Microbial communities such as those residing in the human gut are highly diverse and complex, and many with important implications for health and diseases. The effects and functions of these microbial communities are determined not only by their species compositions and diversities but also by the dynamic intra- and inter-cellular states at the transcriptional level. Powerful and scalable technologies capable of acquiring single-microbe-resolution RNA sequencing information in order to achieve a comprehensive understanding of complex microbial communities together with their hosts are therefore utterly needed. Here we report the development and utilization of a droplet-based smRNA-seq (single-microbe RNA sequencing) method capable of identifying large species varieties in human samples, which we name smRandom-seq2. Together with a triple-module computational pipeline designed for the bacteria and bacteriophage sequencing data by smRandom-seq2 in four human gut samples, we established a single-cell level bacterial transcriptional landscape of human gut microbiome, which included 29,742 single microbes and 329 unique species. Distinct adaptive response states among species in Prevotella and Roseburia genera and intrinsic adaptive strategy heterogeneity in Phascolarctobacterium succinatutens were uncovered. Additionally, we identified hundreds of novel host-phage transcriptional activity associations in the human gut microbiome. Our results indicated that smRandom-seq2 is a high-throughput and high-resolution smRNA-seq technique that is highly adaptable to complex microbial communities in real-world situations and promises new perspectives in the understanding of human microbiomes.
Humans ; Gastrointestinal Microbiome/genetics* ; Bacteriophages/physiology* ; High-Throughput Nucleotide Sequencing ; Sequence Analysis, RNA/methods* ; Bacteria/virology*

Epidemiological evidence suggests that patients with hypertension infected with severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) are at increased risk of acute lung injury. However, it is still not clear whether this increased risk is related to the usage of renin-angiotensin system (RAS) blockers. We collected medical records of coronavirus disease 2019 (COVID-19) patients from the First Affiliated Hospital, Zhejiang University School of Medicine (Hangzhou, China), and evaluated the potential impact of an angiotensin II receptor blocker (ARB) on the clinical outcomes of COVID-19 patients with hypertension. A total of 30 hypertensive COVID-19 patients were enrolled, of which 17 were classified as non-ARB group and the remaining 13 as ARB group based on the antihypertensive therapies they received. Compared with the non-ARB group, patients in the ARB group had a lower proportion of severe cases and intensive care unit (ICU) admission as well as shortened length of hospital stay, and manifested favorable results in most of the laboratory testing. Viral loads in the ARB group were lower than those in the non-ARB group throughout the disease course. No significant difference in the time of seroconversion or antibody levels was observed between the two groups. The median levels of soluble angiotensin-converting enzyme 2 (sACE2) in serum and urine samples were similar in both groups, and there were no significant correlations between serum sACE2 and biomarkers of disease severity. Transcriptional analysis showed 125 differentially expressed genes which mainly were enriched in oxygen transport, bicarbonate transport, and blood coagulation. Our results suggest that ARB usage is not associated with aggravation of COVID-19. These findings support the maintenance of ARB treatment in hypertensive patients diagnosed with COVID-19.
Aged ; Aged, 80 and over ; Angiotensin Receptor Antagonists/therapeutic use* ; Angiotensin-Converting Enzyme 2/blood* ; Antibodies, Viral/blood* ; Antihypertensive Agents/therapeutic use* ; Biomarkers ; COVID-19/complications* ; China ; Female ; Humans ; Hypertension/drug therapy* ; Intensive Care Units ; Length of Stay ; Male ; Middle Aged ; Retrospective Studies ; Transcriptome ; Viral Load

Objective: To establish a high performance liquid chromatography-tandem triple quadrupole mass spectrometry method for the detection of human plasma paraquat concentration. Methods: The plasma samples were pretreated with methanol to precipitate plasma protein, and then were separated by a Waters XBridge BEH HILIC column (2.5 μm, 2.1 mm × 100 mm) with acetonitrile-water containing 200 mmol/L of ammonium formate and 0.1% of formic acid as mobile phase and 0.4 mL/min of flow rate. The paraquat was monitored by ESI positive ion mode, multi-reaction ion monitoring (MRM) scanning, and m/z 186.1-171.1 as quantitative transition ion-pair. The plasma paraquat concentrations in patients were determined by the established method, and the clinical values of plasma paraquat concentration and severity index of paraquat poisoning (SIPP) were evaluated by the receiver operating characteristic (ROC) curve. Results: When the plasma paraquat concentration ranged from 50 to 10 000 ng/mL, the linearity was good (R 2 =0.997), and the lower limit of quantification was 50 ng/mL. The recovery rates and imprecisions of three quality control products at low (100 ng/mL), medium (2 000 ng/mL) and high (8 000 ng/mL) concentration levels all met the requirements, and no matrix effect was found. The pretreated samples were stable at room temperature for 6 hours, and the results were not affected by repeated freezing and thawing for 3 times. The SIPP of 31 poisoned patients was 17.76 (0.30-90.91) h·mg/L. The SIPP in dying patients was significantly higher than that in survival patients (P＜0.05). The area under the ROC curve of SIPP was 0.889, and the optimal cut-off value was 11.679 h·mg/L. Conclusion The established method is sensitive, accurate, rapid and specific, and suitable for the detection of plasma paraquat concentration in patients.

Influenza virus , the causative agent of influenza , has characteristics of fast transmission and strong infectivity, posing a major threat to public health .Currently, with widely use of the first-line antiviral neuraminidase inhibitors , drug-resistant strains have appeared and brought difficulties to the precaution and treatment of influenza .This article reviews influenza virus structural characteristics , drug-resistance mechanisms , resistance situation and detection methods of drug-resistant virus strains , aiming to provide a reference for future research on influenza virus drug-resistance and clinical medication .

Objective To explore the molecular epidemiological characteristics of norovirus isolated from the patients with acute gastroenteritis in Zhejiang province during 2016.Methods The stool samples and clinical data of 1 308 patients with acute gastroenteritis were collected from January to December in 2016.The type Ⅰ and Ⅱ of norovirus in stool samples were detected by one-step double real-time RT-PCR.Some of the positive specimens were selected by stratified sampling and amplified by conventional RT-PCR,and the PCR products were sequenced for genotype identification and phylogenetic analysis.Results Among the samples studied the positive rate of norovirus was 10.55％ (138/1 308) in which 12 cases were GⅠ genotype,118 cases were G Ⅱ genotype and 8 cases were mixed infection of G Ⅰ/G Ⅱ genotypes.The positive rate of norovirus in different age groups decreased with the increased age of patients,and became the lowest in the patient group of more than or equal to 60 years old.There was no significant difference for the positive rates of norovirus in different genders.Norovirus infection was distributed throughout all the year with the peak value of positive rate (37.50％) in December.The sequence analysis demonstrated that G Ⅱ.4 and G Ⅱ.17 genotypes were the prevalent strains of G Ⅱ genotypes with proportions of 40.91％ (18/44) and 34.09％ (15/44),while GⅠ.6 genotype was the prevalent strain of GⅠ genotypes.Conclusion Norovirus should be the important pathogen causing acute gastroenteritis in Zhejiang province during 2016.G Ⅱ.4 and G Ⅱ.17 of norovirus may be the predominant epidemic genotypes.

Objective: To investigate the etiology composition of enterovirus (EV) in patients with severe hand, foot, and mouth disease (HFMD) in children. To assess the diagnostic value of cerebrospinal fluid (CSF) tests in severe HFMD, and to find the key laboratory tests for severe HFMD. Methods: A total of 288 hospitalized cases of children clinically diagnosed with severe HFMD in Hangzhou Children′s Hospital were included from March to July 2016. Throat swabs were collected and enterovirus nucleic acids were detected by fluorescence quantitative reverse transcription (RT)-PCR. Synchronous CSF and serum samples were collected for EV-A71 and CV-sackievirus A16 (CV-A16)-IgM antibody detection. CSF samples underwent routine and biochemical tests. Normally distributed continuous variables were compared using t test. Non-normal distribution continuous variables were compared using Mann-Whitney U test. Differences between categorical variables were compared with χ² test. Results: The total positive rate of enterovirus nucleic acid EV-A71/CV-A16/EV by fluorescence quantitative RT-PCR in the 288 cases of children clinically diagnosed with HFMD was 83.7% (241/288), including EV-A71 55.2% (159/288), CV-A16 4.9% (14/288) and the other enterovirus 23.6% (68/288). Among the other enterovirus group, there were 29.4% CV-A6 (20/68) , 16.2% CV-A4 (11/68) and CV-A10 10.3% (7/68). The total positive rate of combined serum and CSF detection of EV nucleic acid and EV-A71 and CV-A16 IgM antibody was 98.3% (283/288). EV nucleic acid positive rate was 83.7% (24/288). The positive rates were statistically different (χ² =37.289, P=0.000). The CSF nucleated cells count in EV-A71 positive subgroup was higher than those in CV-A16 positive subgroup and other enteroviruses subgroup (Z=-4.472 and -9.991, respectively, both P<0.05). The CSF nucleated cells positive rate in EV-A71 positive subgroup was higher than those in CV-A16 positive subgroup and other enteroviruses subgroup (χ²=43.857 and 133.078, respectively, both P<0.05). The CSF protein level in EV-A71 positive subgroup was higher than those in CV-A16 positive subgroup and other enteroviruses subgroup (Z=-3.151 and -5.255, respectively, both P<0.05). The CSF protein positive (>400 mg/L) rate in EV-A71 positive subgroup was higher than those in CV-A16 positive subgroup and other enteroviruses subgroup (χ²=4.956 and 11.795, respectively, both P<0.05). The CSF nucleated cell counts and positive rates in EV-A71 IgM antibody-positive subgroup were both higher than those in antibody-negative subgroup (both P<0.05). The CSF protein level and elevated proportion in antibody-positive subgroup were both higher than those in antibody-negative subgroup (both P<0.05). The lactate dehydrogenase concentration in antibody-positive subgroup was significantly higher than those in antibody-negative subgroup (P<0.05). The EV-A71 IgM antibody in serum was significantly correlated with the antibody in CSF (r=0.600, P<0.05). Conclusions EV-A71 is still the most important pathogen of severe HFMD in Hangzhou in 2016. Other enterovirus such as CV-A6, CV-A4 increases compared to those in 2014 and 2015. The CSF routine and biochemical tests and the IgM antibody levels can serve as an important indicator for the diagnosis of children with severe HFMD.

Objective To study the effect of propofol intravenous anesthesia on T helper cells of patients with primary liver cancer during perioperative period.Methods A total of 86 patients with primary liver cancer in our hospital from November 2014 to October 2015 were selected,who were divided into observation group and control group according to the method of random numbers,43 cases in each group.The observation group were taken propofol intravenous anesthesia,and the control group were treated with sevoflurane inhalation anesthesia.The Th1 cells percentage,Th2 cells percentage and the ratio changes of the Th1/Th2 cells of two groups before anesthesia and postoperative 1 day were compared.The plasma cortisol levels of two groups before anesthesia,after anesthesia,intraoperative and 1 day after operation were observed.Results The percentage of Th2 cells in observation group and control group 1 day after surgery had no significant difference (P ＞ 0.05).The percentage of Th1 cells and Th1/Th2 cells ratio of the observation group were higher than those of the control group [(16.32 ± 1.76) ％ vs.(14.16 ± 1.03),(8.48 ± 0.92) vs.(7.11 ± 0.72)],the differences were significant (P ＜ 0.05).The plasma cortisol levels of observation group during operation and 1 day after operation were lower than those of the control group[(12.34 ± 1.02) μg/dL vs.(16.13 ± 1.26) μg/dL,(12.01 ± 0.94) μg/dL vs.(15.25 ± 1.08) μg/dL],the differences were significant(P ＜ 0.05).Conclusion The propofol intravenous anesthesia can encourage more Th to differentiate into Th1 cells,which plays a protective role in the patient's immune function.

Objective To evaluate the effect of Critical Incident Reporting System on the quality of clinical anesthesia.Methods Anesthesia-related critical incidents happened in the perioperative period were reported in voluntary,anonymous,no punishment and confidential manners.The data was collected,classified and documented by assigned professionals on a regular basis from September 2012 to August 2016.The critical incidents were retrospectively analyzed after the risk was assessed.The 4-year reporting rate was collected.The risk of critical incidents was assessed using severity and probability analysis,and the critical incidents-inducing risk factors were analyzed.Results The 4-year reporting rate of critical incidents was 0.551％.From 1st to 4th year,the reporting rates were 0.729％,0.598％,0.819％ and 0.368％,respectively,and the incidence of injury incidents was 0.112％,0.106％,0.133％ and 0.031％,respectively.The reporting rate of critical incidents and incidence and reporting rate of the injury incidents showed a decreasing trend for 1st and 2nd year,significantly increased for 3rd year and decreased for 4th year (P＜0.05).The first three critical incident categories were equipment use and respiratory system-and workflowrelated incidents.Patient injury during surgery was considered an extremely high risk incident;the factor of the medical staff in the department of anesthesiology is the first critical incidents-inducing risk factor.Conclusion Critical Incident Reporting System can discover and correct the system-related risk and the inducing factors in the department of anesthesiology and is an effective method of improving the service quality of clinical anesthesia.

Objective To understand the changes on patterns of sleep duration of the China Health and Nutrition Survey (CHNS) cohort in 9 provinces from 2004 to 2011.Methods Four rounds of CHNS data were used.Urban/rural,age and gender specific insufficient sleeping rates and excessive sleeping rates were analyzed.Results In 2004,2006,2009 and 2011,a total of 274,281,329 and 304 children aged 3-5 years;874,806,768 and 742 children aged 6-12 years;789,529,426 and 367 children aged 13-17 years;9 568,9 530,9 942 and 9 609 adults aged ≥18 years were surveyed respectively.The lowest insufficient sleeping rate was 53.9％ (200/371) in 3-17 years old children in rural area in 2006,the highest insufficient sleeping rate was 77.2％ (44/57) in 3-5 years old children in urban area in 2004.The insufficient sleeping rate increased in rural 3-5 years old children from 2004 to 2011.For the adults aged ≥ 18 years,the insufficient sleeping rate ranged from 4.2％ (82/ 1 954) in females aged 18-44 years in 2004 and 2009 to 20.8％ (211/1 015) in urban residents aged ＞ 60 years in 2011.The insufficient sleeping rate in age-groups 44-59 years and ≥60 years increased in both males and females and in both urban area and rural area from 2004 to 2011.The gender specific excessive sleeping rate in 3-17 years old children was very low in both urban area and rural area and no difference was found in different rounds of survey.The excessive sleeping rate in adults ranged from 18.4％ (569/3 093) in urban population in 2011 to 32.5％ (1 617/4 969) in females in 2004.The excessive sleeping rate of adult decreased from 2004 to 2011.Conclusion We should pay attention to the fact that the insufficient sleeping rate in adolescents is high and in increase in rural 3-5 years old children and adults aged ≥45 years.

Objective To explore pathogen spectrum constitution of acute diarrhea in outpatient and emergency of Zhejiang Province, and provide basis for treatment, prevention and control of the disease. Methods During January 2009 to December 2014, we selected seven sentinel hospitals in different regions of Zhejiang, monitored and researched on pathogen spectrum in patients with acute diarrhea from outpatient and emergency. We recorded patients' personal basic information, the main symptoms and signs, and collected stool samples (5 g). Eight kinds of bacteria (Vibrio cholerae, Shigella spp., Salmonella spp., Diarrheagenic E. coli, Vibrio parahaemolyticus, Aeromonas hydrophila, Yersinia enterocolitica and Plesiomonas shigelloides) and five kinds of viruses (Rotavirus, Norovirus, Sappovirus, Astrovirus and Adenovirus) were detected. Chi-square test and Fisher's exact probability method were used to compare different characteristics of patients with single bacterial infection, single virus infection and multiple infection (bacteria-bacteria, bacteria-viruses, virus-virus). Results During 2009 to 2014, 9 364 fecal
samples from acute diarrhea patients were collected and tested, among which 3 500 cases were tested positive, with total positive rate of 37.38%. Positive rates of bacteria and viruses were 13.14%(1 230 cases) and 20.75%(1 943 cases), respectively. Mixed infection positive rate of multiple pathogens was 3.49%(327 cases). Positive rate of Vibrio parahaemolyticus (5.96%, 558 cases) was the highest among bacterial pathogens, followed by pathogenic Escherichia coli (3.86%, 361 cases). Viruses were mainly Norovirus (10.73%, 1 005 cases) and rotavirus (8.35%, 782 cases). A big difference existed in diarrheogenic pathogen spectrum between patients less than 15 years old and patients equal or older than 15 years old. Pathogens for patients less than 15 years old were mainly virus, with the positive rate of 32.69%(1 014 cases). However, the positive rate of bacteria was 16.86% (1 056 cases) in patients equal or older than 15 years old. Single bacterial infection was highest in age group of 25-34 years old (18.62%, 302 cases) , single virus infection was highest in age group of 1-4 years old (41.12%, 435 cases) , and mixed infections of multiple pathogens were mainly existed in age group of 1-4 years old (7.37%, 78 cases) . Pathogen positive rate were increasing year by year. Pathogen positive rate of patients with acute diarrhea has obvious seasonality, with single bacterial infection being highest during July to September and single virus infection being highest during December to March. Pathogen spectrum of outpatient and emergency patients with acute diarrhea in Zhejiang Province changed a little from 2009 to 2014, mainly rotavirus (22.34% (782/3 500)), norovirus (28.71%(1 005/3 500)), vibrio parahaemolyticus (15.92%(558/3 500)) and Escherichia coli (10.31%(361/3 500)). However, pathogen spectrums in different years owned different features. Conclusion Common pathogens in outpatient and emergency patients with acute diarrhea in Zhejiang Province were tested with significant seasonal epidemic law. The composition of pathogenic spectrum was variant in different age group. Constitutes of major pathogen spectrum in different years differed a little.