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

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 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 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.

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

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 the expression and clinical significance of apoptosis inhibitory protein, Livin and Smac,in pancreatic carcinoma. Methods The expressions of Livin and Smac protein in 46 cases of pancreatic carcinoma tissues and 15 cases of insulinoma tissues and 14 cases of normal pancreatic tissues were examined by using immunohistochemical SP staining, and its relationship with clinicopathological parameters was analyzed. Results The positive expression rates of Llivin protein were 73.9％ ( 34/46),73.3％ (11/15) and 14.3％ (2/14) in pancreatic carcinoma, insulinoma and normal pancreatic tissue. Livin was highly expressed in pancreatic carcinoma and insulinoma, but there was no significant difference between the two groups, however, both were significantly higher than that in normal pancreatic tissues group ( P ＜ 0.05 ). The expression of Livin was significantly correlated with lymph node metastasis, histopathological grading and clinical staging (P ＜ 0.05 or P ＜0.01 ). The positive expression rates of Smac protein were 39.1％ (18/46), 100％ ( 15/15 ) and 92.9％ (13/14) in pancreatic carcinoma, insulinoma and normal pancreatic tissue. Smac was highly expressed in normal pancreatic tissues and insulinoma, but there was no significant difference between the two groups, however, both were significantly higher than that in pancreatic cancer group (P ＜ 0.05 ). The expression rote of Smac protein was significantly correlated with lymph node metastasis, histopathological grading, chnical staging and patients' age (P ＜0.05 or P ＜0.01 ).Conclusions Livin protein may play an important role in genesis and development of pancreatic carcinoma,but Smac protein may play a role in preventing the development of pancreatic carcinoma.

ObjectiveTo investigate the effects of diet-induced obesity on the developmental process of testes in pubertal Sprague Dawley (SD) rats and explore the possible reversibility. MethodsSixty one-month-old male SD rats were randomly divided into a control group ( n =10) and a model group ( n =50 ), which were fed on a normal diet and a high-fat diet, respectively. After 8 weeks, all the rats in the control group and 10 rats randomly picked out from the model group were killed. The serum testosterone and estradiol levels were measured by enzymelinked inununosorbent assay. Their left testes sections were stained by HE method, and the histology was observed under optical microscope and the spermatogenic activity was evaluated by Johnsen scoring system. The remaining 40 rats in the model group were further randomly divided into 3 subgroups: continued high-fat diet subgroup ( n =13), resume normal diet subgroup (n =13), and weight-loss subgroup (n =14). The continued high-fat diet subgroup was fed by high-fat diet, while the other two subgroups were fed by normal diet. Rats in weight-loss group took normal diet with running 20 min/d. After 6 weeks, the same parameters were assessed using the same methods. ResultsAfter 8 weeks, compared with the control group, the testosterone level of the model group significantly decreased (P =0.024) and the estradiol level significantly increased ( P =0. 017). The result of HE staining showed that the spermatogenic cell layers decreased, with part of seminiferous tubule experiencing atrophy.The number of Leydig cell also decreased and lipo vacuole was seen in the interstitial tissue of testis. The Johnsen score of the model group was significantly lower than that of the control group (P =0.000). The testosterone level was significantly lower in the continued high-fat diet subgroup than that in resume normal diet subgroup ( P =0.001 ) and weight-loss subgroup ( P =0.000), and was significantly lower in resume normal diet subgroup than that in weight-loss subgroup ( P =0.001 ). The estradiol level was significantly higher in continued high-fat diet subgroup than that in resume normal diet subgroup ( P =0.001 ) and weight-loss group ( P =0.000 ), and was significantly higher in resume normal diet subgroup than that in weight-loss group ( P =0.001 ). HE staining showed that, pathological changes aggravating and worsening compared with the control group, the model group had significantly decreased seminiferous tubule cell layers, with some seminiferous tubules experiencing atrophy.The Johnsen score was significantly higher in weight-loss subgroup than that in the other two subgroups ( P =0.000and 0.001, respectively). The Johnsen score was negatively correlated with body weight ( r =- 0.962, P =0.000), and positively correhted with the serum testosterone level ( r =0.916, P =0.000 ). Conclusions High-fat diet can induce pubertal obesity in male SD rats, which is featured by testicular hypoplasia, decreased spermatogenesis, and endocrine dysfunction. Physical exercise may improve the conditions. The degree of obesity may be negatively correlated with the spermatogenic function.