Objective To evaluate the performance and clinical utility of Verigene gram-negative blood culture ( BC-GN ) assay for a rapid identification of gram-negative bacteria and resistance genes . Methods Non-repetitive blood culture samples containing gram-negative bacteria were collected from inpa-tients in the Second Affiliated Hospital of Zhejiang University School of Medicine from June to October , 2013 .BC-GN assay was performed to identify the species and genetic resistance determinants of gram -nega-tive bacteria directly from the positive blood culture bottles .VITEK MS and the VITEK 2 Compact were used for species identification and antimicrobial susceptibility test , the results of which were considered as gold standards.The resistance genes were further validated by PCR amplification and sequencing analysis .A comparison of the results and time between the BC-GN assay and routine methods was conducted . Results The detection range of BC-GN assay almost covered all of the common gram-negative bacteria .BC-GN assay showed an advantage of high accuracy in the identification of Escherichia coli (13/13), Klebsiella pneumoniae (19/24), Klebsiella oxytoca (9/9), Pseudomonas aeruginosa (39/39), Serratia marcescens (4/5), Enterobacter spp.(6/8), Citrobacter spp.(11/11), Proteus spp.(6/6) and Acinetobacter spp. (24/24) with an overall accuracy of 94.24%for the identification of mono-microbial blood culture samples . Moreover , BC-GN assay accurately identified all of the bacteria and resistance genes from the two multi -mi-crobial samples .Species identification and resistance profiles could be 42 hours earlier available by using BC-GN assay than those by using routine methods .Conclusion BC-GN assay could simultaneously and ac-curately identify bacteria and resistance determinants from blood cultures within 2 hours.More time for clini-cally effective therapy could be achieved by using BC-GN assay for the reduction of mortality associated with bloodstream infection .

Objective To study a semi-automatic segmentation framework for liver complex lesion in CT images. Methods A multiphase level set method of image segmentation based on C-V model was proposed, which was in connection with the complex information in the pathological changes area of liver. Depending on the overall characteristics of the image, the active curve contour of C-V model stopped on the edge of objects. It was possible for the segmentation of multi-objects because multi-phase level set was drawn into. Simultaneously, the problem of overlapping and hollow caused by more level set function was avoided. Results The method behaved well in the segmentation of CT images of the liver lesion, two different area were separated. Conclusion The segmentation tests prove that the proposed segmenting method makes a good result.

Objective To investigate the molecular background of the New Delhi-metallo-1 (NDM-1)-producing Morganella morganii.Methods Two carbapenem-resistant M.morganii named 1 and 2 were isolated in the Second Hospital of Jiaxing,Zhejiang on October 4th and 29th,respectively.Antimicrobial susceptibility was determined by agar dilution method.Pulsed-field gel electrophoresis (PFGE) was performed to analyse the homololgy of isolates.Amplification with specific primers,DNA sequencing,conjugation experiments and genetic environment analysis were conducted to investigate the molecular mechanisms of resistance.Results The two M.morganii isolates were resistant to carbapenem and fluoroquinolones,while susceptible to aztreonam.PFGE analysis indicated that the two isolates were distinguishable.Amplification and DNA sequencing confirmed the coexistence of blaNDM-1,blasHv-12,qnrS1 and aac(6')-Ib-cr in both isolates.Transconjugants were detected with blaNDM.1 and qnrS1 simultaneously.Genetic environment analysis demonstrated that the blaNDM-1-bleMBL-trpF-dsbC-cutA1 structure was in consistence with those from known blaNDM-1-carrying Klebsiella pneumoniae.Conclusion The blaNDM-1 in M.morganii isolates possiblely obtained from K.pneumoniae through translatable plasmids.

Chronic cough is a very common symptom of children presenting to pediatric practitioners,moreover,chronic wet cough is especially troublesome for children's health.Since protracted bacterial bronchitis (PBB) is a common cause of chronic wet cough in children,it is of great importance to timely identify PBB from children with chronic cough.Now,the clinical features and diagnostic clues of PBB,as well as the diagnostic criteria of PBB based on microbiology are introduced;it is also proposed that comprehensive and reliable consultation,detailed physical examination and related laboratory tests are needed when identifying PBB out of children with chronic wet cough.For infants and young children,in particular those with persistent wet cough and without any specific changes of chest radiography and other specific cough pointers (eg,upper airway cough syndrom,cough type asthma,respiratory tract infection with specific pathogen,foreign body),the diagnosis of PBB should be taken into consideration and clinical observation and follow-up are needed to reevaluate.

Objective To investigate the expression level of lipoprotein lipase (LPL) mRNA in chronic lymphocytic leukemia (CLL) patients and evaluate the prognostic value of LPL in CLL Methods Quantitative real-time RT-PCR (qRT-PCR) was performed in 62 CLL patients, 10 normal controls using Taqman probe system. Association between LPL and other known prognostic factors, such as IgVH mutation status, ZAP-70 and CD38 expression, was determined using the Spearman correlation analysis. ROC curve was used to determine the cut-off value of LPL expression level, the positive and negative predictive value of IgVH mutation status. Results The correlation coefficients of the standard curves in qRT-PCR were not less than 0.990. The coefficients of variation (CV) of interrun assay and intramn assay were < 5%, and the sensitivity can reached 102 copies/μg RNA. The median LPL mRNA expression level was 0.006 0 (0-0.737 0) in 62 CLL patients, whereas in 10 normal controls LPL mRNA expression level was extremely low with the median level of 0 (0-0.000 4). The expression levels of LPL in three CLL samples after miniMACS-sorted CD19 positive B cells were 0.036 0, 0.075 0 and 0.197 0, which were similar to the levels before miniMACS-sorted (0.024 0, 0.074 0 and 0.225 0). LFL expression was significantly associated with IgVH mutation status (r=0.45, P<0.05) . LPL expression level in IgVH unmutated patients [0.006 0 (0.000 7-0.110 0)] was significantly higher than the level in IgVH mutated patients [0.002 0(0.000 2-0.027 0)] (U=96.5, P<0.05). LPL expression was also significantly associated with ZAP-70 (r=0.38, P<0.05), CD38 expressions (r=0.43, P<0.05). According to ROC curve, the cut-off of LPL mRNA expression level was 0.036, with a 66.7% specificity, a 72.4% sensitivity, a 51.8% positive predictive value (IgVH unmutated), and a 83.3% negative predictive value (IgVH mutated) for IgVH mutation status. Conclusions The qRT-PCR assay is reliable and sensitive. LPL mRNA expression significantly correlates with IgVH mutation status, ZAP-70 and CD38 expression, and could be a predictive marker of IgVH mutation status. Our data confirms a role for LPL as a novel prognostic indicator in CLL.

Objective To study the relationship between the expression of Mycobacterium tuberculosis small heat shock protein Hsp16.3 and the apoptosis of infected mouse alveolar macrophages.Methods The laboratory mice were infected with bacterial suspension of the international standard virulent strain of Mycobacterium tuberculosis H37Rv strains (H37Rv),Hsp16.3 gene deletion mutants of the international standard virulent of Mycobacterium tuberculosis H37Rv strains(△H37Rv),or sterile saline solution (normal control)by the tail vein. After successful replication of mouse infection model in each group,we cleaved the alveolus of each group of mice and collected lavage fluid to obtain alveolar macrophages of the infected mice at days 1 ,3 ,5 ,7 ,9 ,1 1 ,1 3 and 1 5 .Then the infection status of macrophages was observed with confocal laser scanning microscopy;flow cytometry was used to detect the apoptosis rate of alveolar macrophages of the infected mice;Caspase-3 and Bcl-2 expressions were examined by Western blot.Results The apoptosis rate of Hsp16.3 gene was higher in deletion strain (△H37Rv)group and H37Rv strains (H37Rv)group than in control group.The apoptosis rate of alveolar macrophages in △ H37Rv group gradually increased,peaked at day 7 ,and then gradually decreased.It was significantly higher in H3 7 Rv group than in H3 7 Rv strain group from day 1 to 7 and from day 1 3 to 1 5 (P<0 .0 5 ).Caspase-3 and Bcl-2 protein expressions in the macrophages of△H37Rv group and H37Rv group were higher than those of control group.Caspase-3 expression in the microphages of △H3 7 Rv group and H3 7 Rv group gradually increased from day 1 to 7 and peaked at day 7;it peaked again at day 13 in H37Rv group.However,Caspase-3 expression remained significantly higher in△H37Rv group than in H3 7 Rv group (P<0 .0 5 ).Bcl-2 expression in △H3 7 Rv group did not change much at the early stage of infection (P<0 .0 5 ),but gradually increased after day 9 .Bcl-2 expression in H3 7 Rv group did did not change much from day 1 to 7 (P<0.05),but gradually increased after day 7.However,it remained lower in△H37Rv group than in H37Rv group,especially after 7 days(P<0.05).Conclusion Mycobacterium tuberculosis small heat shock protein Hsp16.3 can inhibit the apoptosis of macrophages during the early and late stages of infection,and this inhibition may be achieved by inhibiting the expression of apoptotic protease Caspase-3 and promoting the expression of Bcl-2 protein.

Objective To observe the effect of electroacupuncture on early enteral nutrition (EEN) support in patients with severe craniocerebral injury.Methods A prospective, randomized, controlled study was conducted, 50 patients with severe craniocerebral injury admitted to the the Department of Intensive Care Unit of Traditional Chinese Medicine Hospital of Ningbo from January 2014 to October 2016 were enrolled, and they were randomly divided into electroacupuncture experimental group (26 cases) and conventional treatment control group (24 cases) by randome number table. Enteral nutrition (EN) support was implemented in 24 - 48 hours after admission for all the patients, additionally, the electroacupuncture experimental group was treated by electroacupuncture stimulating acupoint from the 1st day after admission, once 30 minutes daily for consecutive 10 days. The levels of EN calorie reaching standard situation and the parenteral nutrition (PN) necessary for addition were recorded on 3 days and 5 days after admission; the nasal feeding amount of the two groups was recorded on the 1, 4, 7 days; the incidences of complications during EN support period were observed in both groups.Results The proportion of EN calorie reaching the standard in electroacupuncture experimental group was significantly higher than that in the conventional treatment control group in 5 days of treatment [92.3% (24/26) vs. 70.8% (17/24),P < 0.05]. The proportions necessary for addition of PN support were lower in electroacupuncture experimental group than those in the conventional treatment control group on 3 days and 5 days of treatment [3 days: 19.2% (5/26) vs. 25.0% (6/24), 5 days: 7.7% (2/26) vs. 16.7% (4/24)], but there were no significant statistical differences between the two groups (bothP > 0.05). The nasal feeding amounts in electroacupuncture experimental group were significantly higher than those in the conventional treatment control group on 4 days and 7 days of treatment [4 days (mL): 1292.31±123.04 vs.1204.17±139.81,7 days (mL):1342.31±113.75 vs.1275.00±103.21, bothP < 0.05]. The incidence of complications of the electroacupuncture experimental group was significantly lower than that of the conventional treatment control group in 10 days of treatment [26.92% (7/26) vs. 41.67% (10/24),P < 0.05].Conclusions Application of electroacupuncture has a certain clinical value during implementing EN support for treatment of patients with severe craniocerebral injury, early intervention of electroacupuncture can effectively improve the patients' gastrointestinal function and elevate the successful rate of EN, and the therapy is simple, effective and safe without any obvious adverse reactions.

Objective To investigate the effects of nutritional therapy under energy metabolic monitoring on nutrition indicators and clinical prognosis of elderly patients with critical severe diseases in the department of Intensive Care Unit (ICU).Methods One hundred and twenty elderly patients admitted to the Department of ICU of Integrate Traditional Chinese Medicine Hospital of Ningbo, from January 2013 to December 2016 were enrolled, and they were divided into a control group (62 cases) and an observation group (58 cases) by randomized block method. The patients in observation group received nutritional support treatment under the guidance of energy metabolic monitoring, the amount of nitrogen needed was measured every day, and appropriate energy was provided according to the amount of nitrogen and the ratio of heat to nitrogen; the patients in the control group were given the nutritional support program according to experience. The clinical efficacy was evaluated after 7 days of treatment in the two groups, the differences in hemoglobin (Hb), serum albumin (Alb), prealbumin (PA), weaning success rate within 7 days, duration of mechanical ventilation, length of stay in ICU, the standard rate of enteral nutrition (EN) in 7 days, parenteral nutrition support rate, reaching EN target calorie time, acute physiology and chronic health evaluation Ⅱ (APACHEⅡ) score on the 7th day after admission and at discharge, the incidence of complications such as abdominal distention, stress ulcer, ventilator associator pneumonia (VAP), heart failure during nutritional treatment and mortality were observed and compared after EN support between the two groups.Results Compared with control group, after treatment Hb (g/L: 136.5±2.5 vs. 90.4±2.3), Alb (g/L: 35.7±4.6 vs. 32.8±4.2), PA contents (g/L: 211.0±20.8 vs. 190.9±30.7), weaning success rate within 7 days [55.2% (32/58) vs. 33.9% (21/62)], the standard rate of EN in 7 days in the observation group were obviously higher [82.8% (48/58) vs. 51.61% (32/62) allP < 0.05], but duration of mechanical ventilation (days: 8.8±3.5 vs. 11.1±4.0), length of stay in ICU (days: 21.2±5.0 vs. 25.9±6.5), parenteral nutrition support rate [29.3% (17/58) vs. 51.6% (32/62)], reaching EN target calorie time (days: 4.4±2.1 vs. 6.2±2.9), APECHE Ⅱ score 7 days after admission (18.7±5.8 vs. 20.8±8.1), APACHEⅡscore at discharge (13.0±5.2 vs. 15.6±4.5) and the incidence of complications such as abdominal distension [10.3% (6/58) vs. 41.9% (26/62)], stress ulcer [3.4% (2/58) vs. 12.9%(8/62)], VAP [22.4% (13/58) vs. 25.8% (16/62)], heart failure [15.5% (9/58) vs. 24.2% (15/62)] etc, were all lower in observation group (allP < 0.05), and 2 weeks later the mortality was significantly lower in the observation group than that in the control group [13.79% (8/58) vs. 22.58% (14/62),P < 0.05].Conclusions Nitrogen required in elderly patients critically ill patients with early determination, the supply of nutrients to guide empirical method is more accurate compared to the nutritional therapy. Nutritional support under energy metabolism monitoring can shorten clinical course, improve nutritional indicators and help reduce the risk of complications and death.

Objective:To evaluate the clinical value of NG-Test Carba5 for rapid detection of carbapenemases produced by carbapenem-resistant Enterobacteriaceae (CRE) strains. Methods:A total of 1 210 CRE strains were collected during 2018-2022 from 77 hospitals in 21 provinces of China and were subjected to NG-Test Carba5 for rapid detection of carbapenemase. The whole genome sequencing (WGS) analysis was referenced as the gold standard method.Results:Overall, the NG-Test Carba5 demonstrated excellent performance in detection of five kinds of carbapenemases [Klebsiella pneumoniae carbapenemase (KPC), New Delhi metallo-β-lactamase (NDM), imipenemase metallo-β-lactamase (IMP), Verona integron-encoded metallo-beta-lactamase (VIM) and oxacillinase-48-type carbapenemases(OXA-48)] from CRE strains, with a sensitivity of 98.47% (1 161/1 179), specificity of 100% (31/31), and positive predictive value of 100% (1 161/1 161). The sensitivity for detection of NDM, IMP, OXA and VIM reached 100% (307/307), and 97.70% (763/781) for KPC. For 11 strains carrying blaKPC-25, blaKPC-78, or blaKPC-93, NG-Test Carba5 reported positive KPC detection (11/11). For strains carrying blaKPC-33 and blaKPC-77, however, NG-Test Carba5 delivered negative results. Additionally, for those strains co-producing two or three kinds of carbapenemases, NG-Test Carba5 was able to report all of the targets with a sensitivity of 100% (91/91). Conclusions:NG-Test Carba5 showed excellent performance in rapid and accurate detection of carbapenemases from CRE strains. Nonetheless, for those strains with negative results, some other phenotypic and genotypic methods should be implemented alongside to avoid missing targets.