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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 estimate the application value of a standard operating procedure (SOP) in the detection of syphilitic anticardiolipin reagin. Methods Clinical laboratories from 9 local hospitals in Shanghai participated the program. Quality control samples with unknown target value were qualitatively and quantitatively examined according to the uniform SOP in these laboratories with the same reagent and facility of horizontal reaction. External quality assessment (EQA) was carried out by using seven serum samples with no, or low (1∶ 128 dilution) to high (1∶1 dilution) concentrations of target before and after the implementation of SOP. The test results were statistically analyzed and the reasons for the detecting error were assessed. Results A total of 388 tests were performed in the 9 clinical laboratories. The total accuracy rate was 93.0%, including 40.2% in the detection of samples with 1 ∶ 8 dilution of target, 49.2% in the detection of samples with 1 ∶ 16 dilution of target, and 3.6% in the detection of samples with 1 ∶ 32 dilution of target. No forward bias was observed in these tests. There was a significant difference in the accuracy rate between the two times of EQA before and after the implementation of SOP (x2 = 4.17, P < 0.05). Conclusions The improved standard procedure for nontreponemal antigen test is beneficial to the decrease of testing error, and may provide a basis for the establishment of SOP and implementation of internal quality assessment.
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Objective To investigate the immune response to and protective effect of a bivalent DNA vaccine expressing interleukin-2(IL-2)and Gpd proteins in New Zealand rabbits.Methods Seventy-two male New Zealand white rabbits were equally and randomly divided into 4 groups to be immunized with recombinant plasmids pcDNA3.1(+)/Gpd-IL-2(pcD/Gpd-IL-2),pcDNA3.1(+)/Gpd(pcD/Gpd),empty plasmid pcDNA3.1(+)(pcD)and phosphate buffered saline(PBS),respectively.Immunization was carried out by intramuscular injection at multiple sites with a 2-week interval for 3 times.On week 10 after the initial immunization,the rabbits were challenged intradermally with T.pallidum(Nichols strain).Enzyme-linked immunosorbent assay(ELISA)was used to quantify the serum level of anti-Gpd antibodies in the rabbits and the level of IL-2 and interferon(IFN-γ)in the supernatant of Gpd protein-stimulated spleen cells from the rabbits at different time pionts.MTT assay was conducted to detect the proliferation response of spleen cells collected from the rabbits on day 0,14,28,140 and 168 after the challenge.Results Compared with pcD and PBS,both the vaccines pcD/Gpd and pcD/Gpd-IL-2 elicited significantly higher levels of anti-Gpd IgG antibodies in rabbits at different time points during the vaccination and infection period,with the titers peaking at 1 ∶ 1024 and 1∶4096,respectively(both P ＜ 0.01).There were also significant differences in the serum levels of anti-Gpd IgG antibodies between the pcD/Gpd-and pcD/Gpd-IL-2-immunized rabbits at different time points(all P ＜0.01).The levels of IL-2 in the supematant of spleen cells from pcD/Gpd-and pcD/Gpd-IL-2-immunized rabbits on week 8 after the immunization were 110 ± 12.6 and 167 ± 15.7 μg/L respectively,and those of IFN-γwere 225 ± 17.6 and 447 ± 22.4 μg/L respectively,significantly higher than those in that from the other two groups of rabbits(all P ＜ 0.01).Furthermore,an apparent proliferation response was observed in spleen cells from pcD/Gpd-and pcD/Gpd-IL-2-immunized rabbits with a higher stimulation index compared with pcD-and PBS-immunized rabbits(all P ＜ 0.01).Dark-field microscopic examination of early-stage infected lesions revealed that pcD/Gpd-IL-2-immunized rabbits had a lower detection rate(17.5％)of Tp from lesions,occurrence of ulcerative lesions(15％)and shorter curing time compared with pcD/Gpd-immunized rabbits.Conclusion The recombinant plasmid pcDNA3.1(+)/Gpd-IL-2 could induce protective humoral and cellular immune response more efficiently in rabbits.
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Objective To construct a recombinant plasmid encoding Tp0821,a membrane lipoprotein of T. pallidum,express and purify this protein,and to evaluate its immunocompetence.Methods The recombinant plasmid pQE32/Tp0821 was constructed and induced to express the corresponding protein.Then,New Zealand rabbits were immunized with purified recombinant protein to prepare polycional antibodies,and the titer of polyclonal antibody was determinated.Indirect ELISA was developed with the recombinant protein of T. pallidum as coating antigen to detect 80 control sera and 150 FTA-ABS-positive sera.Results The recombinant plasmid pQE32/Tp0821 was constructed and a fusion protein with expected molecular weight was expressed.Specific humoral response was elicited by the recombinant protein in New Zealand rabbits and the antibody titer reached 1:6400.Compared with FTA-ABS test,the indirect ELISA showed a sensitivity and specificity of 92.6%and 98.6%,respectively,in the detection of control and clinical sera.Conclusion The recombinant protein Tp0821 shows excellent immunocompetence,which can be applied to the serological diagnosis of syphilis.
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Background & objectives: Fluoroquinolone has a broad spectrum of antimicrobial activity, and is widely used for gonorrhoea treatment. However, its effi cacy can be compromised by the drug-resistance property of Neisseria gonorrhoeae isolates. Most resistant cases of N. gonorrhoeae are associated with mutations in the quinolone-resistance-determining-region (QRDR) within genes of gyrA and parC. This study was undertaken to describe resistance profi le of N. gonorrhoeae to fl uoroquinolones in Shanghai, P.R. of China, and also associated resistance mutations in gyrA and parC. Methods: Eighty N. gonorrhoeae isolates were collected from Shanghai Skin Disease & Sexually Transmitted Disease Hospital or DongFang Hospital during April 2005 to April 2006 in Shanghai, P.R. of China. The minimum inhibitory concentrations (MIC) of fl uoroquinolones for these isolates were determined by an agar dilution method. Mutation patterns within gyrA and parC were determined by direct sequencing or by using established restriction fragment length polymorphisms (RFLP) methods. Results: Ninety fi ve per cent (76 of 80) of isolates were resistant, 3.75 per cent (3 of 80) intermediate resistant, and 1.25 per cent (1 of 80) were sensitive to fl uoroquinolone drug ciprofl oxacin. Sequencing and RFLP analysis of gyrA and parC revealed that all resistant isolates had dual mutations of S91F and D95A/G/N in gyrA. Some isolates had an extra mutation within parC either of D86N, S87N or E91A/G. Mutation patterns for gyrA and parC were signififififi cantly (P<0.05) associated with MICs level. Interpretation & conclusions: Mutations of S91F and D95A/G/N in gyrA combined with S87N in parC was the most prevalent mutation pattern of fl uoroquinolone resistant N. gonorrhoeae isolates. This mutation pattern was associated with a high level of quinolone resistance (MIC >16.0 μg/ml) which can serve as a maker for quinolone-resistance prediction in Shanghai, P.R. of China.
Subject(s)
Anti-Bacterial Agents/pharmacology , Anti-Bacterial Agents/therapeutic use , China , Drug Resistance, Bacterial/genetics , Fluoroquinolones/pharmacology , Fluoroquinolones/therapeutic use , Humans , Microbial Sensitivity Tests , Mutation , Neisseria gonorrhoeae/drug effects , Neisseria gonorrhoeae/genetics , Polymorphism, Restriction Fragment Length , Sequence Analysis, DNAABSTRACT
Objective To study the susceptibility of Neisseria gonorrhoeae to antibiotic agents from 1988 to 2002 in Shanghai. Methods The clinical isolates from patients with gonorrhea were collected and tested for their susceptibility to five antibiotics. Agarose-dilution-method was used to detect minimal inhibitory concentration (MIC) of anti-microbial agents including penicillin, tetracycline, spectinomycin, ciprofloxacin and ceftriaxone, and penicillinase producing Neisseria gonorrhoeae (PPNG) were tested with acidometric method. Results Susceptible strains to penicillin decreased from 11.28% in 1988 to 0 in 2002, MIC50 and MIC90 increased 8 and 4 times, respectively, the resistant rate and proportion of PPNG were 94.29% and 50.95%, respectively in 2002. The strains of high resistance to tetracycline increased from 0 in 1995 to 20.95% in 2002. The susceptible strains to ceftriaxone decreased from 100% in 1995 to 23.80% in 2002. The susceptibility to ciprofloxacin decreased significantly and resistant rate reached 99.05% in 2002. However, these strains were kept highly susceptible to spectionmycin. Concerning the multi-drug resistance, we found that the strains resistant to penicillin, ciprofloxacin and tetracycline simultaneously increased from 20.87% in 2001 to 23.30% in 2002, those resistant to both penicillin and ciprofloxacin reached to 70% in the past 2 years. Conclusions In Shanghai the resistant rates of Neisseria gonorrhoeae to antibiotics increased year by year in the past 15 years. The study indicates that spectinomycin and ceftriaxone should be the first choice for the treatment of gonorrhea at present and new sensitive antibiotic should be developed for the treatment of gonorrhea.
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Objectives To compare the co-infection statues of HSV, HBV and HCV in patients with STD and HIV infection for providing evidence of developing prevention and control strategies. Methods Serum samples confirmed to be infected with HIV/AIDS by Western blot, and serum samples of patients with STDs (syphilis, gonorrhoea and chlamydial infection) were tested for HSV2-IgG, HSV2-IgM, HBsAg and HCV-IgG by ELISA. The detection levels were compared between the two groups. Results Out of 76 specimens in STD group, HSV2-IgG was detected in 24 specimens (31.58%), HSV2-IgM in one specimen (1.32%), HBsAg positive in 8 (10.53%), and HCV antibody positive in 4 (1.32%). In 14 specimens of HIV/AIDS group, HSV2-IgG were detected in 7 (50.00%); HSV2-IgM in 5 (35.71%);8 (578.14%) were positive for HBsAg and 3 (21.43%) for HCV. In a total of 90 specimens, both HSV and HBV were detected in 6 specimens, both HSV-IgM and HBV in 2, and the four above-mentioned antibodies in 2. The infection rates of HSV, HBV and HCV were significantly higher in HIV-infected specimens than those in the STD specimens (P