated that the significant sociodemographic variables explained approximately 8% of the variability. Young age, female gender, having a family member infected with COVID-19, and a history of stressful situations and medical problems were associated with higher anxiety levels.In relation to anxiety, 81.3% (2 (7) = 418.594, p < 0.001). The test of parallel lines was not significant (χ2 (7) = 3.620, p = 0.822). Nagelkerke’s pseudo R2 of 0.200 indicated that the significant sociodemographic variables explained 20% of the variability. Higher levels of negative affect and detachment were significantly associated with increased levels of anxiety. Age was excluded from the final model  = 177.072, p < 0.001). The test of parallel lines was not significant (χ2 (22) = 23.936, p = 0.351). Nagelkerke’s pseudo R2 of 0.079 indicated that the significant sociodemographic variables explained approximately 8% of the variability. Higher levels of stress were associated with young age, female gender, having to go out to work, having an acquaintance infected with COVID-19, and a history of stressful situations and medical problems.In relation to stress, 2012 (72.8%) of respondents were in the average range, 404 (14.6%) were in the high range, and 347 (12.6%) were in the extremely high range. The prediction model for stress showed goodness of fit to our observed data (χ2 (8) = 530.980, p < 0.001). The test of parallel lines was not significant (χ2 (8) = 13.842, p = 0.086). Nagelkerke’s pseudo R2 of 0.222 indicated that the significant sociodemographic variables explained 22% of the variability. Higher levels of negative affect and detachment were significantly associated with increased levels of anxiety. A history of stressful episodes and medical problems was excluded from the final model  indicate that, in Italy, only about 6% of adults aged 18–69 report depressive symptoms, compared to the higher percentages of high and very high levels of distress found here. In line with the aforementioned review [Overall, our results showed an increased percentage of people with high and very high levels of distress compared to the European epidemiological statistics ,21. For The results indicated that female gender was associated with increased anxiety, depression, and stress. This finding is in line with the results of previous studies ,7, whichAnother finding was the association between a history of stressful situations and increased anxiety and depression. This result adheres to the findings of researches highlighting that persons with a history of trauma suffer from long-term effects of the trauma , often iThe present study also found an association between a history of medical issues and increased anxiety and depression. This finding echoes that of previous studies indicating that chronic illness or a self-evaluation of poor health is associated with increased psychological distress ,16. A poIn the present research, only young age was found to be associated with increased stress. The literature reports mixed results for this variable, indicating a greater psychological impact for both young adults and the elderly ,25. SomeOur results showed that having an acquaintance infected with COVID-19 was associated with increased depression and stress, whereas having an infected relative was associated with increased anxiety. In contrast, Wang et al. reported that neither having an infected acquaintance nor having an infected relative was associated with psychological distress, although their participants showed high levels of concern about the health of their family members .To the best of our knowledge, the present study represents the first attempt to provide data on the association between having to leave one’s domicile for work and stress levels during the COVID-19 outbreak. In more detail, the results showed that having to leave one’s domicile for work was associated with increased stress. The health-related measures issued by the Italian government indicate that only a limited number of key workers  are permitted to work outside their domiciles. Such workers tend to have a high level of contact with the public, and are thus at greater likelihood of being infected; this may increase their stress levels.Finally, the present study found that not having children was associated with depression. This result does not adhere to the prior findings , which hOverall, female gender, negative affect, and detachment were associated with higher levels of psychological distress. Having an acquaintance infected with COVID-19 increased both depression and stress, whereas a history of stressful situations and medical problems raised depression and anxiety levels. Finally, having a family member infected with COVID-19 and being young in age and needing to leave one’s domicile to go to work were found to increase anxiety and stress levels, respectively.As the first step, this epidemiological picture could facilitate the identification of persons at greater risk of suffering from psychological distress, which can have psychopathological consequences and impair functioning. However, only 30% (maximum) of the variability in depression, anxiety, and stress was explained by sociodemographic and personality factors. This means that the majority of the increase in distress levels noted in this research was likely related to the maladaptive and traumatic course of the pandemic—the effects of which can only be assessed empirically at the end of the emergency. Thus, while these results provide an important benchmark for identifying subjects at risk, they may also be useful for tailoring psychological interventions targeting the post-traumatic nature of the distress.One limitation of this study is that, not having a DASS-21 baseline of pre-pandemic data, accurate pre–post analyses could not be conducted; therefore, we cannot be sure of any increase in distress levels or whether any increase  was COVID-19 related. However, comparing the mean scores obtained for depression, anxiety, and stress between the DASS-21 normative sample and the sample gathered in this study, we observed a difference: scores of our sample were higher than those of the normative sample, for all three dimensions , whereas in our sample it was 5.34 (4.8)).Our results offer a general picture of the psychological impact of COVID-19 on the Italian population, providing a baseline for future research on the impact of COVID-19 throughout the rest of the pandemic. However, further studies should carry out multilevel analysis , with the aim of differentiating distress level in order to develop more targeted interventions.


Poststroke depression (PSD) is an important complication of stroke, resulting in increased disability and mortality, which is a great threat to stroke survivors and public health. Complementary and alternative medicine (CAM) therapies is widely used in the treatment of PSD, However, the selection strategies of different CAM approaches in clinical practice is still not clear, and the purpose of this protocol is to compare the efficacy and acceptability of different CAM therapies using systematic review and network meta-analysis.According to the strategy, the authors will retrieve a total of seven electronic databases by August 2020, including PubMed, the Cochrane Library, EMbase, China National Knowledge Infrastructure, China Biological Medicine, Chinese Scientific Journals Database, and Wan-fang databases. The network meta-analysis will be performed using Aggregate Data Drug Information System 1.16.8 and Stata 13.0 software. In addition, the Cochrane Collaboration's tool is employed for the methodological quality, and the quality of evidence will be evaluated according to the Grading of Recommendations Assessment, Development, and Evaluation system.This study will provide a reliable evidence for the selection strategy of CAM therapies for PSD.The results of this study will provide references for evaluating the effects of different CAM therapies on PSD, and provide decision-making references for clinical practitioners, patients, and health policy makers.This study does not require ethical approval. the results will be disseminated through a peer-reviewed publication.DOI 10.17605/OSF.IO/TNGH6. The major symptoms of early PSD (within the first 3 months after stroke) are dysphoria, melancholia, and vegetative signs.,6 The current evidence indicates that the neurobiological factors may be the main factors associated with PSD, specifically includes change in ascending monoamine pathways, excess of proinflammatory cytokines, dysfunction of the hypothalamic-pituitary adrenal axis and alterations in neuroplasticity. Studies have demonstrated that PSD can significantly compromise quality of life, including affecting cognitive function, social activity, and stroke rehabilitation. Moreover, it is also associated with increase mortality risk.,9 Current research suggests that disability, personal and family history of a psychiatric illness, and high overall medical burden may be risk factors for PSD.,11 Due to the complexity of diagnosis and the uncertainty of various screening tools, consequently, only a small percentage of PSD patients can be accurately diagnosed and treated. The main therapeutic strategies for PSD include pharmacological and nonpharmacological interventions . In the pharmacological interventions, it has been suggested that Selective Serotonin Reuptake Inhibitors is the first line treatment, such as fuoxetine, sertraline, and citalopram. There is no doubt that the pharmacological therapy for PSD has a positive effect. However, there was also a significant increase in adverse events, such as gastroenterological symptoms, epilepsy/ seizures and hyponatremia. In addition, intolerance of antidepressants by some stroke survivors, and poor treatment adherence may further reduce the impact of drugs in PSD treatment. Thus, better strategies for effective PSD treatment are needed.Poststroke depression (PSD) is the most common neuropsychiatric consequences of stroke, Many CAM modalities have been used by stroke survivors all around the world, including acupuncture, meridian acupressure, light therapy, exercise, repetitive transcranial magnetic stimulation (rTMS), music therapy, herbal medicines and so on. One study reports that 46% of stroke survivors engage in some form of complementary medicine. In Korea, 54% of stroke patients used CAM therapies, and 16% who felt that it can effectively achieve psychological relaxation. In recent years, CAM therapies has been increasingly sought by people with PSD. It is reported that acupuncture is more effective than short-term use of antidepressants in patients with PSD. Deng et al found that rTMS is a beneficial therapeutic method for managing PSD and may even be superior in efficacy to selective serotonin reuptake inhibitors. Kim et al reported the positive roles of music therapy on improvement of depressive mood and anxiety in stroke patients. A study from Kang et al has proven Meridian acupressure benefits in improvement of PSD.Complementary and alternative medicine (CAM) therapies refers to a diverse range of healing techniques that are not considered established or standard practices in western medicine.Despite the numerous CAM therapies for PSD has been evaluated in previous randomized controlled trials (RCTs), However, majority have not been quantitatively analyzed in head-to-head comparisons. Thus, we performed a network meta-analysis (NMA) of all RCTs involving CAM therapies for PSD, to compare and comprehensively rank all available CAM therapies, and assess efficacy and acceptability of different CAM therapies.22.1 and the checklist will be upload as an attachment. The NMA protocol has been registered on Open Science Framework platform (https://osf.io/tngh6/), registration number: DOI 10.17605/OSF.IO/TNGH6.This protocol based on the Preferred Reporting Items for Systematic Reviews and Meta-Analyses Protocols (PRISMA-P) guidelines,2.2This research does not require ethical approval.2.3The inclusion and exclusion criteria of this research were based on the PICO principle .2.3.1All studies including patients with PSD, regardless of age, gender, ethnicity, profession, and educational background. Specific diagnostic criteria include:1.A clinical diagnosis of ischemic or hemorrhagic stroke;2.PSD confirmed by specific criteria, such as Diagnostic and Statistical Manual of Mental Disorders , International Classification of Diseases (ICD-10) or depression scales, such as Hamilton Depression Scale (HAMD) scale.2.3.2Interventions comprised complementary and alternative therapies for treating PSD , These interventions can be used alone or in combination. Controlled interventions included control groups with no treatment, sham/placebo groups, or other conventional therapy.2.3.3Primary outcomes. The primary outcome was the change in overall depressive symptoms, measured by standardized instrument such as the Hamilton Depression Scale (HAMD) or Montgomery-Asberg Depression Rating Scale (MADRS) or Beck Depression Inventory (BDI) or Zung Self-Rating Depression Scale (ZDS) from baseline to end point.Secondary outcomes1.Activities of daily living measured by established and validated assessment tools, for example, Barthel index.2.Quality of life obtained from the corresponding scale, for example, the Stroke Specific Quality of Life Scale.3.Adverse events.2.3.4All relevant RCTs using CAM therapies for the PSD will be included, Quasi-RCTs, duplications, animal trails, review documents, clinical experience, letters, meeting abstracts, and case reports will be excluded. Additionally, only English and Chinese literature will be search for this study.2.4The following 7 databases will be searched by computer for RCTs on complementary and alternative therapies for PSD: PubMed, the Cochrane Library , EMbase, China National Knowledge Infrastructure (CNKI), China Biological Medicine (CBM), Chinese Scientific Journals Database (VIP), and Wan-fang databases. The time limit of document retrieval is from the establishment of each database to August 31, 2020. The language is confined to English and Chinese. Moreover, inclusive literature from the field and references from previous evaluations will be manually retrieved to find other potentially relevant articles. The retrieval mode will be a combination of free words and medical subject headings terms, including: “poststroke depression,” “PSD,” “stroke,” “depression,” “complementary and alternative therapy,” “acupuncture,” “meridian acupressure,” “light therapy,” “Yoga",” “rTMS,” “music therapy”,” “herbal medicines,” and “Tai Chi,” etc. The following terms will be used in the Chinese database retrieval: “yiyu,” “cuzhong,” “zhongfeng,” “buchongtidai,” “zhenjiu,” “guangliaofa,” “caoyao,” “yujia,” “taiji,” “yinyueliaofa,” etc. The initial retrieval strategy for PubMed is shown in Table 2.5The study selection program will follow the Prisma guidelines, As shown in Figure 1.The retrieved literatures will be imported into Endnote X9 software for rechecking, and duplicate references are removed;2.By reading the title and preliminarily screening the abstract, exclude the literature that obviously does not meet the inclusion criteria;3.Download and read the full text for rescreening;4.After the final inclusion, the predesigned data extraction table is used for data extraction, and the results will be cross-checked;5.If there is any disagreement, the third researcher (Ailing Huang) will be asked to assist in the judgment.The main content of data extraction includes basic information of literature , basic situation of the research object , outcome data . At the same time, the key factors of bias risk assessment are extracted.2.6Methodological quality will be assessed based on the Cochrane Collaboration's tool (Cochrane Handbook 5.1.0). Two trained researchers (Kai Song and Ailing Huang) will independently evaluate the risk of bias of the included studies. In case of dispute, submit to corresponding author (Hong Zhang) for arbitration.:Cochrane bias risk assessment tool will be used to assess the risk of RCTs being included in NMA, including1.random sequence generation;2.allocation concealment;3.blinding of participants and personnel;4.blinding of outcome assessment;5.incomplete outcome data;6.selective reporting;7.other bias.2.72.7.1 Aggregate Data Drug Information System software uses markov chain-monte carlo (MCMC) algorithm to for priori evaluation and processing the extracted data based on bayesian framework, so as to provide support for further research and decision. Preset model parameters: 4 chains are used for simulation analysis, with an initial value of 2.5, a step size of 10, 20,000 annealing times, and 50,000 simulation iterations. Firstly, the network evidence plot is generated according to different outcome indicators, standardized mean differences or odds ratios is used as the effect quantity for statistical analysis, both with 95% credible intervals. According to the results of the NMA, rank probability plot of various CAM therapies is generated and sorted by dominance, with Rank1 being the optimal sort. However, if the score standard of the outcome is lower and greater, then the lowest ranked prediction sequence is the optimal sequence.This study will use Aggregate Data Drug Information System 1.16.8 for NMA.2.7.2P > .05) between direct comparison and indirect comparison, the consistency model is used, whereas the inconsistency model is used for analysis. If the consistency model is adopted, then the stability of the results is verified by the inconsistency model: when the inconsistency factors including 0, at the same time inconsistency standard deviation including 1 says the result of consistency model is more stable and reliable. At the same time, various analysis models are iterated with preset parameters, and the convergence of iteration effect is judged by potential scale reduced factor (PSRF). When the PSRF value is close to or equal to 1 (1 ≤ PSRF ≤ 1.05), the convergence is complete, the model has good stability, and the conclusion of analysis is reliable. If the PSRF value is not in this range, the iteration continues manually until the PSRF value reaches the range standard.Node-split model is used to verify the consistency of the corresponding data. If there is no statistical difference (2.7.3I2 value. When I2 > 50%, it indicates that there is heterogeneity between studies, and the source of heterogeneity should be further searched. When I2 < 50%, inter-study heterogeneity is considered to be small or there is no obvious heterogeneity.Before the combination of effect size, the heterogeneity of the included literature is tested using Stata 13.0 software. When inter-study heterogeneity exists, the random effect model is used. For comparison of each pair, heterogeneity is assessed by the statistic 2.7.4If necessary, the sensitivity analysis will be used to assess the effect of each study on the random effects model. The sensitivity of the general combined effect of all outcome indicators is analyzed by the exclusion method. That is, each study is excluded, and the remaining studies will be re-analyzed to identify the stability of the results. If there is no qualitative change in the combined effect showed in the results, the results are stable.2.7.5If necessary, we will conduct a subgroup analysis of duration of treatment, age, the clinical course of PSD, and research quality.2.7.6If 10 or more studies are included in the NMA, a comparison-adjusted funnel plot is developed using Stata to evaluate the presence of small sample effects or publication bias in the intervention network. Descriptive analysis will be carried out through the symmetry of funnel plot. If the plot is asymmetric and there is no inverted funnel shape, it indicates that there may be publication bias. This may be related to the difficulty in the publication of the literature with negative results and the low quality of the inclusion methods.2.7.7If the required data is lost or incomplete, we will contact the corresponding author of the original document or the relevant email address of the first author. If there is no response, the record is excluded.2.7.8 Based on the risk of bias, inconsistency, imprecision, indirection, and publication bias, Grading of Recommendations Assessment, Development and Evaluation grades evidence quality into four levels: high, medium, low, and very low.To grade evidence quality and understand the current situation of evidence rating thereby analyzing possible problems, The Grading of Recommendations Assessment, Development and Evaluation instrumental will be used to assess the quality of evidence in the NMA.2.7.9There was no patient or public involvement in the preparation of this protocol.3 However, there is no decision-making conclusion as to which CAM strategy should be preferred in clinical practice of PSD. Thus, our study employed an NMA of relevant RCTs of CAM therapies for PSD, including acupuncture, meridian acupressure, light therapy, exercise, rTMS, music therapy, herbal medicines etc, to evaluate the efficacy and acceptability of different CAM therapies treatments. As we have seen, this study will be the first NMA in this respect. We hope that the study results will provide useful references for PSD clinical practice or guideline to a certain extent.PSD is a common sequela of stroke, which can reduce the quality of life and increases the risk of natural and suicidal death, and bring heavy burden to patients and their families. Globally, an ever-growing number of stroke survivors with PSD using CAM therapies to treat their symptoms, The advantage of CAM therapy is that it is not only considered as effective as a conventional pharmacological interventions, but also more natural and economical, with fewer side effects and available without need of prescription.However, there are some potential limitations that are predictable in this study. For example, due to the limitations of language ability, we only search for literature in English and Chinese, which may lead to selection bias. In addition, difference of methodological quality in the trials may lead to significant heterogeneity.Nevertheless, the ranking of different CAM therapies can guide clinical decision makers to select the best therapeutic strategies for PSD. The findings and results of this study will be published in a peer-reviewed journal.Conceptualization: Kai Song, Fanjie Xiong, Yating Wang.Data curation: Kai Song, Fanjie Xiong, Ailing Huang.Formal analysis: Fanjie Xiong, Haiyan Wang.Funding acquisition: Hong Zhang.Methodology: Kai Song, Fanjie Xiong, Ailing Huang.Project administration: Kai Song, Yating Wang, Haiyan Wang.Writing – original draft: Kai Song, Fanjie Xiong.Writing – review & editing: Hong Zhang.All the authors have approved the publication of the protocol


The aim of this systematic review and meta-analysis is to assess effectiveness and safety of Chinese medicine (CM) as complementary therapy in treating coronavirus disease 2019 (COVID-19).The following databases will be searched: PubMed, Cochrane, Embase, China National Knowledge Infrastructure, Chinese Science and Technology Periodical Database, and Wanfang database from October 1, 2019 to March 1, 2020. Randomized trials and quasi-randomized or prospective controlled clinical trials of CM that reported data on COVID-19 patients will be included. Study selection, data extraction, quality assessment, and assessment of risk bias will be performed by 2 reviewers independently. Odds ratios and correlative 95% confidence intervals will be calculated to present the association between the CM and CWM using Review Manager version 5.3 when there is sufficient available data.The results will be disseminated through a peer-reviewed journal publication.This systematic review findings will summarize up-to-date evidence for that CM is more effective and safe as adjunctive treatment for patients with COVID-19.Ethics approval and patient consent are not required as this study is a systematic review based on published articles.CRD42020185382. The COVID-19 outbreak has been a serious threat to global public health. It has raised more attention on the epidemic and has caused widespread panic. Recent statistic analysis showed the mortality rate is 2.1% with a median survival time of 6.4 days.–6 Since December 2019, it has led to a rising rate of pneumonia cases due to its characteristic of infectiousness. COVID-19-infected pneumonia is characterized by flu-like symptoms such as fever, cough, severe acute respiratory distress syndrome, and even death, most of them had a positive prognosis.–9 Unfortunately, presymptomatic or asymptomatic transmission of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has been reported.,11 Mass isolation, forceful measures, and advanced treatment play a vital in this event. After the outbreak of COVID-19, the state administration of traditional Chinese medicine (TCM) organized experts to make a TCM therapeutic scheme after careful consideration and discussion. Subsequently, some patients were cured after TCM treatment, which promoted the widespread application of TCM in patients with COVID-19 pneumonia. On January 27, 2020, the General Office of the National Health and Health Commission of China and the Office of the State Administration of TCM issued “Diagnosis and Treatment of Pneumonia Caused by Novel Coronavirus Infection.” This included a scientific and detailed TCM treatment plan and required local health committees to implement and strengthen the integration of TCM and CWM.Coronavirus disease 2019 (COVID-19), a novel and acute infectious disease, is currently producing an outbreak of pandemic proportions, whereby nearly 4.4 million people have already been infected worldwide until May 10, 2020.Nevertheless, due to the “personalized” characteristic of TCM treatment, it is difficult to formulate standard treatment rules, resulting in uncertainty of TCM clinical efficacy. Therefore, it is necessary to conduct rigorous and objective quality evaluation for different types of clinical studies, and the results of analysis obtained on this basis are more convincing. With respect to previous review, the main outcomes were evaluated based on TCM symptoms, which were subjective without nucleic acid analysis results. In addition, there were more retrospective studies and fewer RCTS in previous studies. This review was conducted based on more RCTS with relatively more objective outcome assessment such as the disappearance time of main symptoms and nucleic acid. The review was updated according to the present evolution for providing a robust evidence base for clinical practice in treating COVID-19 pneumonia.2https://www.crd.york.ac.uk/PROSPERO/.This systematic review was registered on PROSPERO (CRD42020185382) on May 11, 2020. The registered website for this protocol is 2.1Six databases including PubMed, Cochrane, Embase, China National Knowledge Infrastructure, Chinese Science and Technology Periodical Database, and Wanfang database were searched from October 1, 2019 to March 1, 2020. If any, we would try to contact the original study authors for the information we need. What is more, we would perform a manual search to track the references of relevant literature. Then we browsed the detail of the abstract and the full text, and selected eligible studies according to the inclusion criteria. The detailed search strategy for PubMed is demonstrated in Table 2.22.2.11.Studies: Randomized trials and quasi-randomized or prospective controlled clinical trials that have tested traditional Chinese herbal medicine with or without western medicine for COVID-19 will be included. There will be no restrictions for blinding, follow-up, or publication status. Publications in English and Chinese will be included.2. without immediately life-threatening comorbidities will be included.Participants: Patients diagnosed with COVID-19 referred to Diagnosis and Treatment of Pneumonia Caused by Novel Coronavirus Infection ,3.Interventions: Traditional Chinese herbal medicine involving a variety of forms, including pill, injection, capsule, and decoction. There will be no restrictions with respect to the type of comparator.4.–21Outcomes: Our primary outcomes were effective rate, rate of severe illness, and adverse events. We will also assess the following secondary outcomes: days to disappearance of fever, days to disappearance of cough, days to be negative of nucleic acid, and length of stay in hospital. If other outcomes were reported in the eligible studies, these will be extracted and reported but we will give particular attention to the possibility of selective reporting bias when using any such outcomes in our review.2.2.21.Patients with life-threatening comorbidities likely to lead to death within the trial follow-up period2.Duplicated data or data that cannot be extracted after contacting original authors3.Case reports, reviews, mechanisms, unqualified interventions, and animal model experiments2.3Relevant information was extracted and cross checked by 2 independent reviewers (MZ and FX). The extracted data included the first author, sample size, age, interventions, outcomes, duration, randomization method, blinding of participants and personnel, allocation concealment, incomplete outcome data, follow-up, dropout and withdrawal, and adverse events. If there were disagreements between 2 reviewers, a 3rd reviewer (QL) was available to check for accuracy. Details of the selection procedure for studies were shown in a Preferred Reporting Item for Systematic review and Meta-analysis protocol (PRISMA-P) flow chart Fig. .2.4The assessment was performed by RevMan 5.3.3 according to the cochrane handbook. The overall assessment was based on details including random sequence generation, blinding of participants and personnel, allocation concealment, incomplete outcome data, selective reporting, and other biases. If we encountered differences, we would discuss with the 3rd reviewer (QL) and finally reach an agreement. The quality assessment was graded as “high” risk, “low” risk, or “unclear” risk depending on the degree of information provided.2.5P < .05 was to be of statistical significance. There was no heterogeneity between studies (I2 < 50%), the fixed effect model was adopted; otherwise the random effect model was used. Considering that the treatment effect may be related to the treatment time, the data were analyzed by subgroup according to different intervening measure. Sensitivity analysis was conducted to evaluate the impact of the included studies on the final outcome. Forest plots and Egger test was conducted to assess potential publication bias. If P < .05, this was considered to be statistically significant.Statistical analysis was conducted by the Review Manager 5.3 and stata 12.0 software. In this meta-analysis, the mean difference and the odds ratio were adopted to evaluate continuous variable outcomes and dichotomous outcomes with a 95% confidence interval. 2.6The quality of evidence of outcomes will be assessed according to the Grading of Recommendations Assessment, Development and Evaluation system. The GRADE system includes 5 items: the risk of bias, inconsistency, indirectness, imprecision, and publication bias. The quality of evidence will be rated as “high,” “moderate,” “low,” or “very low.”3Based on the basis of summarizing the experience of TCM in the treatment of SARS, the General Office of the National Health and Health Commission of China and the Office of the State Administration of TCM encouraged the combination of TCM and CWM. and the control group released adverse reactions only on 8 patients. In this study, the experimental group is treated with Abby dole and Lianhua Qingwen Jiaonang, and the control group is treated by Lianhua Qingwen Jiaonang alone. Abby dole manuals shows drug incidence of adverse events is about 6.2%, mainly for nausea, diarrhea, dizziness, and elevated serum transaminase. In this study, no records of clinical adverse reactions were found in all cases except elevated serum transaminase, suggesting to some extent that the safety of the combined application of the 2 drugs is acceptable, but it also suggests that we need to do a good exploration in pharmaceutical care during the process of medication. On the whole, the data analysis shows that the adverse reactions mainly occur in the application of CWM, while the application of TCM is relatively safe and less adverse reactions occur.This review was conducted based on the basis of the existing COVID-19 disease with an overview of the application of TCM for treating COVID-19 patients. In the treatment of COVID-19, TCM as an adjuvant therapy can significantly improve the effective rate, reduce the rate of severe disease, shorten the disappearance time of fever and cough, as well as the hospital stay of patients, and the most objective index is to significantly shorten the time of nucleic acid to be negative. In terms of adverse reactions, there is no significant difference with western medicine, indicating that TCM is safe as an adjuvant therapy. Nevertheless, the experimental group has released adverse reactions along with the increase of serum transaminase on 14 patients in study,Since the COVID-19 epidemic has not been completely subside and more large-sample clinical studies are still under way, the evidence of the current analysis has not yet been persuasive. After the epidemic is fully controlled and more research results are included in the analysis, we look forward to further updating and supplementing the systematic evaluation.Conceptualization: Qiulan Pei, Hang Wen.Data curation: Qiulan Pei, Hang Wen.Formal analysis: Min Zhou, Fan Xu.Funding acquisition: Qijun Liang.Investigation: Qiulan Pei.Methodology: Min Zhou, Fan Xu.Software: Qiulan Pei, Min Zhou, Fan Xu.Writing – original draft: Min Zhou, Fan Xu.Writing – review & editing: Min Zhou, Fan Xu, Qijun Liang.