OBJECTIVE For the reasonable use of antibacterial drugs, drug resisitance diversity of Pseudomonas aeruginosa was studied in our hospital. METHODS Antibacterial activity to all P. aeruginosa collected in four years from 2004 to 2007 were determined by K-B test, and data were analyzed by WHONET software. RESULTS The resistance rate to ampicllin, cefazolin and cefoteton were 100.0%, but low to ceftrazidime, cefoperazone and cefoperazone/sulbactam(3.2% in 2007). The resistance rate to levofloxacin increased year by year, while that to ciprofloxacin descreased from 15.6% to 9.6%.The resistance rate to gentamicin and amikacin descreased from 20.0% to 16.4%,and from 11.1% to 4.8%, respectively. The resistance rate to imipenem and meropenem accounted for about 10.0%, which was as same as ceftazidine. CONCLUSIONS The resistance of P. aeruginosa in our hospital is stable. Clinician should choose right antibacterial drugs on the basis of the test for antibacterial sensitivity and the pharmacological characteristis to improve curative effect and decrease the resistance of bacteria.

Abstract] Objective To establish an assay for the detection of porcine parvovirus ( PPV) and to verify its application for monitoring cells used for production.Methods A pair of primers and one probe were designed according to the conserved sequence encoding non-structural protein 1 (NS1).Based on the designed primers, a real-time fluorescent quantitative PCR assay for the detection of PPV was developed. Several parameters including the linearity, precision, minimum detection limit and anti-interference of the established assay were evaluated.A stock of PPV strains was prepared by infecting swine testicle ( ST) cells with PPV strains.An assay for the detection of PPV infection was developed by using ST cells as sensitive cells.A combined ST cell infection-PCR test was developed by combining the ST cell infection assay with the real-time fluorescent quantitative PCR assay.The sensitivity of ST cell infection-PCR test was analyzed.The cell samples used for production of biological products were detected by using the established assay.Results The real-time fluorescent quantitative PCR assay was specific for the detection of PPV without cross-reaction to other species of parvovirus virus, SV40 virus and other porcine viruses.The linear range of the assay was 1×109-1×104 copies/μl with a R2 value more than 0.98.The sensitivity of the real-time quantitative PCR assay was 1×104 copies/μl.Both of the intra-and inter-coefficient of variation (CV) were less than 5%in Ct values.The intra-and inter-CV in copies of detection were 5%-15% and 30%-40% respectively.The minimum detection limit of the real-time fluorescent quantitative PCR assay was 1CCID50/ml.The PPV strains were detected in cell samples with no interference.The sensitivity of ST cells infection-PCR test was 0.01CCID50/ml.All of the 22 cell samples were negative for PPV by using the real-time fluorescent quanti-tative PCR assay.Conclusion The real-time fluorescent quantitative PCR and the ST cell infection-PCR test for the detection of PPV in cells were established successfully.The application of the two assays was conducive to further enhance the safety of using cells for production and therapy.

Objective To evaluate five detection methods for the laboratory diagnosis of Clostridium difficile infection in the hospitals of USA, and explore a sensitive, specific, accuracy and rapid regimen for the early diagnosis of Clostridium difficile infection. Methods A total of 174 stool specimens submitted to the clinical microbiology laboratory for Clostridium difficile testing were separately tested by five methods including toxigenic culture (TGC), Premier Toxin A&B EIA( A/B-EIA), C. Diff Quick Chek Complete( DEIA), BD G eneOhm Cdiff assay(BD-PCR) and Laboratory-developed PCR(LD-PCR). The gold standard of TGC was used as a reference criterion, and the sensitivity, specificity, positive predictive value ( PPV )and negative predictive value (NPV) of A/B-EIA, D-EIA, BD-PCR and LD-PCR assays were determined. Results Among the 174 specimens studied, 24 were defined as true positives for Clostridium difficile infection by TGC assay, giving a positive rate of 13.8% (24/174). In comparison to the standard,the sensitivity, specificity, PPV and NPV were 62.5%, 99.3%, 93.8% and 94.3% for A/B-EIA;66.7%, 98.7%, 88.9% and 94.9% for D-EIA; 83.3%, 98.7%, 90.9% and 97.4% for BD-PCR;79.2%, 93.3%, 65.5% and 96.6% for LD-PCR. Among all tested specimens, 34 were positive by atleast one of five methods, and of which 15 were concordant by all five methods. The D-EIA results were divided into three groups depending on results of GDH and (or) toxins A/B: 18 were positive for both GDH and toxins A/B, 23 were positive for only GDH, and 133 were negative for both GDH and toxins A/B. Of 18 positive specimens by D-EIA assay, all were concordant with results of BD-PCR assay and 16 were agreement with results of TGC assay. Twenty-two of 24 positive specimens by TGC assay were included in 41 specimens that were positive for GDH. Among eight false negative specimens by D-EIA assay, four were differentiated as positive results by BD-PCR. According to the present study, the sensitivity, specificity,PPV and NPV of a two-step detection algorithm in combination with D-EIA and BD-PCR assays were 83.3%, 98.7%, 90.9% and 97.4%, respectively. Conclusions From the point of technological evaluation, BD-PCR is preferable. A two-step detection algorithm combining D-EIA with BD-PCR is proposed for the laboratory diagnosis of Clostridium difficile infection. This algorithm has demonstrated an excellent sensitivity and specificity, as well as decreased test turnaround time and test cost.

Objective To evaluate the clinical significance of the changes of microalbunminuria(MAU) levels in mechanical ventilated patients with severe pneumonia. Methods According to the ratio between the microalbunminuria and the urine creatinine (MAU/CR) (ACR), setting 25mg/mmol as the threshold, 78 mechanical ventilated patients with severe pneumonia were divided into two groups :ACR increasing group and ACR Non-increasing group,then the clinical significance of changes of MAU levels in 72 hours on prognosis of these patients was observed. Results MAU increased in 64 cases(82. 1%) ,of which 46 cases in ACR increasing group and 18 cases in ACR non-increasing group. There showed statistically significant differences on APACHE Ⅱ score, CPIS score,PCT、the success of getting out of mechanical ventilation and the mortality between two groups, (t = 3.50、2. 19 、x~2 = 3. 95、6. 70、5.38 ,P = 0.01,0.03,0.04,0.01,0.02, all P ＜ 0.05). Conclusion Changes of MAU levels have the clinical significance on prognosis of the mechanical ventilated patients with severe pneumonia.

Objective:To provide the basic data for the further revision of mycoplasma test method described in Chinese Pharma-copoeia and some references for the operation standardization of drug mycoplasma test. Methods:Two incubation conditions,namely aerobic conditions and microaerophilic conditions,were compared with respect to the growth status of mycoplasma in liquid and solid media. Results:The growth of mycoplasma was obvious difference between the two incubation conditions,and the microaerophilic con-ditions were better than the aerobic conditions. Conclusion:The microaerophilic conditions can be used in the incubation of mycoplas-ma test,which should be defined and standardized in the future Chinese Pharmacopoeia.

Objective:To investigate the growth-promoting properties and applicability of the mycoplasma test culture media pre-scribed in Chinese pharmacopoeia, and provide reference for the standardization of the drug mycoplasma test method. Methods: Re-spectively using four quantitative detection methods including sensitivity, degree of color change, colony counts and colony diameter, and mycoplasma media widely used in the world as the reference media, the growth-promoting properties of 4 batches of mycoplasma broth and 4 batches of arginine mycoplasma broth from four domestic manufacturers were investigated. Results:The results of sensitivity assay and absorbance detection showed that all the media inoculated with below 100 CFU test microorganisms exhibited visible color change. Furthermore, the results of color change degree and colony diameter showed that there were significant differences among the media products from different manufacturers(P<0. 01). Conclusion:Mycoplasma broth and arginine mycoplasma broth both can sup-port the growth of below 100 CFU test microorganisms. Due to the difference in the growth-promoting properties among the media prod-ucts from different manufacturers, the drug mycoplasma test workers should use more sensitive methods to examine the applicability of the media.

Objective To establish an assay for the detection of Torque teno sus virus ( TTSuV) strains and to analyze its preliminary application to biologics.Methods Primers and probe were designed according to the conserved sequences.A fluorescent PCR assay for the detection of TTSuV strains was estab-lished.Several parameters including the specificity, linearity, accuracy, sensitivity and anti-interference of the established assay were verified.The fluorescent PCR assay was performed to detect the samples of por-cine blood, cell substrate and rotavirus vaccine.The porcine blood samples positive for TTSuV strains were further genotyped.Results The established fluorescent PCR assay was confirmed to have high specificity as no cross-reactions with parvovirus virus of various species, SV40 and porcine circovirus strains were detec-ted.The linear range of the assay was 1×109-1×103 copies/μl for TTSuV1 genotype and 1×109-1×102 cop-ies/μl for TTSuV2 genotype with a R2 value more than 0.993.The sensitivity of the fluorescent PCR assay was 1×103 copies/μl for TTSuV1 genotype and 1×102 copies/μl for TTSuV2 genotype.The intra-and inter-CVs were both less than 7%in Ct values and less than 25% and 45% respectively in copies.No interfer-ence was found in the detection of TTSuV nucleic acids from cell samples.8 out of 20 porcine blood samples were positive for TTSuV strains, among which one sample was positive for TTSuV1 genotype, four samples were positive for TTSuV2 genotype and the rest were positive for both TTSuV1 and TTSuV2 genotypes.Com-pared with the reference strain, strains genotyped as TTSuV1 and TTSuV2 were respectively shared 98%-99%and 98%homologies in sequences.All of the cell substrate and rotavirus vaccine samples detected by the fluorescent PCR assay were negative for TTSuV strains.Conclusion The fluorescent PCR assay for the detection of TTSuV was established successfully, the application of which would further improve the safety of biologics.

Objective To conduct extensive quality control tests on Madin-Darby Canine Kidney ( MDCK) cells used for the production of influenza vaccine .Methods Tests for characteristics , extraneous agents, endogenous agents and tumorigenicity were performed on MDCK cells according to Chinese Pharma -copeia Book III .Cell lysate and DNA of MDCK cells were tested for oncogenicity in the light of new interna -tional requirements .Results The MDCK cells extracted from canis were adherent cells with an epithelial morphology, whose average number of chromosome was 80±1.No bacteria, fungi and mycoplasma contami-nation were detected . The detection for extraneous and endogenous virus showed that there was no nonspecific virus causing cytopathic effect , hemadsorption , hemagglutination or animal death .Tests for re-verse transcriptase , bovine viruses and canine viruses were all negative .Each nude mouse was injected with 107 viable cells to observe their tumorigenicity .Twelve weeks after cell injection , no node was found at the injection site and in large organs by gross anatomy .There was no significant difference between test group and negative control group .The test for tumorigenicity of viable cells was negative .Cell lysate and cellular DNA collected from equivalent amount of cells were respectively injected into nude mice , and no node forma-tion was found.There was no significant difference between the test cells and negative controls in pathology indicating that the tested MDCK cells were non-oncogenic .Conclusion It showed the possibility of using MDCK cells for the production of influenza vaccine .

Objective To investigate the effect of liver cirrhosis on the potency of propofol for sedation in rats. Methods Fifty-eight male SD rats, aged 10-12 weeks, weighing 180-220 g, were randomly divided into 3 groups: control group (group C, n = 18), mild liver cirrhosis group (group M1, n =20) and severe liver cirrhosis group (group M2, n = 20). The model of liver cirrhosis was established using four factors described by Chen et al. After successful establishment of the model, propofol was injected intravenously. The dose of propofol was determined by up-and-down sequential method for loss of righting reflex. The dose of propofol was 5.912 mg/kg in the first rat and the ratio of the doses between the two consecutive rats was 0.85. ED50 of propofol was calculated using up-and-down sequential method. Results ED50 of propofol was significantly lower in group M1 and M2 than in group C and in group M2 than in group M1 ( P ＜ 0.05 or 0.01 ). Conclusion The liver cirrhosis can enhance the potency of propofol for sedation in rats.

Objective To study the application of short tandem repeat (STR) profiling in quality control of human cell lines used for biological production. Methods The methods detecting 9 and 16 STR loci to identify human cell lines by PCR-capiilary electrophoresis were established respectively. Human cell lines, which were derived from many corporations and including diploid cell strains used for virus-vaccine production and 293 cell lines used for gene therapy products, were analyzed and compared by these two methods. Results The STR profiling methods used for authentication of human cell lines were established. Most of human diploid cell strains(20/21 ) used for virus-vaccine production from 13 corporations were iden-tiffed as the intended cells and no cross-contamination was found. However, one MRC-5 cells was identified as a false cell line and one MRC-5 had 3 alleles in D13S317 locus. For 12 strains of 293 cell lines, there were significant differences in STR profiling from different manufactures, which was likely be explained that the sources and gene modifications of these 293 cell lines are not well known and their genes are unstable during passage. Conclusion The STR profiling method has the advantages of high sensitivity and specifici-ty, and can be used for authentication of each of human cell lines for biological production.