Objective To investigate antibiotic resistance and resistant trend of Streptococcus pneumonia. Methods To investigate 753 Streptococcus pneumoniae isolated from Tongji Hospital in recent 10 years from January 1st 2000 to December 31st 2009, most of them were from respiratory tract specimens,followed by blood and cerebrospinal fluid. The MIC to penicillin & cefatriaxone were determined by E-test,and other antimicrobial susceptibility were tested by Kirby-Bauer method. Results For non-cerebrospinal fluid specimen, the total rate of PNSSP was 23.8%( 93/392 ), it was significant different between the rate of PNSSP from children ( 26. 4%, 47/178 ) and adults ( 16. 8%, 36/214, χ2 = 7. 642, P ＜ 0. 01 ). All of 10 strains isolated from cerebrospinal fluid were PRSP. Most isolates were high-susceptive to moxifloxacin and levofloxacin, and the rate of susceptibility were 96. 9% ( 720/743 ) and 90. 5% ( 672/743 )respectively. None of Streptococcus pneumonia was resistant to vancomycin and meropenam. The resistant rate of most tested antibiotics increased in different degree year by year, especially penicillin, erythromycin and clindamycin. The rate of PNSSP was only 19%( 19/99 )in 2006 ,but in 2009 the rate increased to 30%( 35/114 ). The susceptibility rate of erythromycin was 22% ( 28/125 )in 2000, but only 3% ( 3/114 )in 2009 ;and the susceptibility rate of clindamycin decreased from 40% ( 13/32 ) in 2004 to 4% (5/114) in 2009. Conclusions From 2000 to 2009, Streptococcus pneunoniae was more likely resistant to penicillin,erythromycin and clindamycin year by year, especially those isolates recovered from children. It was suggested that antibiotics should be chosen to use according to antimicrobial susceptibility test results.

Objective To identify one runny mucoid-like Gram-negative bacteria with pink pigment isolated from clinical pus sample. Methods The pus sample was aseptically extracted from a deep lesions of one patient, then stored in Amies medium at room temperature for transportation. One sheep blood plate and one chocolate plate were used to detect the possible pathogens from the specimens. After inoculation, the plates were placed in a humidified incubator with 5% CO2 at 35 ℃. To identify the obtained isolates, we used the commercial Vitek2 and API systems, combining some traditional morphological examination and classical biochemical and physiological characteristics. For pure cultures, the cellular fatty acids were extracted, methylated, and determined by gas chromatography method. The 16S rRNA gene was amplified and sequenced by a commercial broad-spectrum PCR primers. The phylogenetic tree based on 16S rRNA gene was constructed by Mega 4.1 software using the neighbour-joining methods with 1 000 bootstrap replications. Results One runny mucoid-like Gram-negative bacterium, named K8756, was isolated both on sheep blood and chocolate plates after 72 h incubation. The API 20NE profile was 1245045 after a 3-day culture, which would be identified as Ochrobactrum anthropi with a good confidence of 98% probability. It was identified as Ralstonia pickettii and Bordetella bronchiseptica by VITEK 2 GN kits. However, further comparative 16S rRNA gene sequences showed that strain K8756 was closely related to the valid published Roseomonas mucosa MDA 5527 with 100% identity. Colonial morphologic features, phenotypic characteristics and major cellular fatty acid composition were also with high similarity to Roseomonas mucosa. Conclusions Strain K8756( = GIMCC 1.0030 ) is identified as Roseomonas mucosa by the polyphasic phenotypic and genotypic characteristics. The comparative analysis based on 16S rRNA gene sequences is a useful method for identifying the problematic and newly named bacteria.

Objective To establish a method for very long chain fatty acids( VLCFA )with liquid chromatography-tandem mass spectrometry( LC-MS/MS ). Methods One hundred and one healthy cases and 35 suspected ALD patients collected from April to June in 2009 were enrolled into this study. Quantitative analyzed the concentrations of VLCFA in serum was performed using liquid chromatography-tandem mass spectrometry. The precision, accuracy and recovery were analyzed, and the stability of VLCFA concentration of sample under room temperature and repeated freeze-thawing were also investigated. Serum levels of VLCFA in 101 normal cases were determined and analyzed statistically. The results for the 35 randomly chosen serum samples were compared with those from MDI in Germany. Results Serum VLCFA were separated well under these gradient condition with small interference. The linear range of C22:0 was from 2 mg/L to 64 mg/L, the recovery was 99. 92% -102. 05%, and the relative standard deviation ( RSD ) of intra-day and inter-day was less than 6% and 9% respectively. For C24:0 they were 2-64 mg/L. 95. 12%-100. 44%. ≤6%, ≤7%,respectively. For C26:0, they were 0-8 mg/L, 92.21%-103.71%, ≤7%, ≤8%, respectively. The accuracy of C22: 0,C24:0 and C26:0 were among 85% to 115%. The samples could be stable within 12 h at room temperature and repeated 10 times freeze-thawing. The values of VLCFA in 101 normal cases followed a normal distribution and the measured values were C22:0 =( 19. 43 ±4.43 ) mg/L,C24:0 =( 19. 10 ±4. 58 )mg/L, C26:0 = ( 0. 21 ± 0. 11 ) mg/L, the ratio of C24: 0/C22:0 and C26:0/C22: 0 were ( 0. 99 ± 0. 13 )and ( 0. 01 ±0. 01 ) respectively. The statistical analysis showed the concentration of C26:0 in adults ( 0. 18±0. 10 ) mg/L and children ( 0. 21 ± 0. 08 ) mg/L, C24: 0/C22:0 in adults ( 1.01 ± 0. 10 ) and children ( 0. 99 ±0. 14 ) has no significant( t values were 1. 439,0. 806, respectively, all P ＞ 0. 05 ); the ratio of C24:0/C22:0 in male (1.05 ± 0. 10 ) and female (0.97 ± 0. 10 ) has significant difference ( t =3. 394,P =0. 001 ). Compared the values determined by MDI laboratory, the results of C22: 0( 16. 93 ±4. 30 ) mg/L,C24: 0( 19. 57 ± 6. 40 ) mg/L by this method and C22:0 ( 13.85 ± 3. 17 ) mg/L, C24:0( 16. 10 ±5.84 ) mg/L by MDI have significant differences( t = 8. 401 ,P =0. 000;t =9. 914,P =0. 000 ),but C26:0( 0.68 ±0.48 ) mg/L, C24:0/C22:0( 1.20 ±0.40 ), C26: 0/C22:0 ( 0.04 ±0.04 )by this method and C26: 0( 0. 65 ± 0. 67 ) mg/L, C24:0/C22: 0( 1.19 ± 0. 43 ), C26:0/C22: 0 ( 0. 05 ± 0. 05 )by MDI have no differences( t values were 0. 372,0. 317,0. 945 ,respectively ,all P ＞0. 05 ). Conclusions The quantitative analysis method for serum very long chain fatty acid using LC-MS/MS is accurate, sensitive,specific and stable. It could provide important biochemistry information for diagnosis in clinic.

Objective To analyze the diagnostic process of a rare case of acute myeloid leukemia with minimal differentiation undergoing a lineage switch to mixed phenotype acute leukemia, NOS-rare types,and to investigate its difference from other acute myeloid leukemia and mixed phenotype acute leukemia. Methods Following tests were performed on the patient with switched mixed phenotype acute leukemia and three control leukemia patients ( including two acute myeloid leukemia with minimal differentiation and one mixed phenotype acute leukemia ). Cell morphology was analyzed by bone marrow smear and related cell chemical staining. Immunophenotyping of bone marrow was performed by flow cytometry ( FCM ). G-banding technique was used for karyotype analysis and RT-PCR was used for fusion gene detection. All the laboratory data of the switched patient were compared to that of three control patients in order to reveal the characteristics of such a rare phenotype switch in acute leukemia. Results Before switching, the morphology of acute myeloid leukemia with minimal differentiation demonstrated 0.82 blasts occurring in bone marrow, distinct nucleoli and absence of Auer rods. Blast cells expressed hematopoieticassociated antigens ( CD38, HLA-DR ), myeloid antigens ( CD13, CD56, CD11b ) and CD7. And these blasts were negative for MPO, CD33, CD15, CD79, CD19, CD22, cytoplasmic CD3, CD4 and CD8. After switching, 0. 42 blasts were found in bone marrow, showed eosinophilia and presence of basophile. Blast cells expressed hematopoietic-associated antigens ( CD38, HLA-DR ), myeloid antigens ( MPO, CD13 ),lymphoid antigens ( CD19, CD79a ,cytoplasmic CD3, and CD7 ). The control group showed typical morphology and immunophenotyping. No abnormal karyotype and fusion gene were detected. Conclusions It is a rare and complicated case that acute myeloid leukemia with minimal differentiation switched to mixed phenotype acute leukemia, NOS-rare types. The laboratory features, especially the change of immunophenotyping play an important role in the diagnosis.

Objective To analyze the plasma free fatty acid (FFA) composition in patients with T2DM. Methods All subjects were from Zhongnan hospital of Wuhan university, and they were divided into three groups: normal control ( n = 94 ), T2DM ( n = 101 ) and T2DM with hyperlipidemia ( n = 77 ). Fasting blood samples were taken from the participants, and plasma FFA were separated using a modified Doles method with the bromoacetophenone, pre-column-derivative. The quantitation of FFA was performed on were (355.63 ± 100. 35) μmol/L, (421.21 ± 200. 83 ) μ mol/L, ( 473.04 ± 213.40 ) μmol/L in healthy controls, T2DM group and T2DM with hyperlipidemia group, respectively. The significant differences were observed among the 3 groups(x2 = 13.08, P <0.01 ). However, there was no significant difference of UFA concentrations among the 3 groups [(206.29± 61.94) μ mol/L, (218.11 ± 110.28) μmol/L and ( 240.94 ± 116.79 ) μmol/L, x2 = 2.17, P > 0.05]. Compared to normal control [( 355.63 ± 100.35 )μmol/L], the FFA concentration[(421.21 ±200.83) μmol/L] in T2DM has significantly increased (x2 =FFA concentrations were higher in T2DM with hyperlipidemia [(473.04 ±213.40) μmol/L] (x2 =27.93,P <0.01 ). The RSD values for intra- and inter-day precision were less than 5%, and the minimal detection limits ranged from 0.05 μmol/L to 0.35 μmol/L The recoveries of high, intermediate and low-level materials were 96.4% -104.8%. Conclusions The total FFA concentration in T2DM has increased, most of which are saturated FFA. The unsaturated FFA has not significantly increased. They seem to be related to the development of T2DM, and might be a new biomarker for clinical monitoring of metabolic disorder of T2DM.

Objective To evaluate the performance of modified Hodge test on the detection of carbapenemases among Enterobacteriaceae with decreased susceptibility to carbapenems. Methods Fortynine Enterobacteriaceae isolates with decreased susceptibility to carbapenems ( MIC of imipenem, meropenem or ertapenem was ≥ 2 μg/ml ) were collected from 16 teaching hospitals from 2004 to 2008. MICs of imipenem, meropenem and etapenem were determined by agar dilution method. Carbapenemases were detected by modified Hodge test. Carbepenemase-causing positive results and AmpCs-causing positive results were differentiated by phenyl boronic acid and oxacillin. Beta-lactamases encoding genes including blaNDM-1were detected by PCR and sequencing. Results Thirty-six of 49 isolates were non-susceptible to imipenem (MIC >4 μg/ml), 31 were non-susceptible to meropenem (MIC > 4 μg/ml) and 47 were non-susceptible to ertapenem (MIC > 2 μg/ml). Twenty-three isolates showed positive modified Hodge test result, including 9 weak-positive results and 14 strong-positive results. Through PCR detection and sequencing, 2 out of 9 isolates showing weak-positive results carried blaKPC-2 and other 7 did not carry any carbapenemase genes but AmpCs/ESBLs genes. Among the 14 isolates showing strong-positive results, 4 carried blaKPC-2, 8 carried blaIMP-4 and 2 caried blaIMP-8. All 26 isolates with negative modified Hodge test result didn't carry any carbapenemase genes. No isolate carried blaNDM-1. Carbapenemases genes PCR detection was regarded as a gold standard, and the sensitivity, specificity, positive predictive value and negative predictive value of modified hodge test was 100%, 79%, 70% and 100% on the detection of carbapenemases among Enterobacteriaceae with decreased susceptibility to carbapenems. Conclusions Modified Hodge test revealed great sensitivity but showed a few false positive results. True and false positive results can be effectively differentiated by phynel boronic acid and oxacillin.

Objective To investigate the relationship between HBV genotypes and YMDD motif mutations or BCP mutations in Xiangtan of Hunan Province. Methods HBV genotypes, YMDD motif mutations and BCP mutations were analyzed in 952 HBV infected patients. Results HBV genotyping showed that 698 HBV type B patients and 115 HBV type C patients accounted for 73.32% and 12.08% respectively of all the participants. The rest 139( 14.60% )were genotype B and C mixed infection( B + C ). The analysis of YMDD motif mutations showed that 844 YMDD wild-type which accounted for 88.66% of all the subjects and the remainder were YMDD mutation types, of which 54( 5.67% ) carried YVDD, 53( 5.57% ) YIDD,and 1 YVDD and YIDD mixed infection. Basic Core Promoter mutations showed that 1762A/1764G ( wild type )accounted for 70.59% and 1762T/1764A( mutant ) accounted for 19.75%. The rest 92 patients were 1762T/1764A and 1762A/1764G mixed infection. This study showed no significant difference in the rate of YMDD mutation( 10.04% vs 10.43% ,χ2 =0.017,P＞0.05 ) ,but a significant difference in the types of YMDD mutation(χ2 = 4.836, P ＜ 0.05 )between HBV types B and C. The YVDD mutation was more commonly seen in genotype C( 9.57% ) than in genotype B( 5.88% ). The BCP mutation rate showed a significant difference( 27.36% vs 46.09%, χ2 = 16.478, P ＜ 0.01 ). Genotype C was more frequent than genotype B. The BCP mutation rate showed no significant difference between YMDD Wild-type and YMDD mutation types( 28.67% vs 35.51%, χ2 = 2.139, P ＞ 0.05 ), but most of BCP mutations happened in YVDD mutant type( 61.11% ). Conclusions ( 1 ) The predominant HBV genotypes in Xiangtan were genotype B and genotype C, the major genotype was type B, which display the characteristics of epidemiology in Southern China. ( 2 ) Determination of HBV genotypes before lamivudine therapy was probably not an important pretreatment investigation to predict antiviral responses. ( 3 ) Detection of HBV genotypes, YMDD motif mutations and BCP mutations will contribute to the correct evaluation of prognosis and timely proper management of HBV patients.

Objective To screen monoclonal antibodies (mAbs) for early diagnosis of invisive Aspergillus. Methods Monoclonal antibodies against different antigens of Aspergillus fumigatus were produced. The two pairs of combinations of monoclonal antibodies were selected accoring the distinct epitopes and double-antibody sandwich ELISA based on mAbs above were established. The sensitivity and specificity of the methods were analyzed by detecting culture supernatants of clinical isolates and environmental isolatesof Aspergillus. spp, Penicillium Marneffei, Candidas, and serum from animal models and patients. The epitopes recognized by mAbs were identified by immunobotting. Results A total of 32 hybridoma cell lines that stably produced MAbs were obtained. Two double- antibody sandwich ELISAs were established. One method was specific for 19 clinical isolates and environmental isolates of Aspergillus. spp, whereas the other one was specific for the clinical and environmental isolates of Aspergillus fumigatus without cross-reation with other Aspergillus. spp. For the same kind of medium of Aspergillus fumigatus, the sensitivity of the first method was 10 fold higher than the second method. Conclusions The specific mAbs for early diagnosis of invisive Aspergillus were obtained. Antigen recognized by the specific mAbs was mannoprotein with molecular weights of approximately 25 000-75 000. This antigen was potential early diagnostic marker for invasive Aspergillus.

Objective To explore a new high-throughput method with internal standards for analyzing the methylation profiles of lung cancer related genes. Methods The promoter sequences of 7 lung cancer related genes were cloned into plasmids and the target segments were amplified by their special primers respectively. The products were treated with M. Sss Ⅰ methylase and bisulfite. The multiplex ligation PCR method was established by designing probes containing CpGpCpG(for methylatedsequence) at the 3' ends and choosing the optimal ligation enzyme, annealing and ligation temperatures. The standard calibrators and clinic samples were tested by fluid chip platform. The results were validated by methylationspecific PCR. Results We successfully set up the standard calibrators for methylation and unmethylaiton of 7 lung cancer related genes and established a multiplex ligation PCR combined with fluid chip method, which was used to detect methylation status of 7 genes simultaneously. The fluorescence value of p16INK4A, APC,DAPK, RARIβ, RASSF1 A, MGMT and GSTP1 methylation standard calibrators were 863,909,703,701,901,1 060 and 885, much higher than that of unmethylation standard calibrators. The results were consistent with the results of methylation-specific PCP. ConclusionThe new high-throughput method can be used to evaluate the methylation status of 7 lung cancer related genes simultaneously and might be useful for clinical practice.

Objective To estimate the application for the rapid identification of common clinical bacteria by MALDI-TOF MS. MethodsFour hundred and twenty-six bacteria, including Salmonella spp strains collected from Zhejiang center for disease control and prevention were collected from blood, sputum,secretion and urine in 2nd Affiliated Hospital of Zhejiang University during December 2008 to August 2009. The isolates included 76 gram positive coccus and 350 gram negative bacilli. Species identification was performed with the Vitek system, and serotypes of Salmonella and Shigella were determined by serum agglutination test. 16s rDNA gene of 91 bacteria were amplified by PCR. The RCP products were sequenced. Then the results were compared with the reported sequences from GenBank. All strains were identified by MALDI-TOF MS. Results of three identification methods were compared with each other. Results Among 426 tested isolates, identification results from Vitek system and MALDI-TOF MS for gram positive coccus and 323 out of 350 gram negative bacilli (exception for Salmonella and Shigella spp.),were identical. For 23 Salmonella and Shigella spp. , only 2 Salmonella enterica subsp, enterica serovar Typhimurium were identified the same results by the three methods. Besides, results from Vitek system and serum agglutination test for 1 Salmonella enterica subsp, enterica serovar Typhi, 3 Salmonella enterica subsp.enterica serovar Paratyphi A, 1 Salmonella enterica subsp, enterica serovar Paratyphi B, 1 Salmonella enterica subsp, enterica serovar Enteritidis, and 1 Salmonella enterica subsp, enterica serovar Bovis-morbificans were consistent with that from 16S rDNA gene sequence. Four isolates which were confirmed as S. flexneri by Vitek system and serum agglutination test were identified as Escherichia coli by both 16S rDNA gene sequence and MALDI-TOF MS. ConclusionMALDI-TOF MS could be used for rapid and accurate identification of common clinical bacteria with good repeatabihty, excepting for the Salmonella and Shigella spp.