To investigate whether prostaglandin analogue (PGA) eyedrops have a significant effect on central corneal thickness (CCT), we conducted a systematic search of literature published from 2000 to 2021. Among the studies conducted on topical PGA therapy in open-angle glaucoma or ocular hypertension patients over 18 years old, prospective studies with CCT change as an outcome were included. A single-arm meta-analysis was conducted to assess the overall effect on CCT, and subgroup analysis according to exposure time of PGA eyedrops was also performed. We counted the number of articles that reported on severe events (CCT reduction of 25 μm or more) and obtained their proportion. The methodological quality was assessed by the McHarm tool. Twenty-two reports of prospective studies were selected. The results of the single-arm meta-analysis showed very high heterogeneity. Still, in subgroup analysis, when PGA was used for more than 6 months, heterogeneity was low, and a significant decrease in CCT was observed. Severe events were reported in two reports and occurred in 3.8% to 14.8% of participants. PGA eyedrop use may cause a clinically significant CCT decrease, requiring CCT follow-up. Prostaglandin (PG) is a lipid-signaling substance in the family of eicosanoids and is found in almost all tissues of humans and animals. Structural differences between PGs result in variable biological actions. Depending on the type of receptor to which even the same PG binds, the opposite action may appear in one tissue than in another. The PG F receptor in the eye is distributed in the ciliary muscle and trabecular meshwork. When it binds to PG F2a, it promotes the outflow of aqueous humor through the uveoscleral pathway, reducing intraocular pressure (IOP). Although the exact mechanism of this action is not well known, the PG F2a analogue (PGA), an agonist of the PG F receptor, upregulates matrix metalloproteinase (MMP) and degrades the extracellular matrix (ECM) of the ciliary muscle and sclera, reducing the resistance of uveoscleral outflow ,2,3,4,5.In 1996, Latanoprost, the first PGA eye drop, was approved for clinical use for glaucoma in the United States. After that, the FDA approved Travoprost, Bimatoprost, and Tafluprost as eye drops for glaucoma. PGA eye drops are currently the most widely used intraocular pressure-lowering agents. According to a meta-analysis, PGA eye drops showed a 24–33% reduction in IOP and, as a single agent, showed a stronger IOP lowering effect than other conventional glaucoma eye drops ,8.In glaucoma, central corneal thickness (CCT) has two important implications. The Ocular Hypertension Treatment Study (OHTS) suggested that CCT is a risk factor for glaucoma. In that study, for every 40 μm decrease in CCT, the incidence of glaucoma increased 1.71 times . Second,Lass et al. (2001) performed a randomized controlled trial (RCT) of the effects of PGA eyedrops on the cornea. In that study, CCT increased by 0.2 ± 3.1% in the group using Timolol eyedrops for one year, whereas CCT decreased by 1.1 ± 2.5% in the group using Latanoprost eye drops. Although the researchers concluded that there was no significant difference between the two groups because this reduction in CCT did not meet the pre-established criteria for significant change, this was the first report of a decrease in CCT with PGA eye drops . Since tIf the CCT is significantly reduced with PGA eye drops, clinical consideration is necessary when prescribing it and during follow-up. Therefore, through a systematic review, the purpose of this study is to investigate whether the use of PGA eye drops has a significant effect on central corneal thickness change.We conducted a systematic search in MEDLINE, EMBASE, and Cochrane CENTRAL on literature published from 1 January 2000, to 14 June 2021. Although Latanoprost was first marketed in 1996, Lass et al. (2001) first raised the possibility that PGA eye drops could cause CCT reduction. Therefore, the literature search in this study started in 2000. In addition, reports found through Website search results (Google) and reports that met the criteria from the reference list of related SRs were included. Among the studies conducted on topical prostaglandin analogue therapy  in glaucoma (open-angle glaucoma (OAG), ocular hypertension (OHT)) those with patients over 18 years of age, and with central corneal thickness (CCT) change as an outcome were included. PGA eyedrops are used to lower intraocular pressure. Since CCT reduction corresponding to adverse effects is a safety issue, randomized controlled trials (RCTs), non-randomized studies , case-control, cross-sectional study), a systematic review (SR), a case report, and a case series were all included. The search terms and search formulas for each DB are attached in This systematic review is subject to IRB review exemption, and the study protocol was exempted from participant consent from the Institutional Review Board of the Catholic University of Korea in May 2021. This systematic review protocol has been registered with PROSPERO, CRD42021258359.For the primary outcome of CCT change after medication use, which is a safety-related subject, we conducted analysis and synthesis of only prospective studies , and the related case reports were reported separately.Inclusion criteria were studies of the effect of PGA eyedrops on CCT for patients with OAG and OHT over 18 years of age. Studies were excluded for: (1) written in languages other than English, (2) not measuring CCT change, (3) non-prospective studies , (4) co-intervention , (5) incomplete or unclear data, and (6) subjects with corneal abnormalities.Changes in CCT when using PGA eye drops were adverse events. Since CCT is a continuous variable, the mean, standard deviation (SD), and total number (N) were extracted, and the unit was micrometers. Finally, the mean difference was calculated and compared . There aThe concept of clinically significant CCT reduction is not yet established, but a change of 25 μm can be considered. With an average CCT of 550 μm, 25 μm corresponds to about 5% of the total thickness. In such a case, for every 25 μm decrease, the intraocular pressure is underestimated by about 1 mmHg . TherefoTwo reviewers independently conducted the literature selection process for the identified records and the retrieved reports according to preset criteria. In the event of disagreement in the literature selection process, a third researcher was consulted, and a consensus was achieved.https://automeris.io/WebPlotDigitizer/, accessed on 8 June 2021.), and the value was estimated and extracted.Data collection was performed independently by two reviewers. From each document, authors, publication year, journal name, study design, study duration, total number of participants, inclusion criteria, clinical setting of the study, country, mean age, sex, number of intervention groups, primary outcome of the study, measurement method of CCT, time-point of measurement, scale of measurement, information of inspector, information of missing participants, key conclusions of authors, and funding source were extracted. The ingredient of eye drops used, exposure time to eyedrops, baseline IOP, number of participants allocated, baseline CCT, and change in CCT  were collected from the PGA treatment group. For Meda et al. (2017), in which CCT variation was presented only as a graph rather than a numerical value, the graph was directly measured with WebPlotDigitizer version 4.4  tool . Among tThe extracted data were narratively synthesized and analyzed to assess the overall effect of PGA eyedrops on CCT change. Baseline CCT and follow-up CCT value, SD, and total N of the PGA treatment group underwent single-arm meta-analysis. A forest plot was drawn and visually shown to explore heterogeneity, and the i-square test was also performed. If the i-square value was greater than 25%, meta-analysis was performed using a random-effect model. To determine the effect of the duration of PGA eyedrop use on CCT change, a subgroup analysis was performed on studies that presented results within 4 months, 6 months, 12 months, and 24 months or more of eyedrop use. Since this study aimed to show the effect of PGA eye drops on CCT change, if each of the four kinds of PGA drugs was used as an independent treatment arm, the results of each PGA eyedrop were merged into one outcome and analyzed. Second, as a safety issue, we counted the reports that separately reported a reduction of 25 μm or more, which is considered here a clinically significant change in CCT. The proportion of subjects who showed a decrease of 25 μm or more among the total number of participants in the PGA treatment group from the counted reports was calculated. To evaluate publication bias for studies included in the meta-analysis, funnel plots were drawn. Statistical analysis was performed using Comprehensive Meta-Analysis (CMA) version 3 .Of 14,148 records searched via database and registers, 8913 were chosen. Among them, eligibility was assessed for 1018 reports, among which 20 reports were selected. Two reports were selected by assessing eligibility for ten reports out of eleven records retrieved from other sources. A systematic review was conducted on 22 prospective studies .n = 3880 participants). Among them, seven were RCTs, and one of the seven compared PGA with TML. Two of fifteen NRSs were secondary analyses of RCTs. These studies were conducted in 12 countries in Asia, Europe, and North and South America. Five studies were funded by pharmaceutical companies that manufacture PGA eye drops. In the 22 studies, the duration of medication use ranged from 1 month to 8 years. The components of PGA eyedrops used in the studies were LTP in 16 studies, TVP in 9, BMT in 7, and TAF in 1. However, three studies did not mention the eyedrop ingredients. For CCT measuring devices, USP was used in 15 studies, and non-contact type devices were used in six. Only Johny et al. (2020) did not mention the measurement instrument, or method, or the investigator who measured it [There were 22 prospective studies  and Viswanathan et al. (2013) analyzed the CCT change with aging. In a previous cross-sectional study of OHTS, CCT decreased by 0.63 μm per year , while tA clinically significant decrease of 25 μm or more, a severe event, would take 40 years or more to occur naturally, considering a reduction of 0.63 μm/year with aging. Clinically significant corneal thinning was not reported among 668 RCTs with PGA eyedrops, and severe events were reported by only 2 NRS among 22 published prospective studies on CCT changes of PGA eye drops. The proportion of subjects who showed a clinically significant decrease of 25 μm or more in these 2 studies ranged from 5.1 ± 0.6% to 14.8 ± 1.6% among PGA eyedrops users for 12 months. Considering that 1 mmHg of IOP is underestimated for every 25 μm decrease in CCT , 1 mmHg CCT reduction by exposure to PGA eyedrops appears to be reversible. In Meda et al. (2017), CCT increased after 6 weeks of discontinuation in subjects who had been using PGA eyedrops for more than 1 year and decreased again after 6 weeks of re-use. There were no baseline data before using PGA eyedrops, so we cannot know whether CCT had recovered to the level before the first use of the eyedrop. However, after 6 weeks of re-use, CCT decreased to the level of that achieved with use for more than 1 year . AnotherThere are three proposed mechanisms for CCT changes following PGA eyedrop instillation. First, PGA eyedrops increase the expression of IL (interleukin)-1 and IL-6, which are inflammatory cytokines on the ocular surface, and changes in these cytokines increase the production of MMP ,28,29. IIn a cross-sectional study using Casia OCT by Amano et al. in 2019, when PGA eyedrops were used in patients with open-angle glaucoma without corneal disease or surgery, there were changes in the biomechanical factors of the cornea, including CCT. Still, there was no change in the shape itself . HoweverThe limitations of this study are that, first, the analysis was not able to be conducted on comparative studies between the two groups. In the studies for PGA eye drops, TML eyedrops were usually used as a control group, which is appropriate to evaluate the effect of lowering IOP. However, in studies for CCT change, since TML is also known to affect CCT change, it is not suitable as a control. Therefore, this study performed a single-arm meta-analysis only on the PGA treatment group. Second, there is a lack of studies on CCT changes with PGA eyedrops. Two studies which reported severe events were NRS, and no RCT reported severe events. In future prospective studies on PGA eye drops, it is necessary to consider CCT changes in advance. Third, in this SR, analysis according to type of PGA medication could not be performed. Finally, although this review’s pooled estimate of CCT change was small compared to the average corneal thickness, only 2 NRS studies reported the proportion of subjects who showed a clinically significant decrease. Therefore, it is necessary to conduct a large-scale RCT for CCT change following PGA eyedrop use over 2 years. In that RCT, it will be essential to identify the percentage of subjects showing a clinically significant decrease in CCT after classifying the subjects according to whether CCT has decreased and the amount of decline.


There is evidence that the gut microbiota play a regulatory role in the occurrence and progression of tuberculosis. The purpose of the current study was to explore the alterations in gut microbiome under different tuberculosis disease stages in the Uyghur population, clarify the composition of microbial taxonomy, search for microbial biomarkers and provide innovative ideas for individual immune prevention and for control strategies.A case–control study of Uyghur individuals was performed using 56 cases of pulmonary tuberculosis (PTB), 36 cases of latent tuberculosis infection (LTBI) and 50 healthy controls (HC), from which stool samples were collected for 16S rRNA gene sequencing.Bifidobacterium and Bacteroides increased, while Roseburia and Faecalibacterium decreased in the PTB group, when compared with the other two groups, but the changes between the LTBI and HC groups were not significant. The classifier in the test set showed that the ability of the combined genus to distinguish between each two groups was 81.73, 87.26, and 86.88%, respectively, and the validation efficiency was higher than that of a single screened genus.The results showed that the alpha diversity indexes of the PTB group were lower than those of the other two groups (P <0.001), while only observed species were different between LTBI and HC (P <0.05). Beta diversity showed differences among the three groups (P = 0.001). At the genus level, the relative abundance of The gut microbiota of PTB patients was significantly disordered compared with LTBI and HC, while the changes of LTBI and HC were not significant. In the future, gut microbiota could be used as a non-invasive biomarker to assess disease activity. Mycobacterium tuberculosis (Mtb). It is a major global public health problem and a serious threat to human life and health. The World Health Organization (WHO) indicated that tuberculosis was the leading cause of death from a single infectious source and ranked the 13th leading cause of death worldwide. There were 9.87 million new cases worldwide in 2020, with an incidence rate of 127 per 100,000  is an ancient respiratory infectious disease caused by  100,000 . Latent rculosis . The glorculosis , so the rculosis . The occrculosis , but TB rculosis  and can rculosis . Other erculosis , malnutrrculosis , smokingrculosis , and diarculosis . The immrculosis . Howeverrculosis . There irculosis , so hostechanism .Mtb .Fresh stool samples from the participants were collected using sterile containment and stored at −80°C immediately for further analysis.Total genomic DNA samples were extracted using the QIAamp DNA Stool Mini kit , following the instructions of the manufacturer and stored at −20 °C prior to analysis. PCR was used to amplify the hypervariable V3–V4 region of bacterial 16SrRNA genes with the forward primer, 338F: 5’-ACTCCTACGGGAGGCAGCA-3’ and the reverse primer, 806R: 5’-GGACTACHVGGGTWTCTAAT-3’. The libraries were constructed by for quality inspection. The qualified and quantified PE250 libraries were sequenced using the Illumina NovaSeq platform .Microbiome bioinformatics were performed using Quantitative Insights Into Microbial Ecology (QIIME2 V.2019.4) . Raw seqThe 16SrRNA sequencing data analyses were performed using QIIME2 and R packages (v4.0.2). Alpha diversity indices were characterized by observed species, Pielou’s evenness, the Shannon and Simpson indexes. The Kruskal–Wallis H test was used to assess the differences between the three groups. Beta diversity was described by Nonmetric multidimensional scaling (NMDS), using UniFrac distance metrics and the Analysis of similarities (Anosim) was used to examine differences between groups. After singletons were removed, the feature table was counted to visualize the composition distribution of each sample at the phylum, class, order, family, genus, and species levels, and presented in a histogram. Linear discriminant analysis effect size (LEfSe) is a method of analyzing discrepancy, which directly analyzes the difference between all taxonomic levels simultaneously. The analysis used a cladogram showing the taxonomic hierarchy distribution of biomarker species of each group sample. Microbial potential functions were predicted by phylogenetic investigation of communities by the reconstruction of unobserved states (PICRUSt2) on KEGG databases to get predictive metabolic pathways, and conducted correlation analysis with different species demonstrated by heat maps. The sequence data of this study have been deposited in the GenBank Sequence Read Archive (SRA) of NCBI under the accession code BioProject PRJNA795263.The Spss21.0 software and R Packages (V4.0.2) were used for data analysis. Individual characteristics of the interviewee were statistically described by the median and mean ± standard deviation (SDs) for continuous variables, while the Kruskal–Wallis test and analysis of variance (ANOVA) were used for inter-group differences. Frequency was used for categorical variables and the chi-square test was used for inter-group differences.The Mann–Whitney U test was used for each two-group comparison of the top 50 genus level species relative abundance and species with differences among groups were selected out. The research object was divided into training and testing sets, and the random forest model was used to perform five repetitions of 10-fold cross validation and rank variables by importance based on previously screened differences variables, to find the optimal number of variables and their importance order. Filtered variables were used as predictors to draw receiving operating characteristic (ROC) curves, calculating the area under curve (AUC) and its 95% confidence interval (CI). The ROC curve of a single species of the combined species in each group was evaluated. Rank correlation was used to analyze the correlation between prediction pathways and different species. A P <0.05 was considered statistically significant.There were 56 PTB, 36 LTBI, and 50 HC cases included in this study. Age, gender, and BMI were comparable after statistical evaluation (P >0.05), as shown in A total of 8,193,170 usable high-quality sequence reads were generated from 142 samples by quality filtering, denoising, merging and removing chimeras. These had an average of 57,698 sequences per sample, the sequence length ranged from 50 to 437 bp and the average sequence length was 413 bp. A total of 18,085 ASVs were obtained from the PBT group, 25,506 ASVs from the LBTI group and 27,152 ASVs from the HC groups. A total of 2,846 ASVs were shared among the three groups, where the PBT group contained 12,113 unique ASVs, the LBTI group contained 17,871unique ASVs and were compared to 19,615 unique ASVs from the HC group Figure 1’s evenness index, and the Shannon and Simpson indexes. Overall, the Alpha diversity was decreased in the PTB group, when compared with LTBI and HC groups. The observed species were increased in the LTBI group compared with the HC group (P <0.001)  analysis, using UniFrac distance metrics, with the results in According to the species taxonomic annotation, there were 28 microbial phyla, 88 microbial class, 160 microbial orders, 286 microbial families, 623 microbial genera, and 872 microbial species identified Table S2Firmicutes, Verrucomicrobia, Tenericutes and Cyanobacteria differed among groups. The abundance of Firmicutes and Tenericutes decreased in PTB, comparing with the LTBI and HC groups  was significantly enriched and had the highest abundance in the PTB group. In the LTBI group, Dialister, Oscillospira, and Lactobacillus were enriched at the genus level, while Lactobacillaceae was the highest accumulated at the family level. The phylum of Firmicutes, the class of Clostridia, the order of Clostridiales, the family of Firmicutes and the genus of Roseburia were significantly enriched in the HC group  showed a significant downward trend in the PTB group compared with the other two groups and the relative abundance of Bacteroidetes (B) in PTB and LTBI increased slightly. The F/B ratio is related to the susceptibility of disease occurrence  are both considered to exhibit pro-inflammatory property  and has 87.0% ability to discriminate PTB and LTBI, which is a single genus with high discriminatory ability. Luo et al. noted that Lachnospira decreased in abundance in patients with new and relapsing TB and was negatively correlated with CD4+T cell counts, which are important immune cells for TB , had 75.9% ability to distinguish LTBI from HC. This genus currently contains only one member, the Akkermansia Muciniphila species . The patients/participants provided their written informed consent to participate in this study.YW: Conceptualization, Formal analysis, Data curation, Visualization, Writing – original draft, Writing – review & editing. YD: Supervision, Data curation, Writing – original draft. NL: Conceptualization, Supervision, Data curation. YaC: Conceptualization, Data curation. YJ: Formal analysis, Visualization. ZT: Data curation, Formal analysis. ZM: Data curation, Formal analysis. Yu-C: Conceptualization, Formal analysis. YX: Conceptualization, Supervision, Data curation, Writing—original draft, Writing—review & editing. All authors listed have made a substantial, direct, and intellectual contribution to the work and approved it for publication.This study was funded by National Natural Science Foundation of China (No. 81860589). The funders had no role in the study design, data collection and analysis, decision to publish, or preparation of the manuscript.Authors YW and YD are in Xinjiang Production and Construction Corps Center for Disease Control and Prevention (CDC) not a commercial affiliation.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.


Background: Qingfei Paidu decoction (QFPDD) has been widely used in treating coronavirus disease 2019 (COVID-19) in China. However, studies on the treatment effect of COVID-19 patients and other respiratory diseases have not been well demonstrated. Our study aims to determine the treatment effect of QFPDD in combination with conventional treatment on COVID-19 patients and other respiratory diseases.Methods: This retrospective study recruited COVID-19 patients who were treated with QFPDD for at least two courses (6 days) from seven hospitals in five provinces from January 21 to March 18 2020. Demographic, epidemiological, clinical, laboratory, computed tomography characteristics, treatment, and outcome data were collected and analyzed. The improvements in clinical symptoms before and after QFPDD treatment were compared.Results: Eight COVID-19 patients were included in this study. Of them, six were males (75.0%). The median age of the patients was 66 (60–82) years. Four patients were classified as mild and moderate cases (50.0%); there were two severe cases (25.0%) and critical cases (25.0%). The most common symptom was cough (7 [87.5%]), followed by fever (6 [75.0%]), fatigue (4 [50.0%]), asthma (4 [50.0%]), and anorexia (3 [37.5%]). Abnormal findings included decrease in neutrophils (3 [37.5%]), lymphocytes (2 [25.0%]), alkaline phosphatase (3 [37.5%]), lactic dehydrogenase (4 [50.0%]), erythrocyte sedimentation rate (2 [25.0%]), and C-reactive protein (5 [83.3%]) at admission. After one course (3 days) of QFPDD, nasal obstruction and sore throat completely disappeared, and fever (5 [83.3%]), fatigue (2 [50.0%]), and cough (2 [28.6%]) were improved. After two courses (6 days), the fever disappeared completely in all patients, and the other symptoms showed a tendency to improve. In non-severe patients, 87.5% baseline symptoms completely disappeared. In severe patients, 61.1% of the baseline symptoms completely disappeared after patients were administered QFPDD for two courses. Of the abnormal indicators, 55.6% returned to normal levels. The median duration to complete fever recovery was 1.0 day. The median durations of viral shedding and hospitalization were 10.5 and 21.5 days, respectively. None of the patients worsened and died, and no serious adverse events occurred related to QFPDD during hospitalization.Conclusion: QFPDD combined with conventional treatment improved clinical symptoms in COVID-19 patients with other respiratory diseases, and no serious adverse reactions associated with QFPDD were observed. Larger sample studies confirm our findings in the future. Coronavirus disease 2019 (COVID-19) is an acute respiratory infection caused by the novel coronavirus (severe acute respiratory syndrome coronavirus 2) and has become a global health emergency . As of NA recent study has shown that COVID-19 cases combined with other respiratory diseases account for 2.3% of all cases, and patients with combined diseases are more likely to have a worse prognosis than patients without combined diseases . AnotherTo date, few studies have detailed the clinical characteristics, laboratory and computed tomography funings, and treatment outcomes of COVID-19 patients with other diseases of the respiratory system. To further explore the effectiveness of QFPDD, we aimed to retrospectively analyze the data of patients treated with QFPDD combined with conventional treatment in seven hospitals in five provinces from January 21 to March 18 2020. The findings of this study will provide a reference for integrating TCM into the management of COVID-19.Eight patients with confirmed COVID-19 and other respiratory diseases who were treated with QFPDD in combination with conventional treatment were enrolled from seven hospitals in five provinces between January 21 and March 18 2020. The inclusion criteria were as follows: 1) laboratory-confirmed COVID-19 cases; 2) comorbid with other respiratory diseases, including but not limited to bronchiectasis, COPD, tuberculosis, lobar pneumonia, chronic bronchitis, and emphysema; and 3) administered with at least two courses (6 days) of QFPDD.Diagnosis and Treatment Guidelines for 2019 Novel Coronavirus Pneumonia (referred to as the Guidelines) issued by the National Health Commission of the People’s Republic of China  shortness of breath, respiratory rate ≥ 30 times/min; 2) oxygen saturation ≤93% at rest; and 3) arterial partial pressure of oxygen (PaO2)/fraction of inspired oxygen (FiO2)≤300 mmHg (1 mmHg = 0.133 kPa). Critical cases were defined as patients with respiratory failure requiring mechanical or noninvasive ventilation, shock, or combined with other organ failure, requiring intensive unit care. We further categorized mild and moderate patients as non-severe cases and severe and critical patients as severe cases. We defined the time to clinical symptom resolution as the time from using QFPDD to the first clinical symptom resolution, lasting at least 24 h. Absorption of lung lesions refers to the absorption of lung lesions shown by lung imaging at discharge.The diagnostic criteria for COVID-19 were based on the of China . AccordiIn the present study, all patients were treated with QFPDD combined with conventional treatment. QFPDD comprises 21 herbs. Chinese, English, and Latin names and the dose of these herbs are shown in We retrospectively collected demographic, epidemiological, clinical, laboratory, computed tomography characteristics, treatment, and outcome data from medical records. The eligibility of the patients for this study and their data were independently checked and extracted by two researchers. If the core data, such as clinical, laboratory and CT findings, and treatment outcomes were unclear or missing, we would contact doctors in charge of the patients to supplement the data as soon as possible.Adverse events included nausea, vomiting, and abdominal pain. If symptoms are relieved after stopping QFPDD treatment, this adverse event may be related to QFPDD. If an adverse reaction persists after stopping QFPDD treatment, then it is less likely to be associated with QFPDD.Continuous measurements as median (IQR) or mean (SD) is taken accordingly to know whether they are normally distributed or not. Categorical variables were described as frequency and percentages. All statistical analyses were performed with SPSS software, version 13.0 (SPSS Inc.).Case 1 involved a 62-year-old man with mild COVID-19 combined with hypertension, diabetes, and bronchiectasis. He had symptoms of a sore throat. After admission to the hospital on February 7 2020, he was administered with interferon alpha, arbidol, lopinavir, and other antivirals, and cefuroxime, cefoperazone, two antibiotics, and glucocorticoids. But the symptoms of sore throat were not relieved significantly. Three days after admission, several TCM doctors were invited to participate in the consultation of this patient, and then he was treated with one dose of QFPDD three times a day. Five days after admission, his sore throat improved. The nucleic acid test result turned negative. Computed tomography (CT) results of the lungs showed no exudation or signs of pneumonia, and no serious adverse events were observed. Nine days after administration, his condition further improved, and he was discharged from the hospital on February 16 2020.Case 2 was a 66-year-old woman with moderate COVID-19, hypertension, coronary heart disease, and diabetes. She visited the hospital because of cough, sputum, blood in the sputum, and fever. After admission on February 5 2020, as the patient’s symptoms of cough did not improve, it was thought suitable to administer QFPDD through a joint consultation including TCM doctors. Therefore, in addition to taking lopinavir–ritonavir and arbidol, she was administered one dose of QFPDD three times a day. After one course of the treatment (3 days), symptoms of fever, anorexia, and nasal obstruction improved. After 7 days of QFPDD treatment, her cough was completely cured. After administering QFPDD for 23 days, the nucleic acid test result became negative. No serious adverse events occurred during the treatment, and the patient was discharged on March 10 2020.Case 3 was a 67-year-old woman with moderate COVID-19 and tuberculosis. She was admitted to the hospital on February 2 2020 with symptoms of cough. After group consultations with doctors practicing Chinese and Western medicine, she was treated with one dose of QFPDD three times a day. In addition, we administered interferon alpha and arbidol. After taking QFPDD for 2 days, her symptoms of cough significantly improved, and the nucleic acid test result turned negative after admission. Furthermore, serious adverse events were not observed. She recovered and was discharged on February 19 2020.Case 4 involved a 72-year-old man with moderate COVID-19 and tuberculosis. He was admitted to the hospital on January 22 2020, and presented with fever and cough. The symptoms were recurrent after conventional treatment and there was no significant improvement. After inviting doctors of TCM to participate in the consultation, he was advised to be treated with one dose of QFPDD three times a day. After taking QFPDD, the fever was quickly relieved. The cough was completely cured after 4 days of TCM treatment. Sixteen days after admission, the symptoms significantly improved and the nucleic acid test result was negative. Serious adverse events were not observed. The patient recovered and was discharged on February 14 2020.Case 5 involved a 60-year-old man with severe COVID-19 who also had type I respiratory failure and community-acquired pneumonia. He was admitted on February 15 2020 and presented with fever, fatigue, cough, and asthma. Once he was admitted, doctors held a group consultation with doctors practicing Chinese and Western medicine to decide whether to use QFPDD. After the evaluation by TCM doctors, he was treated with one dose of QFPDD three times a day. Seven days after admission, the symptoms significantly improved, and the nucleic acid test result was negative. On February 24, chest CT showed improvement in the lung lesions. No serious adverse events occurred during the treatment. The patient was cured and discharged on February 26 2020.Case 6 involved a 66-year-old man with severe COVID-19 and lobar pneumonia. He was admitted to the hospital on February 16 2020 and presented with fatigue, anorexia, and asthma. The doctors held a group consultation with doctors practicing Chinese and Western medicine to decide whether to use QFPDD. This patient was treated with one dose of QFPDD three times a day after admission. After taking two courses of QFPDD, the symptoms were quickly relieved; scattered ground glass, strip, and dot film shadows appeared on both sides of the lung, and the absorption of lung lesions was reduced compared with that measured on the day of admission. Serious adverse events were not observed. We compared chest CT images before and after taking QFPDD . After 2Case 7 involved a 65-year-old man in critically ill condition. The patient exhibited a fever of unknown origin, accompanied by cough, paroxysmal dry cough, fatigue, and asthma. On February 16 2020, he was transferred to the hospital because of fever and cough for more than 20 days. No significant improvement was observed after conventional treatment. On February 21, relevant experts and TCM doctors were organized for joint consultation and decided to administer QFPDD. After QFPDD administration, the symptoms rapidly improved, and chest CT showed partial absorption on February 25. Serious adverse events were not observed. The patient was cured and discharged on March 8 2020.Case 8 was an 82-year-old man who was critically ill. On February 8 2020, he was admitted to the hospital because of a cough for 8 days and fever for 7 days, accompanied by chills, sore throat, muscle soreness, fatigue, and other discomforts. Respiratory diseases, including respiratory failure, chronic bronchitis with emphysema, and electrolyte disorders, were observed. Chest CT showed scattered ground glass and mesh shadows in lungs, subpleural distribution, interstitial involvement, and scattered strip shadows in both lungs, mainly in the lower lobe. He was considered critically ill on February 11. After treatment, his airtightness was relieved and his blood oxygen saturation was approximately 97%. Interferon and acetylcysteine were administered, and the 13-day treatment had no significant effect on chest CT. On February 21, relevant experts and TCM doctors were organized for a consultation and decided to administer QFPDD after discussion. After two courses , symptoms such as cough and asthma were significantly reduced, and the nucleic acid test result turned negative. Before being discharged, chest CT showed scattered spots, plaques, ground glass, and cable shadows in the multi-lobar segments of both lungs, which were much better than those measured before admission. No serious adverse events occurred during QFPDD administration, and the patient was discharged on March 10 2020.Detailed information of the aforementioned eight cases is shown in A total of eight patients (six men and two women) were included in this study, and their demographic and baseline characteristics are given in The trend of clinical symptom improvements in the eight patients during the two courses of QFPDD treatment is shown in After one course of QFPDD (3 days), nasal obstruction and sore throat were completely cured, and clinical symptoms such as fever (5 [83.3%]), fatigue (2 [50.0%]), and cough (2 [28.6%]) improved. After two courses (6 days), the fever disappeared completely in all patients, and the other symptoms showed a tendency to improve. In non-severe patients, 87.5% baseline symptoms completely disappeared. In severe patients, 61.1% of the baseline symptoms completely disappeared after patients were administered QFPDD for two courses. Of the abnormal indicators, 55.6% returned to normal levels. In addition to QFPDD, most patients received antiviral treatment, among which five (62.5%) received antibiotics and two (25.0%) received corticosteroids. The treatment process and related information for the eight patients are presented in The median duration of fever recovery was 1 day, fatigue recovery was 7  days, cough recovery was 5  days, anorexia recovery was 2  days, and asthma recovery was 4  days . None ofOf the eight patients, seven (87.5%) presented with bilateral macular or ground-glass lung lesions on admission, and one (12.5%) showed no abnormal lung lesions. After they received more than two courses of QFPDD, all seven patients showed different degrees of absorption .the Guidelines, COVID-19 patients should revisit hospitals for a follow-up at 2 and 4 weeks after discharge from the hospital. During the follow-up period, no adverse events such as liver and kidney damage were observed in all eight cases.No adverse events were observed during the treatment. As required by In this pre-post study, eight patients were additionally offered QFPDD after group consultation by Western medicine and TCM doctors. QFPDD may facilitate the improvement of clinical symptoms such as fever, anorexia, cough, asthma, etc., and the absorption of pulmonary lesions improves. During hospitalization, none of the patients’ health worsened or died, and all were cured and discharged. No serious adverse events related to QFPDD were observed. The results indicated that QFPDD had favorable potential effectiveness and safety in treating COVID-19 with other respiratory diseases.sanjiao   , which mnctions) . In addinctions) . The curTreatise on Cold Pathogenic and Miscellaneous Diseases. QFPDD comprises 21 herbs, which can be divided into four formulas: 1) Ma Xing Gan Shi decoction, which can clear lung Qi and relieve symptoms of asthma, and has a positive effect on the symptoms of fever and cough; 2) Shegan Mahuang decoction, which can warm the lungs, resolve phlegm, and relieve phlegm and asthma symptoms; 3) Minor decoction of Bupleurum, which can harmonize and relieve Shaoyang, and improve the metabolic capacity of the human body; and 4) Wu Ling decoction, which can warm yang and damp drains. It has a diuretic effect. The combined effect of these herbs could thus relieve patients’ cough, fever, nasal obstruction, sore throat, and other symptoms significantly compared with before the use of QFPDD. The combination with QFPDD could significantly shorten the duration of symptom improvement according to existing research .The theory of QFPDD can be traced back to one of the classical TCM works, Studies have shown that the components of QFPDD have a certain theoretical basis for improving the symptoms of other respiratory diseases in TCM. According to experienced TCM doctors, the basis of COPD and bronchiectasis is phlegm, heat, static blood, and insufficiency. In the acute attack period of the disease, Ma Xing Gan Shi decoction can be used to clear lung heat, which can be regarded as a representative prescription to relieve cough and asthma to some extent. In addition, the use of QFPDD in the treatment of bacterial pneumonia has been reported . TherefoRelated studies have shown that patients with COVID-19 with other respiratory diseases, particularly COPD, are more likely to have severe outcomes than common patients with COVID-19 , with a Clinical studies have confirmed that the main clinical symptoms, such as fever, cough, asthma, and fatigue, were significantly improved after treatment with QFPDD, and the symptoms of fever, cough, fatigue, and headache improved faster than before .In terms of improving clinical symptoms, we found that the clinical features of these patients were slightly different from those of common patients with COVID-19. The main clinical symptoms of COVID-19 are fever, cough, and fatigue . This maMoreover, we found that QFPDD effectively relieved the clinical symptoms of patients with other respiratory diseases, such as fever and cough. The lung lesions in these patients showed varying degrees of absorption. During the two courses of the treatment, the number of patients with major clinical symptoms showed a general decreasing trend. Most of the hospitalized symptoms completely disappeared after two courses of decoction.Related studies have shown that morbidity and mortality rates of COVID-19 are higher in older people than in younger people, with a mortality rate of 80% in people aged more than 60 years, particularly in those with underlying medical conditions . NotablyIn this study, we concluded that QFPDD might help improve the clinical symptoms of COVID-19 patients with other respiratory diseases; no serious adverse events related to QFPDD were observed, which was in line with previous studies . The resHowever, this study has two main limitations. First, this was a retrospective study with small sample size, and the data were collected at the time of emergency treatment, leading to the conclusion that some data were not collected. Second, no parallel control group was used in this study. In the future, large-sample, multi-center, randomized controlled studies will be conducted to further clarify the clinical efficacy of QFPDD in the treatment of COVID-19 combined with other diseases of the respiratory system.


Pistacia vera L.) is a nut with a good adaptability to the Mediterranean conditions of cultivation, specifically in the Andalusian region, becoming an emerging crop. Moreover, it has been getting attention in the past years for the great content of bioactive compounds such as polyphenols. Although some studies have reported the polyphenolic profile of pistachios, most of them have analyzed the hull part, considered as a residue, and not the kernel which is the edible part. Therefore, characterization of eleven varieties of pistachios kernels cultivated in Andalusia and harvested in 2019 and 2020 was carried out by UHPLC-MS (ultra-high-performance liquid chromatography high-resolution mass spectrometry). The identification and quantification of 56 polyphenolic compounds was performed, being the hydroxybenzoic acids group the most abundant with a 71–86% of the total amount followed by flavan-3-ols group that accounted for 8–24%. Moreover, 3,4-dihydroxybenzoic acid was the main compound in most of the varieties, followed by vanillic acid hexoside. Larnaka, Avdat, Aegina, and Mateur presented the highest amount of total polyphenols, while Kalehghouchi, Joley, Lost Hills, Kerman, and Golden Hills were the varieties with the lowest content. Regarding the harvest season, no significant differences (p < 0.01) were found in the total amount of polyphenols between 2019 and 2020. In addition, the antioxidant activity was measured by DPPH , ABTS ), and ORAC  assays, showing a similar trend as that of the polyphenols.Pistachio ( Pistacia vera L.) is a nut widely consumed throughout the world, originated from arid regions of Central and West Asia. This crop belongs to the Anacardiaceae family, and its health benefits and nutritional characteristics have been recently recognized by some studies [The pistachio , the total world pistachio production in 2020 was estimated at 1,125,305 tons  whereas Pistachio cultivation depends on several factors, hence not all parts of the country (Spain) are suitable for its production. It requires a minimum number of hours of cold weather which influence subsequent flowering and fertility. Likewise, the temperature, humidity, and soil characteristics of the Andalusian region favor the production and ripening of pistachio in a homogeneous way . Hence, Pistachio nut is considered as a very important nutritional product and a good source of unsaturated fatty acids, of which 40–85% are MUFA and PUFA, being linoleic acid the most representative. It is also an important source of dietary fiber and protein. In addition, pistachio is rich in micronutrients such as magnesium, potassium, vitamin K, and tocopherol that contribute to a better glycemic index and the reduction of cardiovascular diseases ,11,12.Moreover, some studies have focused their attention on the analysis of the antioxidant activity and bioactive compounds present in pistachios, especially polyphenols, which are well-known for their ability to prevent the formation of pro-oxidants by blocking the action of reactive oxygen species and playing an important role in positive health effects such as cardioprotective, anti-diabetic, and anti-inflammatory effects ,14. ManyAnalyses related to the characterization of polyphenolic compounds in pistachio kernels are limited. Most research studied the antioxidant activity of the compounds found in the hull, and identified less compounds in the kernels compared to those found in the hull, and analyzed only one or a few varieties. According to the literature, the most commonly studied varieties of pistachio are Bronte , reporting mostly the presence of polyphenols such as quercetin and eriodictyol ,25, as wIn the same way, several others publications were found in the literature with limited information. Arena et al.  reportedTherefore, due to the limited information found in literature regarding phenolic compounds of pistachio kernels, the aim of this study was to obtain an exhaustive polyphenolic profile by UHPLC-MS and antioxidant activity of eleven different varieties of pistachios cultivated in Andalusia (Spain) during two harvesting years (2019 and 2020).Methanol (MeOH) HPLC grade, acetone, and potassium hydroxide were acquired from Panreac Applichem ITW Reagents . Sodium chloride and magnesium chloride hexahydrate were purchased from Fisher Scientific ; sodium bicarbonate and ammonium carbonate were supplied by Sigma-Aldrich ; and potassium dihydrogen phosphate, sodium hydrogen carbonate, magnesium sulfate monohydrate, and potassium hydrogen phosphate were obtained from VWR International Eurolab . Reference standard compounds including gallic acid, pyrogallol , ellagic acid, benzoic acid, chlorogenic acid, ferulic acid, sinapic acid, p-coumaric acid, luteolin, rutin, myricetin, quercetin, isorhamnetin, catechin, epigallocatechin gallate, and epicatechin gallate were purchased from Sigma-Aldrich . The acetonitrile and methanol were of LC-MS grade.Pistacia vera L.) were kindly provided from an experimental field of pistachio varieties of IFAPA in Guadix  which were planted in 2012. Eleven varieties were collected during two consecutive seasons (2019 and 2020): Aegina, Avdat, Golden Hills, Joley, Kalehghouchi, Kastel, Kerman, Larnaka, Lost Hills, Mateur and Sirora. The experimental design consisted in four blocks of random repetitions per variety. Fresh pistachios were peeled and ground using a homogenizer  and stored at −80 °C until analysis.Pistachios (v/v) with 0.1 % formic acid. The samples were centrifuged at 5000 rpm for 15 min at 4 °C, and supernatants were collected. The pellet was re-extracted following the same indications abovementioned. All the supernatants were pooled and made up to a final volume of 25 mL and stored at −80 °C until analysis.The extraction of polyphenols from pistachio samples was adapted from Ordóñez-Díaz et al.  with som·+) was produced by reacting ABTS·+ with a 2.45 mM of potassium persulfate solution stirring in dark at room temperature for 12–16 h before use. The ABTS·+ solution was diluted with ethanol to get a final absorbance of 0.8 ± 0.02 at 730 nm. Fresh ABTS·+ solution was prepared for each assay and day. Samples were resuspended in 500 µL of 7% methylated β-cyclodextrin (RMCD) in acetone/water  solution and 25 µL was added to 190 µL of ABTS·+ solution in a 96-well microplate measuring the absorbance every 20 s at 30 °C for over 6 min in a Synergy HTX Multi-Mode Microplate Reader . The antioxidant activity was expressed as mmol of Trolox equivalents per 100 g of sample (mmol TE/100 g). Each value is the average of three determinations.Free radical scavenging activity was measured using the ABTS  decolorization method  with somFree radical DPPH  scavenging capacity was determined using the previously described methods by Sánchez-Moreno et al. . The antv/v) solution. Then, 25 µL of either Trolox or sample extract or 75 mM phosphate buffer as a blank was added to a 96-well microplate followed by the addition of 150 µL of fluorescein work solution (8.5 × 10−5 mM) prepared in 75 mM phosphate buffer (pH 7.4). The microplate reader ) was programmed to record every two minutes for 120 min at 485 and 528 nm excitation and emission wavelengths, respectively, the fluorescence after the addition of 30 µL of AAPH (153 mM) as peroxyl radical generator, prepared in 75 mM phosphate buffer (pH 7.4). ORAC values are expressed as mmol Trolox equivalents per 100 g of sample (mmol TE/100 g).Oxygen radical absorbance capacity (ORAC) assay was measured according to the method previously published by Huang et al.  and modiTotal phenolic content (TPC) was determined by the Folin–Ciocalteu assay following the methodology of Slinkard and Singleton , with moIdentification and quantification of polyphenols in the pistachio extracted samples were carried out by using UHPLC-HRMS mass spectrometer system  comprising a UHPLC pump, a PDA detector scanning from 200 to 600 nm, and an autosampler operating at 4 °C .m/z range from 100 to 1200 with a resolution of 50,000 Hz and with a full AGC target of 100,000 charges, using 2 microscans. Analyses were also based on scans with in-source collision-induced dissociation (CID) at 25.0 eV. The capillary temperature of the MS experiment with HESI in negative ionization mode was at 320 °C, the sheath gas was 35 units, the heater temperature was 150 °C, the auxiliary gas was 10 units, and the spray voltage was 4.0 kV. Data acquisition and processing were carried out using Xcalibur 3.0 software .Separation of phenolic compounds was performed on a Zorbax SB-C18 RRHD column  preceded by a guard pre-column of the same stationary phase and maintained at 40 °C. The flow rate was set to 0.2 mL/min with a 26 min gradient of phase A: deionized water with 0.1% formic acid and B: acetonitrile with 0.1% formic acid. The gradient started at 3% B, was maintained for 2 min, then rose to 65% B in 18 min, before rising to 80% B in 1 min and being maintained for 6 min with a 26 min gradient. After that, the column was equilibrated to the previous conditions within 10 min. After passing through the flow cell of the PDA detector part of the column, the eluate (0.2 mL/min) went into an Exactive Orbitrap mass spectrometer  fitted with a heated electrospray ionization probe (HESI) operating in negative ionization mode for the determination of polyphenols. Full scans were recorder in Targeted identification of phenolic compounds was achieved comparing the exact mass and the retention time with available standards. In the absence of standards, compounds were tentatively identified by comparing the theoretical exact mass of the molecular ion with the measured accurate mass of the molecular ion and searched against metabolite databases including Metlin, Phenol Explorer and more general chemical databases such as PubChem and ChemSpider. Compounds having molecular masses within the pre-specified tolerance (≤10 ppm) of the query masses are retrieved from these databases. Quantification of phenolic compounds was carried out by selecting the theoretical exact mass of the molecular ion with reference to standard curves. In absence of reference compounds, they were quantified by reference to the calibration curve of a closely related parent compound.Two-way ANOVA and Tukey post-hoc tests were applied to identify the differences among samples using R software . A principal component analysis (PCA) was carried out for data exploration.p < 0.01) were observed among varieties. Larnaka, Mateur, Avdat, and Aegina were the varieties with the highest antioxidant capacity measured by ABTS assay. Regarding DPPH methodology, Larnaka presented the highest antioxidant activity, followed by Mateur, Avdat, and Aegina, while Golden Hills and Kalehghouchi varieties showing the lowest values. In the same way, Larnaka also presented higher values of antioxidant capacity by the ORAC assay, followed by Mateur, Avdat, and Aegina, while Joley, Kalehghouchi, and Golden Hills showed the lowest ones. The measurement of the total phenolic content (TPC) also showed Larnaka as the variety with the highest amount, followed by Aegina, Mateur, and Advat. In general, Larnaka, Mateur, Avdat, and Aegina showed the highest antioxidant activity values for the three scavenging methods and the total phenolic content. This finding agreed with Ojeda-Amador et al. [p < 0.01) in the antioxidant activity values measured by ABTS and DPPH, as well as in the TPC were found for the harvesting year, showing higher values for the samples harvested during 2020. This fact could be explained due to the different climatic conditions between years. No significant differences were shown for data measured by the ORAC method. To sum up, Larnaka variety presented the highest antioxidant activity in two of the three methods analyzed.Total antioxidant activity was evaluated for the eleven varieties of pistachios following three scavenging methods: ABTS, DPPH, and ORAC assays. Results are displayed in r et al.  who repor et al.  found siA total of 56 polyphenolic compounds were identified and quantified by UHPLC-HRMS in the eleven varieties of pistachios from two seasons of harvest (2019 and 2020). Details of the characteristics for the identification of compounds, including the retention time (Rt), the experimental accurate mass, and the error (ppm) between the exact accurate mass and the mass found of the detected compounds are described in p < 0.01) among the eleven varieties were observed for the total content of polyphenols  among varieties for each group of polyphenols, (V) were observed, except for the flavanonols. It could be highlighted that varieties with the lower values for the hydroxybenzoic acid group  presented the highest values for the flavan-3-ols group, which may be due to the genetic synthesis mechanisms of polyphenols.Significant differences (p < 0.01) from 2019 to 2020, whereas for the rest of the groups increased. Regarding the harvesting season (S), in terms of the total polyphenols content , no signp < 0.01) were observed for all the compounds for the varieties except for the benzoic acid derivative III. In this way, we can conclude that Larnaka, Avdat, Aegina, and Mateur presented three-fold concentration than the rest of varieties. The main compound was the 3,4-dihydroxybenzoic acid for most of the studied varieties, ranging from 43 to 67% of the total content of this group, followed by vanillic acid hexoside (28–51%) except for Kalehghouchi and Kerman for which the major one was the vanillic acid hexoside and 3,4-dihydroxybenzoic acid respectively. The rest of the compounds were found in minor amounts. Results are in agreement with the literature, where 3,4-dihydroxybenzoic acid was described as one of the principal compounds detected, for instance, in the shells of Bronte variety [O-rutinoside as the most abundant polyphenol in the kernels from the pistachio studied variety (Bronte) while eriodictyol-7-O-glucoside and gallic acid were described as the main compounds in the skins. Considering that hydroxybenzoic acids group is the main contributor to the total polyphenols content,  variety  and in u variety . In cont variety , found qp < 0.01) between the two harvest years (2019 and 2020) because of 3,4-dihydroxybenzoic acid which is the major compound, since the tendency of most compounds is to increase. Nonetheless, no significant differences were found for theogallin, galloylshikimic acid, digallic acid, and benzoic acid derivative based on the harvesting year factor. As mentioned before, the total amount of hydroxybenzoic acids decreased significantly  are presented in p < 0.01) increase was observed between 2019 and 2020 in flavan-3-ols. The trend in most of the compounds was the same, but for Afzelechin I, for which no significant differences were found. In contrast with the hydroxybenzoic group, a significant , which explained the 71% of the total variance, was plotted .Different clusters related to the different families of polyphenols in pistachios were observed. In the direction of PC1, which explained around 57% of the total variance, Larnaka, Avdat, Aegina, and Mateur varieties which are the most abundant in polyphenols were clearly separated from the ones with less content of compounds . The main families of polyphenols involved in such differences were hydroxybenzoic acids, flavan3-ols, stilbenes, flavonols, and flavanones which could be primarily responsible for the high antioxidant activity measured by ABTS, DPPH, and ORAC assays, being present in the same square . AdditioMoreover, for more detailed information related to the distribution of the 56 polyphenols, a hierarchical cluster analysis (HCA) was performed . This hip < 0.01) were found between 2019 and 2020 in the total amount of polyphenols. In addition, antioxidant activity was measured, showing Larnaka as the highest variety in two of the three methods analyzed without significant differences in the remaining varieties. Results suggest that antioxidant activity is mainly attributable to the content of the family of hydroxybenzoic acids. Moreover, the different results obtained among the antioxidant methods may be linked to the contribution of other antioxidant compounds in the hydrophobic fraction which react with the radicals. This study was carried out to characterize the polyphenols presented in the different varieties of pistachios and the variability of the compounds, since it is the basis for subsequent digestibility analyses, which are necessary to evaluate the bioaccessibility and bioavailability of these compounds.Results of this study report an exhaustive characterization of 56 polyphenolic compounds present in pistachio kernels from eleven varieties cultivated in Andalusia, describing hydroxybenzoic acids and flavan-3-ols as the most abundant groups. Likewise, the main polyphenolic compound was 3,4-dihydroxybenzoic acid for most of the varieties, followed by vanillic acid hexoside. Larnaka, Avdat, Aegina, and Mateur presented the highest amount of the total polyphenols, while Kalehghouchi, Joley, Lost Hills, Kerman, and Golden Hills were the varieties with the lowest content. Regarding the harvest season, no significant differences (


Huntingtin-associated protein 1 (HAP1) is a determinant marker for the stigmoid body (STB), a neurocytoplasmic physiological inclusion. STB/HAP1 enriched areas in the brain/spinal cord are usually protected from neurodegenerative diseases, whereas the regions with tiny amounts or no STB/HAP1 are affected. In addition to the brain/spinal cord, HAP1 is highly expressed in the myenteric/submucosal plexuses of the enteric nervous system in the gastrointestinal tract. The tongue is attached to the pharynx by the hyoid bone as an extension of the gastrointestinal system. To date, the immunohistochemical distribution and neurochemical characterization of HAP1 have not been elucidated in the lingual ganglia. Using immunohistochemistry and light microscopy, our current study demonstrates the expression and immunohistochemical phenotype of HAP1 in the lingual ganglia of adult mice. We showed that HAP1 was profoundly distributed in the intralingual ganglion (ILG) and the ganglia near the root of the tongue . Neurons in ILG and LRG exhibited high coexpression of HAP1 with NOS or ChAT. Furthermore, most HAP1-immunoreactive neurons contained SP, CGRP, and VIP immunoreactivity in both ILG and LRG. The current results might serve as an essential base for future studies to elucidate the pathological/physiological functions of HAP1 in the lingual ganglia. Huntingtin-associated protein 1 (HAP1) was first recognized for its binding to polyglutamine (polyQ)-expanded huntingtin, the causative protein for Huntington’s disease ,3,4. HAPEvidence suggests that neurodegenerative diseases impact the enteric nervous system (ENS) in addition to the CNS . ENS comThe tongue is connected to the pharynx by the hyoid bone and is an extension of the gastrointestinal system. The tongue comprises intrinsic  and extrinsic  muscles. It is well-known that the tongue muscles are innervated by several cranial nerves ,22. MotoOther than cranial innervation, the intrinsic neurons may also play a vital role in tongue functions . The preAdult C57BL/6J were bred and housed at the animal facility of the Faculty of Medicine, Yamaguchi University, Japan. The mice were maintained in standard conditions  with food, water, and nesting martial available ad libitum. For the current experiments, adult male mice were used. The primary antibodies used in the current study are listed in Anesthetized  adult mice were transcardially perfused with PBS before introducing 4% paraformaldehyde in 0.1 M phosphate buffer (pH 7.4). Then, the tongue was dissected and post-fixed in 4% paraformaldehyde overnight at 4 °C. The tongues were kept in 30% sucrose solution for 5–6 d. The tissue was mounted in a cryostat embedding medium and snap-frozen in powdered dry ice. The tongues were sectioned using a cryostat (thickness of 30 µm) and kept at 4 °C in PBS for storage until used for immunohistochemistry. For the myenteric plexus, the whole mount of the duodenum was prepared as described in our previous study .Immunoperoxidase staining was performed as narrated in our earlier reports ,30,31,32Immunofluorescence staining was performed following our earlier studies ,29. In bLight microscopic photomicrographs were taken with a Nikon Eclipse E80i photomicroscope  attached to a Lumenera USB 2.0 camera . Confocal photomicrographs were taken with an LSM510 confocal laser scanning microscope . Single optical sections were captured (1024 × 1024 pixels). Minimal bright/contrast adjustments and plates of photomicrographs were performed with Photoshop 2021 . For cell counting, 20× objective immunofluorescence images were taken and transferred into ImageJ software, version 1.51i . Coexpression ratios for HAP1/ChAT, ChAT/HAP1, HAP1/NOS, NOS/HAP1, HAP1/SP, SP/HAP1, HAP1/CGRP, CGRP/HAP1, HAP1/VIP, VIP/HAP1, ChAT/NOS, NOS/ChAT were calculated from the total number of HAP1, ChAT, NOS, SP, VIP, or CGRP-ir cells, as well as those double immunostained for HAP1 with ChAT, NOS, SP, VIP, CGRP, and ChAT with NOS ,33. Six HAP1 immunoreactivity was detected in the ILG cells scattered mostly in the anterior one-third of the tongue A. HoweveBased on histochemical studies, the ganglion cells in the tongue have been considered to have parasympathetic functions . To elucThese results were intriguing in that HAP1 was coexpressed with excitatory parasympathetic neuronal marker ChAT and inhibitory parasympathetic neuronal marker NOS. The excitatory parasympathetic and inhibitory neurons are usually different entities in the myenteric plexuses of ENS A–C. To aIt has been reported that neuropeptides, such as CGRP, SP, and VIP, are expressed in the lingual ganglionic cells. The functional significance of these neuropeptides in the lingual ganglia needs to be better clarified, but they are believed to modulate sensory functions in the tongue ,28. To eThe immunohistochemical expression and neuroanatomical distribution of HAP1 have been analyzed previously in the brain and spinal cord in CNS ,40,41,42In both ILG and LRG, immunoreactivity for HAP1 was diffusely expressed in the cytoplasm of neurons. It is well-known that HAP1 is the determinant marker of STB. However, the STBs were not clearly detected in ILG or LRG in our current immunostaining. HAP1 has two isoforms, HAP1A and HAP1B ,36,39,43The tongue is innervated by sympathetic, parasympathetic, and sensory fibers via different pathways of cranial nerves. Motor innervation for all extrinsic and intrinsic muscles is provided via the CN X and CN XII. For sensory innervation, general sensation is supplied by the CNV , and taste sensation is provided by CN VII and CN IX. Autonomic sensations are mediated via parasympathetic and sensory nerve fibers ,22. In aIt is believed that HAP1 has putative protective functions against apoptosis/cell death in stress conditions or certain neurodegenerative diseases. Abnormal interactions of mutant protein with HAP1 affect the intracellular transport of selected molecules, which is an essential contributor to the pathogenesis of neurodegenerative diseases. To prevent apoptosis-induced nuclear translocation, HAP1 can also sequester the casual mutant molecules of some neurodegenerative diseases and trap the toxic aggregation in the cytoplasm of the cells ,15,16. IThe present study is the first to elucidate the detailed neuroanatomical distribution and immunohistochemical characterization of HAP1 in the mouse lingual ganglia. Our current study is also the first to show the existence of a new lingual ganglion at the root of the mouse tongue, which we coined as LRG. HAP1 was highly expressed in both ILG and LRG. Our immunohistochemical results exhibit that HAP1-ir neurons are coexpressed with ChAT, NOS, SP, VIP, and CGRP-ir neurons. The present results might indicate the plausible functions of HAP1 in protecting or regulating neurons in ILG and LRG that are related to intrinsic parasympathetic autonomic and sensory functions in the tongue. The present study may serve as an important base for future studies that aim to elucidate the pathological or physiological functions of HAP1 in the lingual ganglia. The tongue has dual innervation, cranial innervation, and intrinsic intralingual innervation. Cranial innervation has been well studied previously ,22, and


Climate change has significantly affected agricultural production. As one of China’s most important agricultural production regions, the North China Plain (NCP) is subject to climate change. This paper examines the influence of climate change on the wheat and maize yields at household and village levels, using the multilevel model based on a large panel survey dataset in the NCP. The results show that: (i) Extreme weather events (drought and flood) would significantly reduce the wheat and maize yields. So, the governments should establish and improve the emergency service system of disaster warning and encourage farmers to mitigate the adverse effects of disasters. (ii) Over the past three decades, the NCP has experienced climate change that affects its grain production. Therefore, it is imperative to build the farmers’ adaptive capacity to climate change. (iii) Spatial variations in crop yield are significantly influenced by the household characteristics and the heterogeneity of village economic conditions. Therefore, in addition to promoting household production, it is necessary to strengthen and promote China’s development of the rural collective economy, especially the construction of rural irrigation and drainage infrastructures. Climatic conditions have always been an important factor shaping agricultural production. Climate change, especially in terms of extreme weather events, has exacerbated the fluctuations in food production and threatened world food security. In most part of China, increase in temperature is the main climate change issue reducing the major crop  yields ,2,3,4. IResearchers have used econometric approaches to analyze the impact of climate change on grain production. Mendelsohn et al.  first prTo address the limitations of the Ricardian approach, the production function approach was used to reveal an empirical relationship between climate factors and grain output in agricultural production, particularly in China ,20,21,22To achieve the above goal, it is necessary to use a multilevel model to analyze large-scale survey data of farm households which typically adopt stratified multistage clustered sampling designs (with household level and village level). The multilevel analysis can model the clusters occurring at different levels of the sampling with nested random effects . This stThe rest of this paper is organized as follows: The stratified sampling data with clustered characteristics show significant differences between different levels of data and high similarity among data at the same level. In this case, a regular ordinary least squares (OLS) model may result in misspecification by ignoring the average variation between groups. Therefore, a multi-level model (MLM) should be developed to deal with the heteroscedasticity caused by inter-dependent error terms and to estimate group-level averages by both fixed and random effects . MLM decThe unconditional means model is an “empty model” that does not include any independent variables. It is reasonable to adopt MLM if individual respondents are clustered within groups and the variance of outcome in two levels are significantly different in a data structure. Assuming that in yield .The characteristics of farm households and villages that affect the grain yield remain unmeasured in Equation (3). Therefore, the variables of such characteristics are introduced to determine whether the between and within components of variation can be explained at the household and village levels. The random intercept model can be expressed as:Equation (5) is the general model of Equation (4). Where, Y is the observation variable; X is the design matrix of constant parameter Maximum likelihood estimation of parameters can be obtained by maximizing Equation (6). That is, The NCP is one of China’s major grain production areas, accounting for approximately 75% and 35% of China’s wheat and maize outputs, respectively . This rehttp://cdc.cma.gov.cn accessed on 15 May 2021), including the daily maximum temperature, minimum temperature, average temperature, and 24 h average precipitation recorded by the meteorological observatory in sample or adjacent counties.To collect the data, stratified multi-stage cluster sampling was implemented. First, three counties were randomly chosen in each province using the following criterion. (i) The counties had experienced at least one episode of either severe drought or flood between 2010 and 2012. China’s national standard for natural disasters  categoriAs a result, the samples of winter wheat included 2261 plots of 1216 households, which were distributed in 123 villages  of five provinces . The samThe 12 main growth stages of winter wheat are seedling emergence, three-leaf, tillering, overwintering, reviving, jointing, booting, heading, anthesis, grain-filling, wax ripeness and mature. This study separated the overwintering stage from the vegetative stage ; it alsProduction inputs and economic and social institutional factors should be incorporated into the model; meanwhile, the factors of long-term climate change and extreme weather events should be included in the production function model. The C-D-C production function equation, which is the extension of Equation (4), is specified as:ij refers to crop yield, which is measured as the wheat or maize output per hectare. As shown in This study independently investigated the effects of climate change and the household and village attributes on the yields of winter wheat and summer maize by Equation (6), respectively. The dependent variable Yij examined in this paper includes average daily temperature and precipitation over the past three decades (1981–2010). For winter wheat, the daily average temperature was only 5.2 °C in the over-wintering stage, and it was 20.4 °C in the reproductive stage. For summer maize, the daily average temperature could be above 20 °C, and the precipitation was more than 100 mm at different growth stages , 24.7% of counties that grew wheat suffered from drought, and 25.1% and only 8.2% of counties that grew maize suffered from drought and flood, respectively. There are four types of farm plot disasters, which are DLD for farm plots suffering from drought, DLF for farm plots suffering from flood, DLR for farm plots suffering from continuous rain, and DLw for farm plots suffering from strong wind. In the past three years, 40.5% of farm plots growing wheat suffered from drought, 7–8% suffered from continuous rain or strong wind, while 2.7% suffered from flood, indicating that drought was the most frequent disaster during wheat planting. During the same period, 36% of farm plots that grew maize suffered from drought, 16% suffered from strong wind, and 15.5% suffered from flood. This shows that drought was the most frequent disaster during maize planting, and the risk of strong wind and flood should not be underestimated.The second climate indicator is extreme weather event, including the variables of county-level disaster D1 were relatively small, with an average farm area of only 0.21 ha and 0.19 ha for wheat and maize  Compared with the overall land quality of village, the farmland quality L3 is divided into three categories, which are low-quality, middle-quality and high-quality land. The majority of plots (68%) were of medium quality, 10% were of low-quality, and 20% were of high-quality.Three variables represent farmland plot characteristics. (i) The farmland areas Lnd maize , respect1, pesticide cost I2, machinery cost I3, labor input I4, and irrigation water I5 at the plot level. 3 and 1730.1 m3 for wheat and maize, respectively, indicating that the grain grown in the NCP is mainly irrigated rather than rainfed.ij) include variables as follows. (i) The assets possessed by the household (H1), which are measured as the value of the durable goods. The average value of durable goods of sample households was 9700 yuan; (ii) H2 represents the education level of the household head, where the average education was 6.9 years. (iii) H3 represents the production and technical training, and about 25% of household members received such training.Farm household’s characteristics  V2 refers to the ratio of irrigation area to total cultivated area in the village, which is more than 80%. (iii) V3 is the distance between the village committee and the nearest road above the township level with an average distance of 1.4 km.Three variables were used to measure village characteristics. (i) V2011  and T2012 , to control technological advances or other unobservable factors that change over time.In addition, this study used year dummies, TAccording to Equation (6), the estimated results of the influence of climate factors and other factors on wheat and maize yields are shown in R2) − Ln(LR1)] = 143.5, is greater than critical R3) − Ln(LR2)] = 54.17 is greater than critical Similarly, Model III shows a better overall fitting than Model II. The likelihood function ratio LR = 2[Ln(LFirstly, the heterogeneity of wheat yield was not only caused by household characteristics, but also determined by differences in village economies, except for farmland characteristics and production input factors. For example, the variance of village level is  = 0.103 , row 36  = 0.118 , row 1 iSecondly, the effect of long-term climate change on wheat yield varied across different wheat growth stages. The increase of average temperature significantly promoted wheat production during the overwintering stage. For example, wheat yield would significantly increase by about 8% if temperature increased by 1 °C row 1, . HoweverThirdly, the occurrence of extreme weather events had significantly negative impact on wheat yield. County-level drought significantly reduced wheat yield. Farm-level disasters also had led to obvious reduction in wheat yield. In particular, drought, flood, continuous rain, and strong wind at the farm level would reduce wheat yield by about 10%, 6%, 16%, and 9%, respectively, holding constant of other variables (rows 8–11, Fourthly, production input significantly affected the wheat yield. The elasticity of labor is −0.016 with other input factors unchanged row 21, . The sigFifthly, the social and economic characteristics of households had a significant impact on wheat yield. As expected, education and participation in production and technology training programs played an important role in promoting wheat yield. In particular, the wheat yield would significantly increase by 0.2% if the schooling year of the household head increased by one row 24, . SimilarFinally, the social and economic characteristics of village could mitigate the loss of wheat yield under extreme weather events, especially drought disaster. For example, the wheat yield decreased by 19.7% if farm plot occurred drought disaster, but it only decreased by 1% if the distance shortened by 1 km between the village committee and the nearest township-level road. Moreover, the wheat yield decreased by 8.4% and 19.7%, respectively, when county-level drought and farm plot drought occurred simultaneously. However, in the case of drought, the wheat yield could significantly improve by 0.1% if irrigation proportion rise by 1%.Firstly, the heterogeneity of maize yield was shaped by village attributes. In particular, the village level variance of  row 36,  with int3 row 1,  without Secondly, the occurrence of extreme weather events had a significantly negative impact on maize yield. For example, the maize yield decreased by 12.7% and 14.2% under drought and flood disasters at the county level, respectively. The disasters occurring on farmland plot led to more loss in maize yield than those at the county level. Specifically, drought, flood, continuous rain, and strong wind reduced maize yield by 13.6%, 22.4%, 12.2%, and 9.8%, respectively.Thirdly, production input significantly affected the maize yield. The sign of labor elasticity is negative, and the reasons have been mentioned above. Pesticide and irrigation water input could significantly promote maize yield. Specifically, the maize yield would increase by 0.032% and 0.018% for every 1% increase in pesticide input and irrigation water, respectively (rows 20 and 23, Finally, the social and economic characteristics of village could mitigate the loss of maize yield under extreme weather events. The coefficients of the cross term between village collective enterprise and drought disaster reveal that the negative impact of drought disaster on maize yield would decrease dramatically if the village had more collective enterprises. For example, the maize yield declined by 48.9% if drought occurred on the farmland plot, but it only decreased by 11% for one additional collective enterprise. Furthermore, the maize yield could decrease by 9.1% under county-level drought, but it could increase by 12.8% for an additional collective enterprise.Based on the data of 6749 wheat plots and 5212 maize plots of farm households over 2010–2012, this paper adopted a multilevel model to analyze the impact of long-term climate change and extreme weather events on the wheat and maize yields in the NCP. It also considered village social and economic conditions, social and economic characteristics of the household, production inputs, and farmland plot characteristics as the influencing factors of wheat and maize yields. The findings of this study suggest the following conclusion and discussion.There are three main findings and conclusions in this study.Firstly, spatial variations in crop yield are significantly influenced by the heterogeneity of village economic conditions. The explained variation in crop yield is much higher at the village level than at the household level. The social and economic characteristics of the village have a positive effect on crop yield and mitigate the loss of crop yield under extreme weather events. Therefore, under China’s dual-level management system of integration of unification and separation in rural area, it is necessary to strengthen household production behavior and improve village collective economy.Secondly, the arid and semi-arid region of NCP has been experiencing climate change, affecting grain production over the past three decades. The effects of long-term climate variables on winter wheat and summer maize yields vary across the growth stages. Therefore, it might be time to think of making agricultural production adapt to climate change. For example, it may need to adjust the planting system, change crop varieties, and build households’ adaptive capacity to climate change.Thirdly, extreme weather events are more likely than long-term climate change to reduce the wheat and maize yields. The negative impact of extreme weather events on crop yield is more serious and immediate. These findings suggest that the governments should establish and improve the disaster service and coping system for grass-roots units. Specifically, it is essential to use modern information technology to improve the monitoring, forecasting, and warning of agricultural disasters and provide and publicize disaster early warning and response information timely. At the same time, agricultural technical guidance and financial support should be provided for disaster prevention and control, enabling farmers to minimize crop production loss. The households also need to prepare for extreme weather events.In dialogue with the existing literature, we respond to, and confirm, the scholarly view that the role of the village collective economy plays a key role in ensuring food security ,26. We hAs usual, this paper still has some limitation. Due to data unavailable, for example, the impact of the township or even the county economic levels cannot be taken into consideration; long-term temperature and precipitation at county-level data have to be used for plot-level modelling. For the future study, it be better incorporating long-term plot-level temperature and precipitation into plot-level modelling by taking into consideration of county- and -township-level economic impacts.


The likelihood of coinfection increases in regions where HBV is endemic because of the similar transmission route. China is another endemic nation, with 5.9% of the population being HBsAg-positive. This study aimed to evaluate the prevalence of HCV antibody positivity in HBsAg-positive subjects, HCV RNA positivity in anti-HCV positive subjects, and HBV/HCV coinfection with the hope of exploring hepatitis C microelimination using currently available therapies.  12,500 HBsAg-positive serum samples were collected. All samples were screened for anti-HCV. Furthermore, positive samples were screened for HCV RNA. All patients with positive HCV RNA were followed up for suspicious transmission routes of HCV and linkage to care.  44 out of 10,560 (0.4%) patients with positive HBsAg had detectable anti-HCV. There were 32 males and 12 females, with a statistical difference. 17 out of 44 were HCV RNA positive. Among them, 15 out of 38 patients were HCV RNA positive; 8 patients had started anti-HCV treatment with the DAA regimen, while the other 7 patients had not. After patient education, one patient had begun treatment and reached SVR12, while three patients still refused anti-HCV treatment. NCT03794791. The HCV/HBV coinfection prevalence was found to be lower in this study. Even though HBV and HCV share a somewhat similar transmission route, HBsAg-positive subjects may not be at high risk for HCV infection. The process of hepatitis C's microelimination could be accelerated by increasing patient awareness and education. This trail is registered with  Viral hepatitis C is a kind of infectious disease and a global endemic caused by the hepatitis C virus (HCV), to which the majority of the population is vulnerable. Blood is one of the main routes of pathogen transmission. After contracting an HCV infection, there is a significant risk of developing chronic hepatitis C, with estimates ranging from 55%–85%. Although it is possible that some people would not exhibit any evident signs, the general population has a 5% to 15% risk of cirrhosis if the infection has lasted for more than 20 years [Recent research estimated that over 71 million people around the world are chronically infected with HCV, with approximately 10 million of these subjects residing in China , 3. NotaThe goal of the microelimination strategy for hepatitis C is to eradicate the disease in order to reduce the burden it places on individuals, communities, and institutions. This will be accomplished through a variety of strategies, some of which include incorporating HCV drugs into the health insurance system, increasing screening for high-risk and ordinary people in each region and in special places , expanding health care propaganda about HCV, and increasing the rate of HCV diagnosis and linkage to care.Taking all of these things into account, this study evaluated serum anti-HCV antibodies in HBsAg-positive patients and interviewed those who tested positive by telephone. Following that, all HCV-infected patients in this subset were additionally tested for HCV RNA. Patients who tested positive for HCV RNA were contacted by telephone to discuss their prognosis and treatment options. This was done in an effort to encourage them to undergo anti-HCV therapy.A total of 12,500 outpatients and inpatients with positive hepatitis B surface antigen who visited the Second Affiliated Hospital of Chongqing Medical University, Chongqing, China, between June 1, 2018, and November 24, 2020, were enrolled. A blood sample of about 2.0 milliliters (ml) was collected from each patient. Repeat visits were excluded based on the patients' medical records. This research was approved by the ethical committee of the second affiliated hospital of Chongqing Medical University. (201837).All the tests followed the methods of Cai et al. . First oT-test was used for continuous variables, and P < 0.05 was considered statistically significant.SPSS26.0 was used for statistical analysis, and the Chi-square test was used for rate comparison, while the independent sample P = 0.003). The average age of males was 46.34 ± 7.58 and females was 48.67 ± 11.59, and the differences were statistically significant (P = 0.032). 38 of the 44 cases who were contacted or whose data were available had suspected routes of getting HCV infection. These include: 13/38(34.2%) surgery history, 10/38 (26.3%) intravenous drug use, 8/38 (13.9%) blood transfusion use, 1/38(2.6%) tooth extraction history, 1/38 (2.6%) paid blood donation, 1/38 (2.6%) hemodialysis, 2/38(5.3%) sexual transmission, 1/38(2.6%) dental treatment history, and 11/38(28.9%) others  others .23 of the 38 participants, whose treatment information was collected or with whom contact was made were negative for HCV RNA, whereas 15 were positive. 12/23 HCV RNA-negative patients had a history of anti-HCV treatment, while 11/23 HCV RNA-negative patients had no history of antiviral treatment and had spontaneous virus clearance. 15 out of 38 participants, whose information could be obtained or contacted, tested positive for HCV RNA. Eight of these patients had started HCV treatment, whereas seven had no prior HCV treatment history. One of the seven patients with no history of HCV treatment succumbed to HCC, while four failed to grasp the importance of hepatitis C treatment. After HCV-related education, one patient had already begun HCV treatment with SOF/VEL + RBV and completed treatment, whose HCV RNA at 12 weeks was negative. However, three patients still declined treatment owing to financial hardships, terminal-stage lung cancer, or lack of care. The other two patients did not begin HCV therapy because they did not know they were afflicted with HCV. However, after getting information about HCV and expressing a desire to begin treatment, DAA was planned for them at an appropriate time.Twenty (52.6%) of the 38 patients had a history of HCV treatment, while 18 had no history of HCV treatment. Among the patients with a history of treatment, nine were treated with Indian generic drugs, 2 with sofosbuvir + darunavir ± ribavirin, 3 with sofosbuvir/vipatavir ± ribavirin, 2 with Elbasvir/Gravir, 1 with interferon + ribavirin, 2 with sofosbuvir + ribavirin, and 1 with Ledipasvir/Sofosbuvir.One of these 38 patients tested positive for HBsAg and anti-HBc. Three were positive for HBsAg, HBeAg, or anti-HBc. 34 were positive for HBsAg, HBeAb, or anti-HBc. Three out of 38 HBV DNA test samples were positive. For the three individuals with HBV DNA positivity and negative HCV RNA, no HCV treatment was necessary. 8/20 patients with a history of anti-HCV therapy had HBV treatment prior to initiating anti-HCV medication, while 3/20 patients combined HBV treatment with anti-HCV therapy. 2 out of 20 patients initiated HBV therapy at the end of HCV therapy, with abnormal liver function and confirmed hepatocellular cancer (HCC). 7 out of 20 did not initiate HBV treatment at all.There were four patients who developed HCC. They were all positive for HBsAg, HBeAb, and anti-HBc positive, but negative for HBV DNA. One-fourth of the population consisted of females, while three-fourths consisted of males. One-fourth of HCV RNA-positive patients have died in the absence of anti-HBV and anti-HCV treatment. One-fourth had uremia and negative HCV RNA without a history of anti-HBV and anti-HCV treatment, and they were also deceased. Two-fourths were HCV RNA negative and had consumed alcohol over 80 g per day for more than 20 years, one of them had anti-HCV therapy, and both of them started anti-HBV therapy after HCC developed.The treatment of chronic hepatitis C infections has been revolutionized by the recent advent of highly efficient direct-acting antivirals with cure rates above 95%, making the physical eradication of viral hepatitis C possible. Nevertheless, a substantial proportion of individuals with HCV infections might stay asymptomatic for decades, placing them at risk for a slow progression to severe liver disease and death if they do not obtain timely testing and treatment. In light of the efficacy of DAAs, the strategy for eradicating HCV has shifted from treatment to the identification of subjects with chronic HCV infection. Africa and the Western Pacific region have the highest prevalence of hepatitis B infection, between 7.5% and 5.9%, according to the World Health Organization. China, which is located in eastern Asia along the Western Pacific region and has a population of more than 1.4 billion, has the highest chronic HBV infection population in the world, with approximately 82.6 million individuals [This study showed that the anti-HCV antibody positive rate was 0.4% and the HCV RNA positive rate was only 0.16% in this special population of 10,560 HBsAg-positive patients in our hospital, which was not significantly different from the anti-HCV antibody positive rate of 0.43% in the 1–59 years old Chinese population in 2006 [Ruiling et al.  found thAmong the 38 patients who tested positive for anti-HCV antibodies, the results showed that 20 of them  had previously received anti-HCV treatment, while the remaining 18 patients had no history of anti-HCV treatment. Furthermore, 7 of the patients tested positive for HCV RNA, while 11 of the patients tested negative for HCV RNA. This suggests that the 11 patients who tested negative for HCV RNA had a history of HCV infection but may have undergone spontaneous HCV clearance. In adults, the rate of chronicity following HCV infection has been estimated to range anywhere between 50 and 80 percent in previous studies. According to the findings of our research, 27 out of 38 (71.1%) individuals had chronic hepatitis C, which was in line with findings from previous studies. One of the seven patients who tested positive for HCV RNA but had no prior history of therapy passed away, whereas the other four out of seven did not have any clinical manifestations.The identification of patients alone is not sufficient for the microelimination of HCV infection. This was understood through patient telephoned interviews when patients and their families knew nothing about HCV infection and its clinical consequences, or whether there was therapy available. This was found to be the reason why patients refused anti-HCV therapy. In this study, among the 4 untreated patients who did not realize the importance of hepatitis C treatment, 1 patient had accepted DAA therapy for hepatitis C, had completed the treatment, and achieved SVR12 after the patient education (in the form of telephone interview), while 3 patients still refused treatment due to financial constraints, terminal stages of lung cancer, or the belief that there were no symptoms, therefore, there were no diseases. This revealed that the lack of HCV infection symptoms and patients' understanding of the disease's chronic nature are the two main factors impeding the eradication of the virus. Therefore, it is important to raise public awareness of the disease and publicize the benefits and costs of the current treatment for hepatitis C. In this study, it was also found that two other patients did not start HCV treatment because they did not know they had contracted HCV. After being informed of the virus nature, HCV, and the disease, hepatitis C, during the telephone interview, they had decided to take DAA for hepatitis C treatment in the near future. It is obvious that now that there are so many patients with HBV infection in China and they should undergo a routine blood test, it is possible to have the test of HCV infection included in the routine HBV test to improve the detection rate and treatment rate of hepatitis C in China.The majority of the 20 patients with a history of HCV therapy opted for less expensive generic medications rather than the original formulations. From January 2020 on, sofosbuvir/velpatasvir and elbasvir/grazoprevir have been included in the national medical insurance in China, and the individual burden for the cost of anti-HCV treatment has been approximately one-seventh of the previous level, allowing more patients with HCV infection to access standard HCV care. There is no doubt that patient education is the most effective method for providing them with information.In this study, 15 out of 38 individuals tested positive for HCV RNA. Among these 15 individuals, eleven did not have a history of HBV or HCV treatment despite having negative HBV DNA. Compared to a single infection, HBV/HCV coinfection may result in a higher incidence of liver cirrhosis, liver decompensation, and liver cancer . It has There are also some limitations to this study. First of all, this study explored the status of HBV/HCV coinfection mainly in hospital visited patients. Those who did not visit a doctor were not included. Secondly, some medical history data of patients with positive anti-HCV antibodies and HCV RNA was missing due to a wrong phone number or the uncooperative attitude of those patients, and thus we failed to educate them about HCV through the telephone.In conclusion, China has the most HCV RNA-positive cases of any country in the world, with about 200,000 cases reported each year. Hepatitis C can now be cured. To microeliminate hepatitis C, it is important to raise awareness, lower the cost of treatment, and manage and track patients' treatment plans. This meets the WHO's 2030 viral hepatitis elimination objective . In addi