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Objective To determine the prevalence of penicillinase-producing Neisseria gonorrhoeae(PPNG) and the distribution of blaTEM-135 gene variants in PPNG at several gonococcal antimicrobial surveillance sites in China, to compare N. gonorrhoeae multi-antigen sequence typing(NG-MAST)types of PPNG and its blaTEM-135 gene variants, and to assess the difference and association in NG-MAST types of blaTEM-135 gene variants among different regions. Methods A total of 572 N. gonorrhoeae isolates were collected at 6 gonococcal antimicrobial surveillance sites from Jiangsu, Shanghai, Zhejiang, Tianjin, Guangdong and Guangxi in 2012. After isolation, purification, and identification, cefalotin paper discs were used for detection of PPNG. DNA was extracted by QIAxtractor DX kits after cultivation of the PPNG strains. Then, mismatch amplification mutation assay (MAMA) PCR was performed to identify blaTEM-135 variants, and NG-MAST analysis to determine N. gonorrhoeae genotypes. Results Among the 572 N. gonorrhoeae strains, 38.1%(218/572) were identified as PPNG, and of the PPNG strains, 52.3% (114/218) were blaTEM-135 variants. The detection rate of PPNG at these surveillance sites from high to low was as follows: 51.7% (45/87, Zhejiang), 45.6%(36/79, Shanghai), 38.0% (78/205, Guangdong), 37.5% (12/32, Guangxi), 31.2% (24/77, Jiangsu) and 25.0%(23/92, Tianjin), and that of blaTEM-135 variants was as follows: 68.9%(31/45, Zhejiang), 58.3%(14/24, Jiangsu), 50.0%(39/78, Guangdong), 47.2%(17/36, Shanghai), 39.1%(9/23, Tianjin)and 33.3%(4/12, Guangxi). NG-MAST analysis showed that the ST2318, ST1768, ST1866, ST1053 and ST8726 types predominated among these bla TEM-135 variants, and a strong correlation was found between blaTEM-135 variants and some NG-MAST types, such as ST1768, ST1053 and ST8726 types. The distribution of NG-MAST types was significantly different between the surveillance site in Tianjin (in the Northern part of China) and the other sites (in the Southern part of China), but highly similar among the surveillance sites in Jiangsu, Zhejiang and Shanghai regions. Conclusions There is a high prevalence of PPNG and its blaTEM-135 variants at several gonococcal antimicrobial surveillance sites in China, with significant differences in NG-MAST genotype distribution of PPNG and its blaTEM-135 variants among different regions.
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Objective To assess the vitro susceptibility to 6 antimicrobial agents and genotypes of clinical isolates of Chlamydia trachomatis (Ct) from Guangzhou region. Methods Ct was isolated from clinical specimens by using McCoy cell culture and subjected to propagation. The minimum inhibitory concentration (MIC) and minimal bactericidal concentration (MBC) of 6 antimicrobial agents (clarithromycin, roxithromycin, azithromycin, doxycycline, tetracycline, ofloxacin) against Ct isolates were determined in McCoy cell culture. Nested PCR was performed to amplify the outer membrane protein 1 (omp1) VS1-2 gene followed by sequencing. Results Seventy-six Ct strains were isolated from 346 urogenital specimens, and 40 strains met the require ments for susceptibility testing after serial propagation. The MIC50/MIC90 of clarithromycin, azithromycin, roxi thromycin, doxycycline, tetracycline and ofloxacin were as follows: 0.008/0.032, 0.080/0.160, 0.125/0.500, 0.032/0.064, 0.250/0.500 and 0.500/1.000 mg/L. Seven genotypes were observed. The most prevalent geno types in decreasing order were E (14, 35%), J (10, 25%)and F (6, 15%). The MIC50 was consistent for azithromycin among the 7 genotypes, but varied by 1 - 4 folds for doxycycline, ofloxacin and roxithromycin. Conclusions Clarithromycin, doxycycline and azithromycin exhibit an excellent activity against Ct, and the activity of azithromycin is consistent among the 7 genotypes of Ct.
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Dynamic fucosylation of glycoprotein especially 80 ku which bound to UEA and LCA during the course of rat hepatocarcinogenesis was investigated. In patient hepatocellular carcinoma, more UEA- and LCA-bound proteins were also observed in patients with high metastatic potential than those with low metastatic potential. Fueosylated glycans constitute important adhesion molecules such as Lewis antigens. A differential expression pattern of Lewis antigens was further conf'Lrmed on various metastasis potential hepatocellular carcinoma cells (HCC). High metastatic hepatocellular carcinoma cell line (HMCC97H) expressed much more Lewis x and b than low metastasis HMCC97L cells. Moreover, surface Lewis x, or b expression level declined significantly after the cells were treated by retinoie acid. Not only in experimental metastasis foei, but in HCC as well, both α1,3/1,2 and α1,6 fucosyltransferase activities were quite high. After retinoie acid treatment, α1,3/1,2 fueosyltransferase activities were significantly inhibited, Lewis x on epidermal growth factor receptor reduced, and the EGFR was less phosphorylated. These results suggested that fucosylated glycans such as Lewis x played an important role in HCC development and metastasis.
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Objective To develop a PCR-mverse dot blot hybridization(RDB)assay to rapidly detect pathogenic mycoplasmas in genitourinary tract.Methods Universal primers were designed and applied to amplify the 16S rRNA gone of ureaplasma parvum(Up),ureaplasma urealyticum(Uu),Mycoplasma genitalium(Mg),Mycoplasma hominis(Mh)by using nestcd PCR.Specific nucleotide probes of Up,Uu,Mg and Mh Were constructed and immobilized onto nylon membranes.PCR products were denatured and hybridized、with specific oligonucleofide probes on nylon membrane.The sensitivity and specificity of the PCR-RDB assay were evaluated based.on the hybddizafion results.Also,PCR-RDB Was utilized to detect pathogenic mycoplasmas from 60 clinical samples.Results The four probes selectively hybridized with the PCR product of corresponding mycoplasmas,and no cross hybridization was observed.The detection limit of PCR-RDB Was one colony forming unit(CFU)of mycoplasma.Out of the 60 clinical samples、19were positive for mycoplasm,Mixed infections were found in three samples,including two coinfected with Up and Uu and one with Uu and Mg.Conclusion PCR-RDB is a rapid,specific and sensitive approach to the identification of pathogenic mycoplasmas in urogenital tract.
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Objective To assess the performance capability of syphilis serology among STI laborato-ries in Guangdong Province. Methods Positive and negative sera samples were prepared and delivered toSTI laboratories. Non-treponemal and treponemal quantitative and qualitative tests were performed with these samples based on standardized procedures. Quality and proficiency of syphilis serology among these labora-tories were evaluated. Results A total of 225 STI laboratories in Guangdong Province participated in this survey, 3696 serological tests for syphilis were performed from 2004-2006. The outcomes showed that the total reproducibility among participating laboratories increased from 82.5% in 2004 to 90.0% in 2006 (x2 =16.8, P ＜ 0.05), and the percentage of laboratories with a performance level higher than the provincial average level rose from 64.7% in 2004 to 76.8% in 2006. The total reproducibility was 95.5% for non-treponemal qualitative tests,95.0% for treponemal qualitative tests, 82.1% for non-treponemal quantitative tests, and 81.1% for treponemal quantitative tests. The false-negativity and false-positivity rates were 4.7% and 4.0%,respectively, for non-treponemal qualitative tests, and 7.2% and 0.4%, respectively, for treponemal qualitative tests. Conclusions It is important to do capability building and improve the performance of STI laborato-ries, in order to prevent and control syphilis.
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Objective To develop a nested PCR for the detection of early syphilis and genotyping of Treponema pallidum (TP), and to investigate the distribution of genotypes of TP in Guangzhou. Methods Specimens were consecutively collected from genital ulcers of patients with suspected chancre during 2002-2004, and were detected by dark-field microscopy and nested PCR. The acidic repeat protein (arp) gene and the T. pallidum repeat (tpr) gene family were amplified with the positive specimens above. The number of repeats presented in the arp gene and the restriction fragment length polymorphism by Mse I in the tpr gene were analyzed by electrophoresis. The strains were genotyped according to Pillay's criteria. Results Out of 62 patients with suspected chancre, 33 cases (53.2%) were positive by dark-field microscopy and 54 cases (87.1%) by nested PCR. Of 47 TP-positive specimens genotyped by arp gene, 36 (76.6%) were type 14, while of 49 cases genotyped by tpr gene 39 (79.6%) were type d. By combining genotypes of arp and tpr genes, 7 genotypes were found, including 14d (31, 66.0%), 13d (5, 10.6%), 14b (4, 8.5%), 12b (3, 6.4%), 12d (2, 4.3%), 15d(l, 2.2%) and 14i (1, 2.2%). Conclusions Nested PCR shows a high sensitivity in early detection of TP. Genotype 14d seems the predominant type of TP in Guangzhou.
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Objective To establish a simple assay for quick scanning of effective agents in growth inhibition and apoptosis induction. Methods After observing the correlation between cell number and acid phosphatase activity, and between cell apoptosis and acid phosphatase activity, we established a microplate densimetry method to measure the acid phosphatase activity. We also compared the sensitivity with MTT assay and confirmed cell apoptosis by flow cytometry. Results In two cell lines of cancer (Hep G2 and CBRH-7919 cells), the acid phosphatase activity was correlated well with the cell number, and was directly proportional to the cell number in the range of 0.5×103～0.7×103. The coefficient reached to 0.994. After stimulation of phorbel ester (TPA) for one hour, the acid phosphatase activity rose significantly. In contrast, after the inhibition of cell growth by As2O3, the acid phosphatase activity decreased significantly. The higher the concentration of As2O3, the lower the acid phophatase activity. The acid phophatase activity went down significantly when apoptosis in Hep G2 cells was only 3.98% and no apoptosis in CBRH-7919 cells after 24-hour treatment with As2O3. Conclusions The acid phophatase activity of cell may be used to measure cell proliferation and apoptosis. Because of the easiness and quickness, acid phophatase assay might be an ideal approach to scan the effective growth inhibitors and apoptosis inducers in a large number of drugs.
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Objective To investigate the prevalence of resistant Neisseria gon orrhoeae and plasmid-mediated resistant strains in Guangzhou from 1996 to 2001. Methods The resistant N.gonorrhoeae and plasmid-mediated resistant strains to tetracycline (TRNG) were determined using agar dilution method, and penicillinas e-producing N. gonorrhoeae (PPNG) by acidometric method. Results A total of 793 gonococcal isolates were tested from 1996 to 2001. The resistant rate for penic illin increased from 57.2%to 81.8%and PPNG from 2%to 27.2%, respectively, du ring the six years. Resistance to tetracycline remained high and stable over the years, while the rates of TRNG were increased from 1.5%to 27.2%. Conclusions The prevalence of plasmid-mediated resistant strains of N. gonorrhoeae increase s year by year in Guangzhou. These results suggest that the clinical isolates of gonococcal strains in this city are highly resistant to penicillin and tetracyc line.