Objective To investigate the carbapenemases and integrons in imipenem-resistant Acinetobacter baumannii. Methods One hundred and three Acinetobacter baumannii were collected from Janurary 2008 to March 2010 in Tianjin Medical University General Hospital. The identification of strains and antimicrobial susceptibility test were performed by using Vitek-2 compact automatic system. Isolates of imipenem-resistant A. baumannii were screened for carbapenemases by modified Hodge test, improved threedimensional test and 2-mercaptopropionic acid synergy test. Isolates were then subjected to the multiplex PCR targeting genes encoding for OXA type carbapenemases, metallo-β-lactamases (MBLs) and integrases. The variable regions of integrons were amplificated and sequenced. Results Among the 103 isolates, 75 (72. 8% ) demonstrated positive in the modified Hodge test, 80 (77.7%)were positive in the improved three-dimensional test. No MBLs was found in the 2-mercaptopropionic acid synergy test. Eightyfour isolates were positive for blaOXA-51-like, blaOXA-23-like, and intI1; five were positive for blaOXA-51-like and blaOXA-23-like ;eight were positive for blaOXA-51-like and int11 ;two were positive for blaOXA-51-like and blaOXA-24-like ;four were only found positive for blaOXA-51-like. The blaOXA-58-like, IMP-1, VIM-2 and intI2 genes were all negative. Eighty-nine(96. 7% )of the intI1 positive strains owned the variable region. Two different cassettes arrangements were identified within class 1 integrons:81 isolates harbored aacA4-catB8-aadAI (2 300 bp) and 8 harbored aacCl-orX-orfX-orX'-aadAla (3 000 bp ) . Conclusion The presence of OXA-23 carbapenermase and class Ⅰ integrons are correlated with Acinetobacter baumannii resistant to carbapenems and multi-drug resistance.

Objective To investigate the resistance of Acinetobacter baumannii to clinical common antibiotics and new drug tigecycline. Methods Six hundred and two Acinetobacter baumannii isolates were collected from 2008 to 2009 in four teaching hospitals in Zhejiang province. Agar dilution method was used to detect the resistance of 13 clinical commom antibiotics, polymyxin B and tigecycline. Homology analysis of 24 multi-drug resistant Acinetobacter baumannii strains was used to investigate the relationship of each strain with the method of pulsed field gel electrophoresis. Results From 2008 to 2009, the Acinetobacter baumannii isolates of four teaching hospitals in Zhejiang province were mainly isolated from respiratory specimens with the number of 277 (86.0%) strains in 2009, the number of blood samples decreased from 15 (5.4%) strains in 2008 to 5 ( 1.5% ) strains in 2009, and there were no obvious change in other specimens. Acinetobacter baumannii strains were resistant to 13 clinical common antibiotics at different degree, fluctuated from 35.0% to 85.0%. Compared with the resistance in 2008, levofloxacin and tobramyxin decreased 0. 9% and 9.0% in 2009, respectively. However, the resistance of other antibiotics increased at different degree, the resistance of ceftriaxone and cefepime increased about 10.0%, and the resistance of imipenem and meropenem increased to 74.2% (239/602) and 70.8% (228/602) in 2009,respectively. Acinetobacter baumannii showed high resistance to tigecycline with the percent of 78.9% (475/602), while it was only 3.7% (22/602) for polymyxin B. There were six cloning types among the 24 Acinetobacter baumannii isolates, and the most common type was type A with the percent of 50%.Conclusions The resistance of tigecycline makes the situation of nosocomial infectious more serious. It is necessary to control the transmission of multi-drug resistant Acinetobacter baumannii immediately.

Objective To observe the relationship of IL-10 gene single nucleotide polymorphism and the susceptibility to ALL. Methods The bone marrow and peripheral blood samples from 115 ALL patients and 323 healthy controls were collected in Peking University First Hospital and Beijing Dao-pei Hospital from January 2007 to December 2009. The DNA were extracted from all samples. The primers of -819C/T and -592A/C in the promoter region of IL-10 gene were designed for the PCR. The restrictive fragment length polymorphism of IL-10 gene was analyzed by using restrictive enzyme Msl Ⅰ and HpyCH4 Ⅲ.Sequencing was done in part of these samples to confirm the results of PCR. The differences of genotypes and allele ratio of -819 and -592 sites were analyzed between the ALL patients and healthy controls. Real-time quantitative PCR was performed to detect the EB virus (EBV) infection and the expression of BCR/ABL fusion gene. The differences of genotypes and allele ratio of -819 and -592 sites were analyzed between the positive and negative group. Results The genotype ratios of -819CC, -819TT, - 819CT, -592AA,- 592CC and - 592AC were 14. 8% ( 17/115 ), 45.2% ( 52/115 ), 40. 0% ( 46/115 ), 43.5% ( 50/115 ),16. 5% ( 19/115 ), 40. 0% ( 46/115 ) in ALL patients, and were 9. 9% ( 32/323 ), 16. 4% ( 53/323 ),73.7% ( 238/323 ), 11.8% ( 38/323 ), 15.5% ( 50/323 ), 72. 8% ( 235/323 ) in the healthy controls,respectively. The genotypes of -819 and -592 sites had statistically significant differences between the two groups(x2 values were 46.000 and 54.550, all P ＜ 0. 05 ). The allele ratio of -819T and -592A were (65.2%, 150/230) and (63.5%, 146/230) in ALL patients, while they were 53.5% (344/646) and 48. 1% (311/646)in the healthy controls. There were statistically significant differences between the two groups (x2 values were 9. 877 and 15.986, all P ＜ 0. 05 ). The EBV DNA were detected in 42 ALL patients,among which 22 were positive and 20 were negative. The genotype ratios of -819CC, -819TT, -819CT,-592AA, - 592CC, - 592AC in EBV positive group were 9. 1% ( 2/22 ), 40. 9% ( 9/22 ), 50. 0%(11/22) ,31.8% ( 7/22 ), 13.6% ( 3/22 ), 54. 5% ( 12/22 ), while they were 35.0% ( 7/20 ), 45.0%(9/20) ,20. 0% (4/20) ,35.0% (7/20) ,45.0% (9/20) ,20. 0% (4/20) in the EBV negative group. The genotypes of -819 and -592 sites showed no statistical differences between the two groups( all P ＞ 0. 05 ).The BCR/ABL fusion gene were detected in 36 ALL patients, among which 20 were positive and 16 were negative. The genotype ratios of - 819CC, - 819TT, - 819CT, - 592AA, - 592CC, - 592AC in BCR/ABL positive group were 0% (0/20) ,45.0% (9/20) ,55.0% ( 11/20), 45. 0% (9/20) ,5.0% (1/20) ,50. 0%( 10/20), while they were 18. 8% ( 3/16 ), 50. 0% ( 8/16), 31.3% ( 5/16 ), 50. 0% ( 8/16 ), 18. 8%(3/16), 31.3 % (5/16)in the BCR/ABL negative group. The genotypes of -819 and -592 sites showed no statistical differences between the two groups ( all P ＞ 0. 05 ). Conclusion The population with - 819TT and - 592AA genotype of IL-10 gene shows susceptibility to ALL.

Objective To establish a REDE-DHPLC method for detecting the EGFR and KRAS mutations in plasma DNA from tumor patients, and investigate its clinical significance. Methods Restriction endonucleases Mse Ⅰ , Msc Ⅰ , BstN Ⅰ and Bgl Ⅰ were used to digest the wild type fragments of exon 19,exon 21 of EGFR gene and coden 12, 13 of KRAS gene for enriching the mutation fragments, and REDE-DHPLC method was established to detect EGFR and KRAS mutations. The sensitivities of REDE-DHPLC and conventional DHPLC were analyzed by using a series of plasmids containing 50%, 10%, 5%, 1% and 0. 1% mutation genes. Then, Plasma samples and paraffin-embedded tissue samples of 120 NSCLC patients and 120 colorectal cancer patients were detected by REDE-DHPLC. Compared with conventional DHPLC and sequencing, the diagnostic efficiency of REDE-DHPLC method was evaluated by detecting the mutation status of 2 genes in plasma of NSCLC and colorectal cancer patients. Results The sensitivity values of REDE-DHPLC and conventional DHPLC for detecting mutations in 4 loci were 0. 1% and 1%respectively. Plasmid DNA containing 0.1% mutation gene was detected to be positive continually for 2 to 3 times by REDE-DHPLC. EGFR mutation rates of 120 plasma from NSCLC patients detected by REDE-DHPLC, conventional DHPLC and sequencing methods were 27. 5%, 16. 7% and 12.5% respectively, and KRAS mutation rates of 120 plasma from colorectal cancer patients were 38. 3%, 25. 8% and 16. 7%,respectively. The positive rates of EGFR and KRAS mutation detected by REDE-DHPLC were significantly higher than conventional DHPLC(x2 = 4. 092, 4. 301, all P ＜ 0. 05 ) and sequencing method (x2= 8. 438,14. 127,all P ＜ 0. 05 ). In comparison with conventional DHPLC, the sensitivities of REDE-DHPLC for detecting EGFR and KRAS mutation were 100% (20/20,31/31), the specificities were 87. 0% (87/100)and 83. 2% (74/89). In comparison with sequencing method, the sensitivities of REDE-DHPLC were 100%( 15/15,20/20), the specificities were 82.9% (87/105)and 74. 0% (74/100). The coincidence rate of the two methods for detecting EGFR and KRAS mutation were 89. 2% ( 107/120, Kappa = 0. 690, P ＜ 0. 05 ) and 87.5% ( 105/120, Kappa= 0. 718, P ＜ 0. 05 ). The Consistency of EGFR and KRAS mutation status in plasma and tissues detected by REDE-DHPLC were 91.7% (33/36, Kappa =0. 939,P ＜0. 05)and 90. 2 %(46/51, Kappa = 0. 914, P ＜ 0. 05 ), respectively. Conclusions The REDE-DHPLC method is highly sensitive and specific for detecting EGFR and KRAS mutations in plasma DNA from tumor patients. The results are easy to be interpreted without missing homozygous point mutation, which indicate that the detection of EGFR and KRAS mutations in plasma DNA by REDE-DHPLC could therefore extend to be usedin clinical laboratory.

Objective To detect the expression level of human zinc finger 23 (ZNF23) in hepatocellular carcinoma tissue samples and HepG2 cell lines and investigate the relationship between hZNF23 expression and clinicopathological characteristics of HCC and cell apoptosis. Methods The expression levels of hZNF23 and GAPDH mRNA in 37 cases of HCC were measured by real-time RT-PCR. The association between the expression of hZNF23 and the clinicopathological characteristics of HCC was analyzed. Cultured HepG2 cells were divided into 4 groups ( control group, 1.25 μg/ml , 2.5 μg/ml and 5 μg/ml cisplatin)or 6 groups( control group, 1.25 μg/ml, 2.5 μg/ml, 5 μg/ml, 10 μg/ml and 20 μg/ml cisplatin). MTT method was employed to evaluate cell proliferation. Annexin V-FITC assay was used to assess percentage of apoptotic HepG2 cells. The expression levels of hZNF23 and GAPDH mRNA of HepG2 cells after apoptosis induced by cisplatin with a series of concentrations were measured by real-time RT-PCR.Results The median ( quartile1, quartile 3) expression levels of hZNF23 mRNA in 37 HCC tissue samples and adjacent tissue samples were 8.84 (3.59-15.05), 22.20 ( 13.85-42.90 ), respectively. There was significant difference ( U = 259.5, P ＜ 0.01 ). The median ( quartile1, quartile 3 ) expression levels of hZNF23 mRNA in cancer tissue samples with Edmondson stage Ⅰ + Ⅱ [12.80(4.80-19.50)] was much higher than those in stage Ⅲ + Ⅳ [5.01 ( 2.88-11.68 ), U = 99.00, P ＜ 0.05] The median ( quartile1,quartile 3 ) expression levels of hZNF23 mRNA in patients with and without hepatic cirrhosis were 9.92(3.80-15.25) , 3.21 (2.78-3.60), respectively. The median ( quartile1, quartile 3 ) expression levels of hZNF23 mRNA in HBV infection and non-infection patients was 9.09(3.72-15.25 ), 2.48 (1.79-12.10),respectively. There was no significant difference between groups with and without hepatic cirrhosis and between HBV infection and non-infection groups( U = 16. 00 and 24.00, P ＞0.05 ). MTT assay indicated that cisplatin significantly inhibited HepG2 cells proliferation in a dose-dependent manner. Annexin V-FITC/PI assay showed that HepG2 cells apoptosis rates were (0.9 ± 0.2 ) %, ( 4. 2 ± 0.3 ) %, ( 9.8 ± 4. 3 ) %,(23.0 ± 6.0)%, respectively. Cisplatin significantly induced HepG2 cells apoptosis in a dose-dependent manner( F = 27.89, P ＜ 0.01 ). The expression levels of hZNF23 mRNA in cisplatin groups [( 10.39 ±3.08) × 10-5, (24.10 ± 2.09) × 10-5, (6.90 ± 2.24) × 10-4] were significantly lower than that of the controlgroup[(94.80±1.80) ×10-5, F=6.027, P＜0.01]. Conclusions The expression level hZNF23 mRNA is related to Edmondson stage of HCC. The apoptosis effect of cisplatin on HepG2 cells may be associated with the upregulation of hZNF23.

Objective To investigate the variations in the pol region of HIV-1 strain in treatment failed patients in Yunnan province's Dehong prefecture and Kunming. Methods Blood samples were collected from 139 patients who experienced treatment failure ( HAART treatment ＞ 1 years and HIV-1 RNA Viral load ＞ 1 000 copies/ml). HIV-1 RNA was extracted from plasma, and nested-PCR was performed for amplification of PR and RT genes on the HIV-1 pol region. The PCR products were then sequenced and submitted to Stanford HIV Drug Resistance Database for comparison. The evolution tree was built up with MEGA 4. 1 system, combined with patients' demographics. Results The most prevalent mutation in Kunming patients were T215F/N/Y/I, M41L/M, and T69G/N/I/S/A/D, the mutation rates were 39%(24/62), 27% (17/62) and 27% (17/62) , respectively, which were higher than the corresponding mutations in the Dehong prefecture [16% ( 11/69), 13% (9/69) and 9% (6/69)]. The rate differences were statistically significant ( x2 = 8.646, 4.242 and 7. 909, all P ＜ 0.05 ). The most common HIV-1 pol region subtype in the Dehong patients were CRF01_AE subtype (32%, 22/69), followed by C subtype (25% ,17/69), and B subtype ( 19%, 13/69). Major subtypes in Kunming patients were 08_BC (60%,37/62 ), CRF01_AE subtype(21% , 13/62 ) and 07_BC ( 15% ,9/62). Conclusions Partial differences of the point mutations of the HIV-1 strain pol region and frequency of their occurrences exist among Dehong and Kunming patients, HIV-1 strains in Dehong prefecture for the NNRTIs mutations at the T215 Y/N/T, M41L and T69G/N/I/S/A/D are significantly higher than those in Kunming. Six isoforms are found respectively:CRF01_AE, B, C, BC, 08_BC and 07_BC from the epidemic strains of HIV-1 pol region subtype in Dehong and Kunming areas.

Objective To investigate the association between the genetic polymorphisms in GSTA1 and the clinical outcome of breast cancer patients treated with cyclophosphamide-based adjuvant chemotherapy. Methods A total of 137 breast cancer patients receiving cyclophosphamide-based adjuvant chemotherapy were recruited ( 124 cases with infiltrative ductal carcinoma, 5 cases with infiltrative lobular carcinoma and 8 cases with other histological types). PCR-LDR method was used to detect the genotypes of GSTA1. Survival curves were generated by the Kaplan-Meier method, and verified by the log-rank test. Cox proportional hazards regression analysis was used to estimate the prognostic factors in multivariate analysis. Results Of the 137 breast cancer patients, the genotypic frequencies of the GSTA1 * A/* A,* A/* B and * B/* B were 67.2% ( 92/137 ), 31.4% ( 43/137 ) and 1.5% ( 2/137 ), respectively. No significant differences were found between the genotypic frequencies and groups categorization according to age, stage, lymph node metastasis, ER or PR status (x2 = 0. 722,1. 967, 3. 303, 0. 226 and 0. 709, all P ＞0. 05 ) ;through Fisher exact test, also no significant differences were found between the genotypic frequencies and group categorization according to tumor size, histological types and grading ( all P ＞ 0. 05 ) . The recurrence rates in patients with GSTA1 * A/* A and * A/* B or * B/* B genotypes were 47. 8% (44/92) and 31.1% ( 14/45 ), respectively, and the mortality rates were 22. 8% ( 21/92 ) and 17. 8% ( 8/45 ),respectively. Patients with GSTA1 * A/* B and * B/* B genotypes were significantly associated with reduced hazard of relapse (x2 =18.723, P＜0. 01)and mortality (x2 =7.352, P＜0.01), compared to cases with the common * A/* A genotypes, according to Kaplan-Meier survival analysis and log-rank test. Moreover,Cox multivariate analysis showed that GSTA1 polymorphisms appeared to be an independent risk factor for recurrence-free survival ( OR =0. 222, 95% CI:0. 108-0. 458, P ＜0. 01 ) and overall survival ( OR =0. 362,95% CI:0. 145-0. 902, P ＜ 0. 05 ). Conclusion These data indicate that GSTA1 polymorphism may be a potential prognostic factor for recurrence-free survival and overall survival in breast cancer patients treated with cyclophosphamide-based adjuvant chemotherapy.

Modern pharmacogenomics demonstrates that genetic polymorphisms account for the most important factor of adverse drug reactions and interindividual variations of drug therapy. Personalized medicine aims to choose the appropriate drugs and dosage for increasing the efficacy and safety with minimal adverse effects based on the patients' genotypes. This article introduce the progresses in pharmacogenomics in China, discusse many factors that influence the application of personalized medicine from bench to bedside, and highlighted the challenges and further development of this field in the future.

Objective To develop an assay to determine Mycobacterium tuberculosis resistance to rifampin, isoniazid, ofloxacin and amikacin by pyrosequencing and evaluate the value of this method in clinical application. Methods Eighty-nine clinical isolates of Mycobacterium tuberculosis from tuberculosis patients were collected from Shanghai Pulmonary Hospital during 2008 to 2009. All strains were isolated from decontaminated sputum, cultured on Lowenstein-Jensen media and identified by traditional biochemical tests with standard methods. Ten Mycobacterium tuberculosis were selected from the strain bank of Shanghai Pulmonary Hospital. The optimal conditions of detection of rpoB, katG, gyrA and rrs gene by pyroseuencing were determined, using the 10 Mycobacterium tuberculosis strains whose drug susceptibility of Bactec 960 and sequence of rpoB, katG, gyrA, rrs gene were known. Then the drug susceptibility of 89 Mycobacterium tuberculosis clinical isolate strains were detected by pyrosequencing using this conditions and the results were compared with that of the Bactec 960 methods. Results The pyrosequencing program of sequence analysis was suitable for the detection of the mutations of rpoB and gyrA genes, and the program of single nucleotide polymorphism was suitable for katG and rrs genes. Among the 89 Mycobacterium tuberculosis clinical isolates,when using the drug susceptibility of Bactec 960 as the standard, the sensitivity of rifampin, isoniazid,ofloxacin and amikacin is 98.0%, 64. 1%, 79.5%, 78. 3% respectively, the specificity is 97.5%,100. 0%, 90. 0%, 100. 0% respectively, the accuracy is 97.8%, 77. 5%, 85.4%, 94. 4% respectively,tested by pyrosequencing. The results of the detection of resistance to rifampin, isoniazid, ofloxacin and amikacin in Mycobacterium tuberculosis using pyrosequencing technique were almost the same with that of Bactec 960, and Kappa ≥0. 7 in each detection. Conclusion Pyrosequencing is thus a rapid, accurate and high throughput method for detecting Mycobacterium tuberculosis resistance to these four drugs.

Objective To investigate the stabilization of betaine on the enzyme activity of ALT in human serum at different temperatures, and to evaluate the matrix effects of human serum with betaine among different analytical systems. Methods The enzyme activity of ALT in human serums with l mol/L betaine stored at 37 ℃, 25 ℃, 4 ℃ and - 20 ℃ respectively were measured. The matrix effect experiment was carried out according to the procedures specified in Clinical and Laboratory Standards Institute (CLSI)document EP14-A. Four analytical systems were the candidates for evaluation, and Roche Diagnostics (Roche)-Hitachi analytical system served as the reference system. The enzyme activity of ALT of 40 patient specimens and 3-level specimens ( high, medium and low) with 1 mol/L betaine were measured using the evaluated methods and the reference method respectively. The linear regression analysis was performed to compare the mean y value of 3-level specimens with the statistically defined limits ( the two-tailed 95% prediction interval) derived from 40 patient specimen data. Results The enzyme activity of ALT could be stabilized with betaine in serum, which remained to be 99% for 48 h at 37 ℃, 98% for4 d at 25 ℃, 97%for 4 weeks at 4 ℃, and 99% for 4 months at - 20 ℃. The enzyme activity of control serum decreased to 42%, 86%, 71%, 79% respectively. The mean y values of three specimens with the betaine were within the 95% prediction interval of the estimated mean y values, which suggested no obvious matrix effects in serum. Conclusion Betaine can be used in the development of enzyme ALT calibrator as its stabilizer.