Objective To improve the speed and accuracy of bacteria detection, and develop the test of 16S rRNA genes PCR amplification plus gene chip hybridization to diagnose neonatal sepsis. Methods Bacterial 16S rRNA genes were detected in blood and CSF samples of 125 suspected neonatal sepsis, and the results were compared with blood culture, CSF culture, and non-specific diagnostic parameters (WBC, PLT, CRP). 30 non-infectious neonates were regarded as the negative control group. Gene chip test were performed by extraction of DNA, primers and probes design, PCR amplification, preparation of gene chip, hybridization, laser scan and reading of the results. 18 specific probes, including universal 1, universal 2, Gram positive bacterial probe, Gram negative bacterial probe 1, Gram negative bacterial probe 2, Staphylococcus aureus, Staphylococcus epidermidis, CoNS (Coagulase Negative Staphylococcus), Escherichia coli, Haemophilus influenzae, Listeria monocytogenes, Streptococcus pneumoniae, Streptococcus agalactiae, Bacteroides fragilis, Bacillus, Meningococcus, Corynebacterium, Propionibacterium, were used in the test. Results The positive rate of PCR test was 51.2% in 125 blood samples, and was significantly higher than the positive blood culture (25.6%), or the indicator of two abnormal non-specific parameters (32.8%) (P

Objective To establish a method for rapid diagnosis and identification of seven species human herpesviruses infection.Methods Primers and oligonucleotide probes were designed and synthesized based on the highly conserved regions of the DNA polymerase genes in human herpesviruses, namely herpes simplex virus type 1 (HSV-1),HSV-2,varicella-zoster virus (VZV),Epstein-Barr virus (EBV),cytomegalovirus (CMV),and human herpes virus 6 (HHV-6A/6B).DNA microarrays were made by printing the oligonucleotide probes on the special glass slides.A total of 282 blood specimens from children with suspected infection were analyzed by this DNA microarray technique,and the results were compared with those of TaqMan PCR.Results The products of the seven human herpesviruses after PCR amplification could be used to identify the virus species with DNA microarrays.The detection limits were 10 copies/?l for HSV-1,HSV-2,VZV,EBV,CMV,HHV-6A,and HHV-6B,respectively.The assay did not show cross-reaction to the DNA extract of hepatitis B virus,staphylococcus aureus,E.coli,Candia albicans and human genome.Among the 282 samples,59 were positive for human herpesviruses DNA.Compared with those of TaqMan PCR,the sensitivity and specificity of the microarray assay were 96.7% and 99.5%,respectively,and the index of accurate diagnosis was 0.962.Conclusions This DNA microarray for identifying human herpesviruses species is specific and sensitive,and may serve as an efficient technique for simultaneous detection and species identification of human herpesviruses in clinical specimen.

Objective To explore a rapid diagnostic method in neonatal sepsis and bacterial me- ningitis. Methods The primers and TaqMan probes were designed and synthesized based on the sequences of bacterial 16S rRNA gene. Nineteen bacterial strains, 3 different viruses, fungus and human genomic DNA were tested by FQ-PCR assay. Blood specimens and CSF from 195 cases of suspected septicemia were detected with both TaqMan PCR assay and blood or/and CSF culture. Results The FQ-PCR showed very high sensitivity and specificity and was able to detect at least 10 copies of 16S rRNA gene equivalent to 1~2 copies bacterium. No cross-reaction was found with human genomic DNA, other fungus and viruses. Among the 195 cases, the positive rate by FQ-PCR was 12.8%(25 cases) and 7.1%(15 cases) by blood culture ( P

0.05 compared with group B);ZT and Youjiangtang Tablets had similar effect in promoting the synthesis of hepatic glucose and the release of serum insulin(P

Objective To assess the value of combined detection of enterovirus nucleic acid and antibody in early etiological diagnosis for hand-foot-and-mouth disease ( HFMD).Methods A case-control study was conducted.A total of 1 066 cases of children clinically diagnosed with HFMD from Hangzhou Children′s Hospital were involved into the research group from January to June 2014, consisting of 401 common cases and 665 severe cases; Throat swabs and serum samples from these children underwent combined detection for EV71/CA16/EV of enterovirus nucleic acid by fluorescence quantitative RT-PCR and for EV71/CA16-IgM by ELISA.All data were analyzed with SPSS 16.0.Results The total positive rate of enterovirus nucleic acid EV71/CA16/EV by fluorescence quantitative RT-PCR in the 1 066 cases of children clinically diagnosed with HFMD was 75.52%( 805/1 066 ) ( 95%CI: 72.80%-78.05%).But the total positive rate of combined detection was 91.46%( 975/1 066 ) ( 95%CI:89%.58-93.04%).The total positive rate of combined detection is higher than that of RT-PCR test(χ2 =98.338,P=0.000).The positive rate of EV71 type of combined detection was 64.63%(689/1 066)(95%CI:61.67%-67.49%),which is 15.38%higher than that of RT-PCR test 49.25%(525/1 066)(95%CI:46.21%-52.29%)(χ2 =51.453, P=0.000).In 665 severe cases of HFMD, the total positive rate of combined detection was 96.69%(643/665)(95%CI:94.95%-97.87%), which is higher than that of RT-PCR test 79.25%(527/665)(95%CI:75.92%-82.22%)(χ2 =95.607, P =0.000).In the severe cases, the positive rate of EV71 type of combined detection was 87.52%( 582/665 ) ( 95%CI:84.71%-89.89%) , which is 18.95% higher than that of RT-PCR test 68.57%(456/665) (95%CI:64.87%-72.06%) (χ2 =69.665, P=0.000).In the fatal cases, the positive rate of EV71 type of combined detection was 95.92%(94/98) (95%CI:89.28%-98.68%).Conclusions The combined detection of enterovirus nucleic acid and specific IgM antibody can significantly increase the positive rate of HFMD, especially for severe cases.The combine detection increases both the total positive rate and EV71 positive rate.Thus it has a high potential for becoming a new guidelines for laboratory diagnosis of HFMD.

Objective To evaluate the diagnostic value of the common test parameters in acute fever without obvious infection focus and sick appearance in children under 5 years.Methods The hospitalized children with fever duration less than 7 days, anal temperature higher than or equal to 38°C, age younger than or equal to 5 years, and without obvious infection focus and sick appearance were recruited, we investigated the diagnosis value of common test parameters including C-reactive protein (CRP), procalcitonin (PCT), the white blood cell count (WBC), and neutrophil percentage (N%) , according to the ifnal diagnostic.Results Of 228 children, 42 children (18.42%) had serious diseases, the difference of CRP, PCT between serious diseases group and non-serious diseases group were statistically signiifcant (P<0.001). The diagnostic cut-off point of CRP was 67.1 mg/L by speciifcity of 0.810 and sensitivity of 0.715, that of PCT was 0.505 ng/L by speciifcity 0.762 and sensitivity 0.672. The speciifcity and sensitivity combining CRP with PCT was respectively 0.918 and 0.617. Of 228 children, 32 children had viral infections, 40 children had bacterial infections, 15 children had mycoplasma infections. The difference of CRP, PCT, WBC, and N% among three groups were statistically signiifcant (P<0.01).The cut-off point of CRP was 38 mg/L by sensitivity 0.900 and spec-iifcity 0.813, that of PCT was 0.450 ng/L by sensitivity 0.700 and speciifcity 0.812, and the speciifcity and sensitivity combining CRP with PCT was respectively 0.965 and 0.630, to distinguish bacterial infections from viral infections. The diagnostic cut-off point of CRP was 80.75 mg/L by sensitivity 0.700 and speciifcity 0.933 distinguishing bacterial infections from mycoplasma infections.Conclusions The parameters CRP and PCT have the diagnostic value for the children with the acute fever and age younger than or equal to 5 years and without obvious infection focus and sick appearance in etiology and serious diseases, espe-cially the value of combining CRP with PCT is better.

BACKGROUND:Studies have demonstrated that chitosan can indirectly promote the proliferation of fibroblast and the synthesis of collagen, Using chitosan and specific fibroblasts together to construct tissue-engineering materials for tendon repair may be an adequate way to promote healing and prevent adhesion during the healing process.OBJECTIVE: To observe the effect of water soluble chitosan (WSC) on the growth and genotype of 3T3TK fibroblasts.DESIGN: Controlled experiment based on observation.SETTING: Jiujiang University Medical College.MATERIALS: 3T3TK fibroblasts were provided by Cell Bank of Chinese Academy of Sciences. WSC (specification: 60 mesh, 30CPS, Jinan Haidebei Marine Bioengineering Co.,Ltd),DMEM (low sugar) (Gibco Company, USA), fetal bovine serum (FBS, Sijiqing Biological Engineering Institute, Hangzhou), penicillin, streptomycin (Biosharp Company, USA),trypsin (BioEev-Tech Scientific & Technical Co.,Ltd, Beijing).METHODS: This experiment was carried out in the Laboratory for Clinical Application of Biology, Center for Medial Scientific Research, Jiujiang University between November 2004 and April 2006. ①Cells in the log phase were digested with 2.5 g/L trypsin to produce single cell suspension and inoculated into a 96-well culture plate at a density of 6 000 cells /200 μL medium. Five groups were prepared, 5 holes per group. 200 μL cell suspension was added into each well.After 24 hours of cultivation, supernatant was discarded, 200 μL DMEM with 1, 0.1, 0.01 and 0.000 1 g/L chitosan was added respectively in the first four groups. The remaining group was taken as the negative control group, in which 200 μL DMEM medium without chitosan was added. The cell suspension was routinely cultured for 72 hours. The cell viability was measured by CellTilter- GloTM Luminescent Cell Viability Assay according to the manufacturer's protocol. Cells in the log phase were split and inoculated into 24-well culture plates. Four groups were prepared (5 holes per group). 1 mL DMEM medium supplemented with 1, 0.1, 0.01 g/L chitosan was added into the first 3 groups respectively, and the 4th group was set as control group. Hydroxyproline and alkaline phosphatase(ALP) kits were used for detecting the contents of collagen and ALP in the supernatant of fibroblasts.MAIN OUTCOME MEASURES : ①Effect of WSC on viability of fibroblasts cultured in vitro.②Contents of collagen and ALP in the cell culture supernatant of fibroblasts.RESULTS:①Detection results of viability of fibroblasts: There were no significant differences in viability of fibroblasts between each chitosan group and control group (P ＞ 0.05).②Contents of collagen and ALP in the cell culture supernatant of fibroblasts: Hydroxyproline content in the cell culture supernatant of fibroblasts of 1 g/L and 0.1 g/L groups was (7.598±0.770) and (8.366±0.308)mg/L, respectively, which was higher than that of control group [(11.269±0.707)mg/L,P ＜ 0.01]; Collagen content in the 1 g/L and 0.1 g/L groups was(56.703±5.748) and(62.437±2.301)mg/L, respectively, which was lower than that of control group [(84.099±5.280)mg/L,P ＜ 0.01]. With the concentration of chitosan increasing, the collagen content was decreased in a dose-dependent manner. There were no significant differences in ALP activity in the cell culture supernatant between each chitosan group and control group (P ＞0.05).CONCLUSION: WSC does not obviously inhibit the viability of 3T3TK fibroblasts, but can markedly reduce the content of secreted collagen. It is indicated that chitosan can be used as tissue engineering material for tendon repair, and inhibit adhesion formation during tendon repair.

Objective To investigate the effects of dilute concentration and acting time of pronase on quality of gastroscopy.Methods A total of 448 patients were randomly divided into two groups : sodium bicarbonate group and pronase with sodium bicarbonate group.Pronase was diluted into 50 ml (400 U/ml)and 100 ml (200 U/ml) using sodium bicarbonate.The patients were pretreated by pronase of different concentrations 10 min, 20 min, 30 min, 60 min and 120 min before gastroscopy.Diluent of same quantity were taken by the control group.Visibility of gastroscopy, procedure times and positive rates of lesions were compared.Results Pretreatment of pronase significantly improved visibility of gastroscopy, raised positive rates of lesions, and reduced procedure times of gastroscopy, compared with the control group (each P ＜ 0.05).The visibility of gastroscopy were over 80％ 20,30, and 60 minutes before the examination with no significant difference(P ＞ 0.05).The visibility of gastroscopy decreased sharply 30 minutes after taking pronase, especially after 60 minutes.There was no significant difference in the quality of gastroscopy between the 200 U/ml and 400 U/ml group 20-60 minutes before gastroscopy (P =0.640).Conclusion Pronase (200 U/ml-400 U/ml) significantly improves visibility of gastroscopy, raises positive detection rates of lesions, and reduces procedure time of gastroscopy 20-60 minutes before pretreatment.

Objective To investigate cerebrospinal fluid characteristics and clinical features in children with severe hand,foot and mouth disease (HFMD) induced by enterovirus 71 (EV71) infection.Methods A total of 114 children with severe HFMD,in whom EV71 was detected by reverse transcription polymerase chain reaction (RT-PCR),were admitted in Hangzhou Children's Hospital during May and August 2013.Seventy-eight children with severe HFMD induced by other enteroviruses admitted at the same period served as controls.The results of cerebrospinal fluids (CSF) routine examination and biochemical tests,and the clinical symptoms were compared between two groups.Differences in enumeration data were compared with x2 test,and measurement data were compared with Mann-Whitney U test.Results The incidences of vomiting and limb shaking in EV71 infection group were 35.1％ and 50.9％,which were higher than those in control group (x2 =7.864 and 19.682,P ＜ 0.05).The incidence of limb shaking in children with nucleated cells count ≥ 100 × 106/L in EV71 group was higher than that with nucleated cells count ＜ 100 × 106/L (72.3％ vs.35.8％,x2 =14.740,P =0.000).The nucleated cells count,protein quantity and their positive rates in EVT1 infected group were higher than those in control group (Z =-9.458 and-6.591,P=0.000; x2=105.421 and 10.932,P =0.000 and 0.001).Conclusion The symptoms of nervous system damage and abnormal CSF examination were more serious in HFMD induced by EV71 infection,and in EV71 infected patients the incidence of limb shaking is correlated with nucleated cell count in CSF.

Objective To develop a method of simultaneous detection and Gram classification for pathogens causing sepsis with gram-specific probes based real-time PCR. Methods A pair of universal primers and a set of probes including Gram-positive probe and Gram-negative probe were designed based on the bacterial highly conserved region of 16SrRNA gene. With the gram-specific probes based real-time PCR, 35 clinical frequently-isolated strains including 17 gram-positive and 18 gram-negative bacteria were identified correctly with the corresponding gram probe. The blood samples from 512 cases of suspected septicemia, who were hospitalized in our neonatal ward and the NICU and developed clinical signs suggestive of infection, were tested with routine culture and bacterial gram-specific probes based real-time PCR separately. Results The detection limit of the gram-specific probes based real-time PCR assay was 10 CFU of the bacteria. The 35 isolates could be detected and classified correctly by gram-specific probes based real-time PCR. The PCR results were all negative for Cytomegalo virus, EB virus, hepatitis B virus, Cryptococcns neoformans, candida albican, human genomic DNA and negative control. The gram-specific probes based real-time PCR appeared to be quite specific. For 512 blood specimens from the patients with suspicious neonatal sepsis, the positive rate of the gram-specific probes based real-time PCR array was 8.20% (42/512,), which is significantly higher than that of blood culture (32/512, 6.25%) (χ2=8.10, P<0.01). When blood culture was used as a standard, the sensitivity of the gram-specific probes based real-time PCR was 100%. The specificity was 97.92% and the accuracy was 98.05%. Canclusions Cram-specific probes based real-time PCR with universal primers and gram-specific probes are developed. This study suggests that the bacterial gram-specific probes based real-time PCR are very useful for the rapid and accurate diagnosis of bacterial infection.