The aim of this study is to establish a rapid,efficient,and sensitive method for detecting the infectious laryngotracheitis virus(ILTV).The DNA of ILTV was extracted and used as a tem-plate to develop a recombinant enzyme-mediated isothermal amplification(RAA)fluorescence de-tection method for ILTV through optimization of conditions,sensitivity analysis,and repeatability assessment.Additionally,the nucleic acids of avian influenza virus(AIV),IBV,and Newcastle dis-ease virus(NDV)were detected to verify the specificity of this method.Finally,this method was applied to analyze 59 clinical samples collected from multiple large-scale chicken farms in Hebei Province,and the results were compared with those obtained from real-time fluorescence quantifi-cation(qPCR)and PCR methods according to national standards.The results showed that the RAA detection method established in this study had a reaction system of 25.0 μL buffer,2.1 μL primer,0.6 μL probe,5.0 μL magnesium acetate,and 5.0 μL template.The reaction temperature was 39 ℃ and the amplification time was within 20 minutes.The sensitivity of this method was 101 copies/μL,and the specificity detection was 100％.Testing of 59 clinical samples showed that 17 were detected positive by both RAA fluorescence and qPCR,and 12 were detected by PCR,and the detection rate of RAA(fluorescence)was consistent with real-time fluorescence quantification and qPCR,which was higher than that of the PCR assay.The research results indicate that the RAA fluorescence method has a short detection time,good specificity and sensitivity,and can be used for rapid detection of ILTV.

The aim of this study is to establish a rapid,efficient,and sensitive method for detecting the infectious laryngotracheitis virus(ILTV).The DNA of ILTV was extracted and used as a tem-plate to develop a recombinant enzyme-mediated isothermal amplification(RAA)fluorescence de-tection method for ILTV through optimization of conditions,sensitivity analysis,and repeatability assessment.Additionally,the nucleic acids of avian influenza virus(AIV),IBV,and Newcastle dis-ease virus(NDV)were detected to verify the specificity of this method.Finally,this method was applied to analyze 59 clinical samples collected from multiple large-scale chicken farms in Hebei Province,and the results were compared with those obtained from real-time fluorescence quantifi-cation(qPCR)and PCR methods according to national standards.The results showed that the RAA detection method established in this study had a reaction system of 25.0 μL buffer,2.1 μL primer,0.6 μL probe,5.0 μL magnesium acetate,and 5.0 μL template.The reaction temperature was 39 ℃ and the amplification time was within 20 minutes.The sensitivity of this method was 101 copies/μL,and the specificity detection was 100％.Testing of 59 clinical samples showed that 17 were detected positive by both RAA fluorescence and qPCR,and 12 were detected by PCR,and the detection rate of RAA(fluorescence)was consistent with real-time fluorescence quantification and qPCR,which was higher than that of the PCR assay.The research results indicate that the RAA fluorescence method has a short detection time,good specificity and sensitivity,and can be used for rapid detection of ILTV.

Objective To evaluate the agreement of four common HEp-2 indirect immunofluorescence assay(IFA)kits in the patients with antinuclear antibody(ANA)-associated rheumatic immune diseases(AARD)and the patients with non-autoimmune diseases(NAD).Methods The experiment in this study included two stages.In stage 1,the serum samples were randomly selected from 134 patients,and ANAs were detected by IFA at Xinhua Hospital Affiliated to Shanghai Jiao Tong University School of Medicine from Janu-ary to June 2023.All of the samples were tested using four kinds of HEp-2 IFA kits,and the consistency of qualitative results was eval-uated by statistical analysis.The kit exhibited highest positive rate was defined as Kit X.In the stageⅡ,a total of 554 serum samples(from 218 AARD and 336 NAD patients)with positive results detected by initial screening of reagent X were selected during the same period,and then the samples were tested by the other three HEp-2 IFA kits.The patterns and titers of ANA were recorded,and a semi-quantitative evaluation system was established.The reproducibility of different patterns of ANA and the consistency of the results among varying clinical characteristics,fluorescence reaction intensities and positive reaction sites in nucleus was statistically analyzed.Results There were no significant differences of qualitative results among the results from four kits(P＞0.05).The highest positive rate ap-peared in the kit m(45.86%)which was deemed as the initial screening kit X.Significant differences in the consistency of ANA pat-terns were observed.The reproducibility scores of centromeric pattern and granular pattern were higher than those of homogeneous pat-tern,dense fine speckled pattern,nuclear cytoplasmic mixed pattern and other mixed pattern with significant difference(P＜0.05).The reproducibility score of simple pattern was higher than that of mixed patterns(P＜0.05).In the nucleoplasmic region,the consistency score of the AARD group was higher than that of NAD group(P＜0.01).The consistency scores of each reaction site increased with the rise of the intensity of reaction.In the three reaction parts(nucleoplasm,nucleolus and equatorial plate),the scores between the weak and strong fluorescence reaction intensity groups showed significant differences(P＜0.001).The lowest consistency score occurred in cytoplasmic region.Conclusion The clinical interpretation for IFA ANA reports should be more cautious for the results showing weak fluorescence intensity,mixed patterns,and staining positive cytoplasmic sites.For the choice for reagents,the clinical laboratories should be also mindful of the impacts of fluorescent secondary antibodies of anti-human immunoglobulin on the test results.The develop-ment of standardized official guidelines for the manufacture of HEp-2 IFA kits should be crucial initiative for enhancing the consistency of ANA detection and promoting mutual recognition for the results between laboratories.

In order to understand the difference and expression of genes in CEK cells before and af-ter baicalin interferes with IBV,further reveal and analyze the mechanism of baicalin interfering IBV replication in CEK cells in vitro.After IBV infected CEK cells for 2 h,9.75 mg/L baicalin liq-uid was added to interfere with CEK cells,which was recorded as the baicalin(H-IBV)group,and three replicate wells were set in the control group and the IBV(IBV)infection group.After 36 h culture,cell samples were collected and subject to transcriptome for sequencing.The results showed that there were 102 differentially expressed genes in H-IBV group compared with IBV in-fection group,among which 48 genes weresignificantly up-regulated and 54 genes were significantly down-regulated.Through functional annotation in GO and KEGG databases,it was found that dif-ferentially expressed genes were mainly annotated in biological processes such as cellular proces-ses,biological regulation,metabolism,and secondary pathways such as viral infectious diseases,signal transduction and interaction.Retinol metabolism pathway,phospholipid transfer to mem-brane,IL-27 mediated signaling pathway,MDA5/RIG-I and Toll-like receptor signaling pathway were significantly enriched in CEK cells,and the production of type Ⅰ interferon and interferon al-pha and the process of antiviral infection were also positively regulated.There were more differen-tial genes enriched in nucleic acid catalysis,immune system,and reaction,and interbiological reac-tion.Through the STRING network interaction map,it was found that most immune-related genes could form a 36-node interaction network centered on IRF7,TLR3 and STAT1.Therefore,com-pared with IBV group,the differentially expressed genes after baicalin treatment were mainly an-notated and enriched in the biological process,and the immune system and response were en-hanced,mainly through the positive regulation of IRF7 in the MDA5/RIG-I receptor signaling pathway and the inhibition of TLR3 signal transduction in the Toll-like receptor signaling path-way.Positive regulation of IL-27 mediated pathway and regulation of JAK-STAT signaling path-way were supplemented by activation of the expression of IRF7,TLR3,STAT1 and other related genes,and interaction with corresponding downstream proteins to promote the expression of IFN-α and regulatory cytokines,coupled with negative regulation of viral(defense)response and viral processes.Thus,baicalin interferes with IBV replication in CEK cells.

OBJECTIVEGroup A β-hemolytic streptococcus (GAS) or Streptococcus pyogenes may be encountered in diverse clinical situations in children. A rising incidence of invasive group A streptococcus (IGAS) infections has been noted in children in the past three decades. The aim of this study was to summarize the clinical characteristics and antimicrobial resistance of IGAS in children, and to raise the level of diagnosis and treatment of this infection.

METHODThe clinical data from 19 cases of IGAS younger than 14 years old seen from January 2004 to December 2011 treated in the authors' hospital were analyzed. IGAS infections are defined as the isolation of GAS from a normally sterile site in patients.

RESULTThe 19 cases were identified as IGAS infections, among whom 15 were male and 4 were female, and the ratio of them was 3.75. The age ranged from 1 day to 14 years, with a median age of 4 years. The course of disease was 4 h-10 days. The average length of stay was 12.2 days. In 13 cases the episodes of the infection occurred in winter and spring. In 18 cases the infection was community-acquired. Overall, 10 cases had neck or foot dorsum abscess, four cases had purulent peritonitis, and 3 cases were diagnosed as streptococcal toxic shock syndrome (STSS) complicated with empyema, pyopneumothorax occurred in 1 case and neonatal septicemia in another. Three cases had an underlying disease, including 2 cases wounded in a car accident and 1 case of congenital esophageal atresia and tracheoesophageal fistula. Before the isolation of GAS, 5 cases had stayed in ICUs, the length of ICU stay was 1-32 days, 4 cases had received intubation and mechanical ventilation, the ventilation time was 8 h-24 days, 2 cases had received major surgery; 5 cases had other pathogen coinfection, including 4 cases of abdominal pus at the same time and Escherichia coli was isolated, and 1 case had parainfluenza virus type I coinfection. Peripheral blood leucocyte increased in 18 cases, one case dropped off. The C-reactive protein (CRP) levels increased in all patients, including 16 cases who had 14-160 mg/L, 3 cases had levels higher than 160 mg/L. Twenty strains of GAS were isolated from 19 cases' sterile sites, of them 10 strains were isolated from abscess, 4 strains were isolated from blood and another 4 from ascites. Two strains were from the same patient at different times of pleural effusion. All 20 strains displayed a full susceptibility to cefazolin, levofloxacin and vancomycin, and the rates of resistance to both cefotaxime and penicillin were 10.0%. The rates of resistance to erythromycin and clindamycin were 55.0% and 70.0% respectively. Among the patients 3 cases were cured, 14 cases improved, and 2 cases died, of whom 1 case died of STSS secondary to multiple organ dysfunction, 1 case died of basic disease secondary to multiple organ dysfunction.

CONCLUSIONSkin and soft tissues were the most common IGAS infection sites in children, and IGAS infection also can lead to serious STSS and even can be life threatening. Penicillin and cephalosporin are still sensitive for children IGAS infections.

Abscess ; drug therapy ; epidemiology ; microbiology ; Adolescent ; Anti-Bacterial Agents ; pharmacology ; therapeutic use ; Cephalosporins ; therapeutic use ; Child ; Child, Preschool ; Clindamycin ; therapeutic use ; Community-Acquired Infections ; drug therapy ; epidemiology ; microbiology ; Drug Resistance, Bacterial ; Female ; Humans ; Infant ; Infant, Newborn ; Male ; Microbial Sensitivity Tests ; Retrospective Studies ; Soft Tissue Infections ; drug therapy ; epidemiology ; microbiology ; Streptococcal Infections ; drug therapy ; epidemiology ; microbiology ; Streptococcus pyogenes ; drug effects ; isolation & purification

Objective To study the change of toll-like receptor 3 (TLR3) and tumor necrosis factoralpha (TNF-α) both at the levels of protein and mRNA in human lung epithelial cells (A549) infected with respiratory syncytial virus (RSV) and the effects of peroxisome proliferator-activated receptor γ (PPARγ) agonists Rosiglitazone and 15-deoxy-delta12,14 prostaglandin J2 (15d-PGJ2).Methods RSV inoculation after A549 cells subcultured,then A549 cells were randomly divided into six groups:15d-PGJ2 + RSV group (group A),Rosiglitazone + RSV group (group B),RSV group (group C),PDTC + RSV group (group D),GW9662 + Rosiglitazone + RSV group (group E) and cell control group (group F).Cells were harvested after incubated for 12 h,24 h and 48 h respectively.The expression of TLR3 and TNF-α at mRNA was determined by real-time RT-PCR,and the protein level of TLR3 and TNF-α was determined by Western Blot and ELISA respectively.Results The expression of TLR3 and TNF-α at mRNA and protein at virous time in group C were significantly higher than those of group F (all P ＜ 0.01).While,the expression of TLR3 and TNF-α mRNA and protein in group A,B and D were significantly lower than than those of group C (all P ＜ 0.01).15d-PGJ2 and Rosiglitazone had the strongest effect to inhibit the expression of TLR3 and TNF-αmRNA and protein at 12 h and the expression of TLR3 and TNF-α mRNA and protein decreased as the increasing dose of 15d-PGJ2 and Rosiglitazone.Conclusion RSV infection induces increased expression of TLR3 and TNF-α mRNA and protein,which can be inhibited by Rosiglitazone and 15d-PGJ2 in a dose-dependent manner.

Objective To observe the expressions of RANTES , FKN and IP-10 at mRNA and pro-tein levels in human lung epithelial cells (A549) infected by respiratory syncytial virus (RSV), and to eval-uate the changes of them interfered with 15-deoxy-delta12,14prostaglandin J2(15d-PGJ2), rosiglitazone or 2-chloro-5-nitrobenzanilide ( GW9662 ) .Methods A549 cells were seeded in 6-well culture plates and cul-tured overnight in F12K culture solution.Then they were randomly divided into five groups , including group A (15d-PGJ2+RSV group), group B (rosiglitazone+RSV group), group C (DMSO+RSV group), group D (GW9662+rosiglitazone+RSV group) and group E (cell control group).Cells and supernatants were harves-ted from each group at different time points (12 h, 24 h and 48 h) of culture.The expressions of RANTES , FKN and IP-10 at mRNA and protein levels were measured by ELISA and real-time quantitative RT-PCR analysis.Results The expressions of RANTES , FKN and IP-10 at mRNA and protein levels in group C were significantly higher than those in group E at time points of 12 h, 24 h and 48 h (all P<0.05).In group C, the expressions of the three chemokines at mRNA level reached a peak at 24 h, but began to de-crease at 48 h, which showed no statistical significance compared with those at 12 h (all P>0.05).Moreo-ver, the expressions at protein level were peaked at 48h, and had significant difference with those expressed at 12 h and 24 h (all P<0.05).Compared with group C, the expressions of the three chemokines both at mRNA level and protein level were decreased in group A and B as the dose was increased (all P<0.05), and the lowest levels were observed with the intervention of 20 μmol/L of 15d-PGJ2 in group A and 30μmol/L of rosiglitazone in group B .Conclusion The expressions of RANTES , FKN and IP-10 at mRNA and protein levels were increased with RSV infection , and the peaks of mRNA level and protein level were respectively achieved at 24 h and 48 h after infection.PPARγagonists played an anti-inflammatory role through inhibiting the expressions of the three chemokines both at mRNA level and protein level in a dose -de-pendent manner .

Objective To analyse the kidney damage situation of the elderly health examination people,and identify its characteristic.Methods A cross-sectional study was held which enrolled 1088 elderly health examination people.Urine routine,random urine albumin/creatinine ratio (ACR),serum creatinine,urea nitrogen were detected by biochemical analyzer,and estimated glomerular filtration rate (GFR) with CKD-EPI formula.Kidney structure change was examined by Color doppler ultrasound detector.Results The prevalence of hypertension,hyperlipemia,diabetes mellitus was 61.5％,62.1％,11.6％,respectively.The abnormal detection rate of urine routine was 19.0％,including 2.6％ proteinuria,14.1％ hematuresis and 5.4％ leucocyturia.The abnormal detection rate in the people with was higher than those without (P＜0.01).However,the albuminuria detection rate with random urine ACR was 25.1％,obviously higher than that of urine routine (P＜0.01).The ultrasound results showed that 6.8％ of the total were examined with elderly characteristic kidney change,the proportion of renal cyst was the highest,accounted for 21.8％.70.7％ of all people were in the level of eGFR more than 60 ml· min-1 · 1.73 m-2.The level of eGFR＜60 ml· min-1 · 1.73m-2 in the people with was higher than those without (P＜0.01).eGFR was declined with age.When age increased every 10 years,eGFR was decreased 7 ml · min-1 · 1.73 m-2.Conclusions No matter in structure or function,the elderly people's kidney damage has its characteristic.We should make it clear to correctly diagnose and cure elderly kidney disease.

Analyzing the complicated variety and specification of drugs and the objective demand of pharmaceutical circulation, to seek out the key factors in improving the efficiency of pharmaceutical circulation, for putting forward suggestions to promote the development of pharmaceutical circulation in China. The conclusion is drawed from industrial organization theory and successful experience of foreign countries, high market attention met with the demand of complicated variety and specification of drugs in pharmaceutical circulation.
China ; Drug Industry ; economics ; organization & administration ; Marketing of Health Services ; economics ; Pharmaceutical Preparations ; economics