Persistence and clearance of viral RNA in 2019 novel coronavirus disease rehabilitation patients / 中华医学杂志(英文版)

BACKGROUND@#A patient's infectivity is determined by the presence of the virus in different body fluids, secretions, and excreta. The persistence and clearance of viral RNA from different specimens of patients with 2019 novel coronavirus disease (COVID-19) remain unclear. This study analyzed the clearance time and factors influencing 2019 novel coronavirus (2019-nCoV) RNA in different samples from patients with COVID-19, providing further evidence to improve the management of patients during convalescence.@*METHODS@#The clinical data and laboratory test results of convalescent patients with COVID-19 who were admitted to from January 20, 2020 to February 10, 2020 were collected retrospectively. The reverse transcription polymerase chain reaction (RT-PCR) results for patients' oropharyngeal swab, stool, urine, and serum samples were collected and analyzed. Convalescent patients refer to recovered non-febrile patients without respiratory symptoms who had two successive (minimum 24 h sampling interval) negative RT-PCR results for viral RNA from oropharyngeal swabs. The effects of cluster of differentiation 4 (CD4)+ T lymphocytes, inflammatory indicators, and glucocorticoid treatment on viral nucleic acid clearance were analyzed.@*RESULTS@#In the 292 confirmed cases, 66 patients recovered after treatment and were included in our study. In total, 28 (42.4%) women and 38 men (57.6%) with a median age of 44.0 (34.0-62.0) years were analyzed. After in-hospital treatment, patients' inflammatory indicators decreased with improved clinical condition. The median time from the onset of symptoms to first negative RT-PCR results for oropharyngeal swabs in convalescent patients was 9.5 (6.0-11.0) days. By February 10, 2020, 11 convalescent patients (16.7%) still tested positive for viral RNA from stool specimens and the other 55 patients' stool specimens were negative for 2019-nCoV following a median duration of 11.0 (9.0-16.0) days after symptom onset. Among these 55 patients, 43 had a longer duration until stool specimens were negative for viral RNA than for throat swabs, with a median delay of 2.0 (1.0-4.0) days. Results for only four (6.9%) urine samples were positive for viral nucleic acid out of 58 cases; viral RNA was still present in three patients' urine specimens after throat swabs were negative. Using a multiple linear regression model (F = 2.669, P = 0.044, and adjusted R = 0.122), the analysis showed that the CD4+ T lymphocyte count may help predict the duration of viral RNA detection in patients' stools (t = -2.699, P = 0.010). The duration of viral RNA detection from oropharyngeal swabs and fecal samples in the glucocorticoid treatment group was longer than that in the non-glucocorticoid treatment group (15 days vs. 8.0 days, respectively; t = 2.550, P = 0.013) and the duration of viral RNA detection in fecal samples in the glucocorticoid treatment group was longer than that in the non-glucocorticoid treatment group (20 days vs. 11 days, respectively; t = 4.631, P  0.05).@*CONCLUSIONS@#In brief, as the clearance of viral RNA in patients' stools was delayed compared to that in oropharyngeal swabs, it is important to identify viral RNA in feces during convalescence. Because of the delayed clearance of viral RNA in the glucocorticoid treatment group, glucocorticoids are not recommended in the treatment of COVID-19, especially for mild disease. The duration of RNA detection may relate to host cell immunity.

Persistence and clearance of viral RNA in 2019 novel coronavirus disease rehabilitation patients / 中华医学杂志(英文版)

Background@#A patient’s infectivity is determined by the presence of the virus in different body fluids, secretions, and excreta. The persistence and clearance of viral RNA from different specimens of patients with 2019 novel coronavirus disease (COVID-19) remain unclear. This study analyzed the clearance time and factors influencing 2019 novel coronavirus (2019-nCoV) RNA in different samples from patients with COVID-19, providing further evidence to improve the management of patients during convalescence.@*Methods@#The clinical data and laboratory test results of convalescent patients with COVID-19 who were admitted to from January 20, 2020 to February 10, 2020 were collected retrospectively. The reverse transcription polymerase chain reaction (RT-PCR) results for patients’ oropharyngeal swab, stool, urine, and serum samples were collected and analyzed. Convalescent patients refer to recovered non-febrile patients without respiratory symptoms who had two successive (minimum 24 h sampling interval) negative RT-PCR results for viral RNA from oropharyngeal swabs. The effects of cluster of differentiation 4 (CD4)+ T lymphocytes, inflammatory indicators, and glucocorticoid treatment on viral nucleic acid clearance were analyzed.@*Results@#In the 292 confirmed cases, 66 patients recovered after treatment and were included in our study. In total, 28 (42.4%) women and 38 men (57.6%) with a median age of 44.0 (34.0–62.0) years were analyzed. After in-hospital treatment, patients’ inflammatory indicators decreased with improved clinical condition. The median time from the onset of symptoms to first negative RT-PCR results for oropharyngeal swabs in convalescent patients was 9.5 (6.0–11.0) days. By February 10, 2020, 11 convalescent patients (16.7%) still tested positive for viral RNA from stool specimens and the other 55 patients’ stool specimens were negative for 2019-nCoV following a median duration of 11.0 (9.0–16.0) days after symptom onset. Among these 55 patients, 43 had a longer duration until stool specimens were negative for viral RNA than for throat swabs, with a median delay of 2.0 (1.0–4.0) days. Results for only four (6.9%) urine samples were positive for viral nucleic acid out of 58 cases; viral RNA was still present in three patients’ urine specimens after throat swabs were negative. Using a multiple linear regression model (F=2.669, P=0.044, and adjusted R2=0.122), the analysis showed that the CD4+ T lymphocyte count may help predict the duration of viral RNA detection in patients’ stools (t=-2.699, P=0.010). The duration of viral RNA detection from oropharyngeal swabs and fecal samples in the glucocorticoid treatment group was longer than that in the non-glucocorticoid treatment group (15 days vs 8.0 days, respectively; t=2.550, P=0.013) and the duration of viral RNA detection in fecal samples in the glucocorticoid treatment group was longer than that in the non-glucocorticoid treatment group (20 days vs 11 days, respectively; t=4.631, P <0.001). There was no statistically significant difference in inflammatory indicators between patients with positive fecal viral RNA test results and those with negative results (P >0.05).@*Conclusions@#In brief, as the clearance of viral RNA in patients’ stools was delayed compared to that in oropharyngeal swabs, it is important to identify viral RNA in feces during convalescence. Because of the delayed clearance of viral RNA in the glucocorticoid treatment group, glucocorticoids are not recommended in the treatment of COVID-19, especially for mild disease. The duration of RNA detection may relate to host cell immunity.

Clinical characteristics of glucocorticoid-induced eye adverse reactions in children with primary nephrotic syndrome / 北京大学学报(医学版)

Objective:To investigate the clinical features and side effects,with regard to glucocorticoid-induced ocular hypertension,glaucoma or cataract in children with primary nephrotic syndrome.Methods:Clinical data were collected and analyzed from 71 cases of primary nephrotic syndrome with glucocorticoid-induced ocular hypertension,glaucoma or cataract from Jun.2014 to Jun.2016.These children were hospitalized in Peking University First Hospital.Results:Totally 1 580 children with primary nephrotic syndrome were collected,glucocorticoid-induced complications in eyes were found in 71 cases,and the incidence was 4.5％.There were 66 cases with ocular hypertension,2 cases with glucocorticoid glaucoma,2 cases with glucocorticoid glaucoma combined with cataract,1 case with high intraocular pressure combined with cataract.There were 41 boys and 30 girls with eye-related side effects caused by glucocorticoid.The average age of onset of glucocorticoid-induced eye adverse reactions in children with primary nephrotic syndrome in our research were 8 (2,16) years.The average duration or interval time from glucocorticoid medication use to eye adverse effects was 157 (6,420) days.No statistical significance was found in intraocular pressure between different genders,types of glucocorticoid,different route of glucocorticoid and whether methylprednisolone pulse treatment (P ＞ 0.05).There was no significant correlation between age,body mass index,blood pressure,cumulative dosage,duration time of glucocorticoid,mean daily dosage and glucocorticoid-induced ocular hypertension (P ＞ 0.05).The ocular hypertension was controlled after treatment.Conclusion:Children with nephrotic syndrome after treatment of glucocorticoid are susceptible to ocular complications,and the occurrence of ocular hypertension is closely related to glucocorticoid susceptibility of the nephrotic children.Regular eye monitor is indispensable for the children suffering from primary nephrotic syndrome.

Effects of patient age, number and quality of embryo transferred on the outcome of in vitro fertilization-embryo transfer / 南方医科大学学报

<p><b>OBJECTIVE</b>To study the effect of patient age, the number and quality of embryo transferred on pregnancy outcome of in vitro fertilization-embryo transfer (IVF-ETs).</p><p><b>METHODS</b>A retrospective study was performed in infertile women who underwent a total of 1800 cycles of IVF-ET and intracytoplasmic sperm injection (ICSI). The patients were divided into three age groups, namely<30 years group, 30-35 years group, and>or=35 years group. The clinical pregnancy rate and the multiple pregnancy rate were compared when 1, 2 or 3 embryos and 0, 1, 2 or 3 good-quality embryos were transferred.</p><p><b>RESULTS</b>In patients<30 years, no significant differences was found in the clinical pregnancy rate between 1, 2 and 3 embryos transfer groups; 2 and 3 good-quality embryos transfer resulted in similar pregnancy rate, which was significantly higher than that resulted from 0 and 1 good-quality embryo transfer. Multiple pregnancy was not found in 1 embryo transfer group. In patients aged 30-35 years, the pregnancy rate showed no significant differences not only between 1 and 2 embryos transfer groups, but also between 2 and 3 good-quality embryos transfer groups; multiple embryo transfer led to significantly increased multiple pregnancy rate. In patients aged>or=35 years, the transfer of 1, 2 and 3 embryos resulted in similar pregnancy rate; transfer of 3 good-quality embryos had obviously higher pregnancy rate than 0, 1 and 2 good-quality embryos transfer groups. Increased numbers of embryos and good-quality embryos transferred were both associated with increased multiple pregnancy rate.</p><p><b>CONCLUSION</b>One good-quality embryo transfer in patients<30 years and 2 good-quality embryos transfer in patients>or=30 years can obtain ideal pregnancy rate and reduce the incidence of multiple pregnancy. For patients aged>or=35 years, transfer of only good-quality embryo is recommended.</p>

Development and genetic polymorphism of abnormal pronuclear zygotes after intracytoplasmic sperm injection / 中华医学遗传学杂志

<p><b>OBJECTIVE</b>To compare the development of abnormal pronuclear zygotes after intracytoplasmic sperm injection (ICSI) and analyze their genetic polymorphism.</p><p><b>METHODS</b>Four hundred and ninety three abnormal pronuclear zygotes after ICSI were divided into three groups based on the number of pronuclei: 347 nonpronuclear oocytes, 71 monopronuclear zygotes and 75 multipronuclear zygotes. All of them were cultured in the medium of Vitrolife G5 series(TM). Sixteen short tandem repeats (STR) of seven blastocysts were then analyzed by ABI3100.</p><p><b>RESULTS</b>The cleavage rate of nonpronuclear group (25.4%) was lower than that of the others (P<0.01), the proportion of blocked embryos in nonpronuclear group (48.9%) was significantly higher than that of the others (P<0.05), but the blastocyst rate showed no significant difference in three groups (P>0.05). The genetic polymorphism of the 16 STRs showed that the blastocysts from the nonpronuclear and multipronuclear were diploid, and one of the blastocysts from nonpronuclear oocyte was Y-bearing.</p><p><b>CONCLUSION</b>The zygotes with abnormal pronuclei after ICSI might have development potential, and the blastocysts from nonpronuclear oocytes and multipronuclear zygotes could be diploid.</p>

Identification of novel genetic markers in Mycobacterium tuberculosis Beijing genotype strains / 中华传染病杂志

Objective To identify a unique protein as a novel genetic marker for rapid molecular typing of Mycobacteriutn tuberculosis Beijing genotype strains by comparing the proteome of Beijing genotype strains with non-Beijing strains.Methods Fifty-six clinical isolates of Mycobacterium tuber- culosis were analyzed by spoligotyping to determine genotypes.The two-dimensional electrophoresis (2 DE)was used to compare the global protein patterns between Beijing genotype strains and non Bei jing strains.Differential expressed proteins were measured by matrix assisted laser desorption ioniza tion lime of flight mass spectrometry(MALDI-TOF-MS).The data obtained from peptide mass fingerprinting were compared in protein database.The genes encoding differential expressed proteins and their upstream were sequenced.Results Forty nine of the 56 isolates were Beijing genotype strains and 7 isolates were non-Beijing strains.A unique protein Rv0927c was identified,which is absent in Beijing genotype strains compared with 7 non Beijing strains and H37Rv.There were two characteristic mutations in Beijing genotype strains,a deletion of AGC at nucleotide position 421 of Rv0927c and a 127 G→A muta- tion in the upstream of Rv0927c.but not in non Beijing strains and H37Rv.Conclusion Characteris tic mutations of Rv0927c in Beijing genotype strains can be used as a novel genetic marker for rapid molecular typing of Mycobacteriuln tuberculosis Beijing genotype strains and non Beijing strains.

Cultivation and karyotype analysis of the human embryonic stem cells HUES4 / 中华医学遗传学杂志

<p><b>OBJECTIVE</b>To establish a protocol for culturing of human embryonic stem cells (HUES4) without any animal-derived feeder cells and to investigate the karyotype stabilities of HUES4 cells after long-term cultivation.</p><p><b>METHODS</b>HUES4 cells were cultured on mitomycin C treated MEFs or human foreskin fibroblast feeder cells. The pluripotency of the ES cells was analyzed by immunocytochemistry staining to detect the expression of pluripotent marker, karyotype of the ES cells at passage 27, 34, 41, 44 and short tandem repeat (STR) at passage 27 were analyzed.</p><p><b>RESULTS</b>The HUES4 cells cultured on human feeder cells were positive for alkaline phosphatase activity, SSEA-4, TRA-1-60 and TRA-1-81 staining, but negative for SSEA-1. Analysis of karyotype at different passages suggested an abnormal karyotype 46, XY, t(9;15)(q22;q26) mosaicism occurred in HUES4, and the ratios of abnormal increased with passage.</p><p><b>CONCLUSION</b>HUES4 could be cultured without animal-derived feeder cells and the incidence of abnormal karyotype might be increased with long-term culture.</p>

A rapid and high throughput method for apoE genotyping / 中华医学遗传学杂志

<p><b>OBJECTIVE</b>To develop a new method that can determine the apolipoprotein E(apoE) genotypes rapidly in high throughput.</p><p><b>METHODS</b>Genome DNA samples were extracted from the anticoagulated peripheral blood samples of 79 patients with Alzheimer's disease(AD) and 63 healthy individuals, and the 492 bp apoE gene fragments including 112 and 158 codons were amplified by polymerase chain reaction (PCR). With one PCR product, three recombined alleles (epsilon 2, epsilon 3 and epsilon 4) of apoE gene as controls were obtained by cloning and site-directed mutagenesis. The excess primers and dNTPs in all PCR products were removed by treatment with clean up reagents, then template-directed dye-terminator incorporation reaction (TDI) was performed and R110 or TAMRA labeled Acyclo-terminators were added into the mutation sites specifically. Fluorescence polarization value (FP) was measured using victor 2 multilabel counter and the polymorphisms in 112 and 158 condons of apoE gene were investigated.</p><p><b>RESULTS</b>The apoE genotypes in recombined plasmid controls and all serum samples were analyzed using the authors' TDI-FP method, and the reliability and specificity were confirmed by DNA sequencing. The frequency of epsilon 4 allele in patients was significantly higher than that in controls, suggesting that apoE epsilon 4 allele gene is a risk factor for late-onset AD.</p><p><b>CONCLUSION</b>TDI-FP is an easy, reliable and high throughput technology in analyzing polymorphism of apoE gene; it can be used in the prediction of susceptibility to AD in elderly individuals. Furthermore, it is an ideal method for large-scale screening and for studying the relationship between the allelic and genotypic frequencies of apoE and other diseases.</p>

Preparation and identification of polyclonal antiserum against angiotensinogen / 生理学报

For studying the expression and distribution of angiotensinogen (AGT), the C-teminus of rat AGT gene was expressed in E.coli. Rabbits were immunized with expressed AGT protein and sera from different rabbits were raised. ELISA showed a high titre (1:25600) of the antiserum. With the antiserum, Western blotting recognized not only the prokaryotic expressed AGT, but also the endogenous AGT protein in liver tissue of both rats and humans. Using this antiserum, immunohistochemistry showed the expression of AGT protein in islet cells of human pancreas as well as in epithelium of human bile duct. These results suggest that the prokaryotic expressed AGT protein is an effective immunogen for the preparation of anti-AGT antiserum. Our present work provides an important tool for study of the pathophysiological role of AGT as well as local renin-angiotensin system.