Objective:To optimize the laboratory methods for isolation, culture, and preservation of Neisseria gonorrhoeae ( N. gonorrhoeae) . Methods:Five quality control bacterial strains ( N. gonorrhoeae strain 1, N. gonorrhoeae strain 2, Neisseria mucosa strain 3, Enterococcus faecalis strain 4, and mixed bacterial strain 5) were separately cultured using a self-made gonococcal selective medium and 7 commercialized gonococcal selective media (gonococcal isolation plates, gonococcal T-M selective medium, gonococcal chocolate agar plates, gonococcal selective medium, modified Thayer-Martin [MTM] agar plates, disposable gonococcus [GC] agar medium, and gonococcal agar plates), and their cultivation performance was evaluated. Sixteen strains of N. gonorrhoeae were cultured both inside and outside a candle jar to screen for CO 2-dependent strains. The preservation performance of 4 self-made gonococcal preservation solutions, including calf serum with 10% dimethyl sulfoxide, calf serum with 10% glycerol, brain-heart infusion broth with 20% glycerol, and trypticase soy broth with 30% glycerol, was evaluated. The survival of N. gonorrhoeae in freeze-dried and non-freeze-dried states was observed. Results:The growth performance of the 5 quality control strains varied across different commercialized gonococcal culture media. Concretely, N. gonorrhoeae strain 1 formed large and numerous colonies on both the self-made culture medium and MTM agar plates, which outperformed the other 6 culture media; the growth performance of N. gonorrhoeae strain 2 on the 7 commercialized culture media was inferior to that on the self-made culture medium; all 7 commercialized culture media had inhibitory effects on the growth of Neisseria mucosa strain 3, among which the self-made culture medium, gonococcal chocolate agar plates, MTM agar plates, disposable GC agar medium, and gonococcal agar plates could completely inhibit its growth; the gonococcal T-M selective medium could completely inhibit the growth of Enterococcus faecalis strain 4; mixed bacterial strain 5 showed better separation performance on the self-made culture medium, gonococcal isolation plates, gonococcal T-M selective medium, gonococcal chocolate agar plates, MTM agar plates, and disposable GC agar medium. Under CO 2-enriched conditions, all 16 strains of N. gonorrhoeae exhibited good growth performance; however, the growth of 11 strains was markedly inhibited without CO 2. No significant differences were observed in the preservation performance of the 4 preservation solutions at -70°C, and confluent colonies could be observed in all preservation solutions after 12 months of strain preservation; at -20 ℃, the trypticase soy broth with 30% glycerol showed the best preservation performance, with a few viable strains remaining after 6 months, while the calf serum with 10% dimethyl sulfoxide performed worst, with partial strains remaining viable after 2 weeks. Non-freeze-dried N. gonorrhoeae survived for varying duration at different temperatures (4 ℃, -18 ℃, -29 ℃, -70 ℃, and liquid nitrogen) ; no N. gonorrhoeae strains survived by day 4 when stored at 4°C; freeze-dried N. gonorrhoeae remained viable with the presence of confluent colonies for 6 months at 4 ℃. Conclusion:The self-made gonococcal selective medium demonstrated superior cultivation and isolation performance compared to commercialized gonococcal selective media; a small amount of CO 2 could promote the growth of N. gonorrhoeae; ultralow temperature and freeze-drying preservation could increase the survival time of N. gonorrhoeae, with freeze-drying at 4 ℃ being the most cost-effective long-term preservation method.

Objective:To optimize the laboratory methods for isolation, culture, and preservation of Neisseria gonorrhoeae ( N. gonorrhoeae) . Methods:Five quality control bacterial strains ( N. gonorrhoeae strain 1, N. gonorrhoeae strain 2, Neisseria mucosa strain 3, Enterococcus faecalis strain 4, and mixed bacterial strain 5) were separately cultured using a self-made gonococcal selective medium and 7 commercialized gonococcal selective media (gonococcal isolation plates, gonococcal T-M selective medium, gonococcal chocolate agar plates, gonococcal selective medium, modified Thayer-Martin [MTM] agar plates, disposable gonococcus [GC] agar medium, and gonococcal agar plates), and their cultivation performance was evaluated. Sixteen strains of N. gonorrhoeae were cultured both inside and outside a candle jar to screen for CO 2-dependent strains. The preservation performance of 4 self-made gonococcal preservation solutions, including calf serum with 10% dimethyl sulfoxide, calf serum with 10% glycerol, brain-heart infusion broth with 20% glycerol, and trypticase soy broth with 30% glycerol, was evaluated. The survival of N. gonorrhoeae in freeze-dried and non-freeze-dried states was observed. Results:The growth performance of the 5 quality control strains varied across different commercialized gonococcal culture media. Concretely, N. gonorrhoeae strain 1 formed large and numerous colonies on both the self-made culture medium and MTM agar plates, which outperformed the other 6 culture media; the growth performance of N. gonorrhoeae strain 2 on the 7 commercialized culture media was inferior to that on the self-made culture medium; all 7 commercialized culture media had inhibitory effects on the growth of Neisseria mucosa strain 3, among which the self-made culture medium, gonococcal chocolate agar plates, MTM agar plates, disposable GC agar medium, and gonococcal agar plates could completely inhibit its growth; the gonococcal T-M selective medium could completely inhibit the growth of Enterococcus faecalis strain 4; mixed bacterial strain 5 showed better separation performance on the self-made culture medium, gonococcal isolation plates, gonococcal T-M selective medium, gonococcal chocolate agar plates, MTM agar plates, and disposable GC agar medium. Under CO 2-enriched conditions, all 16 strains of N. gonorrhoeae exhibited good growth performance; however, the growth of 11 strains was markedly inhibited without CO 2. No significant differences were observed in the preservation performance of the 4 preservation solutions at -70°C, and confluent colonies could be observed in all preservation solutions after 12 months of strain preservation; at -20 ℃, the trypticase soy broth with 30% glycerol showed the best preservation performance, with a few viable strains remaining after 6 months, while the calf serum with 10% dimethyl sulfoxide performed worst, with partial strains remaining viable after 2 weeks. Non-freeze-dried N. gonorrhoeae survived for varying duration at different temperatures (4 ℃, -18 ℃, -29 ℃, -70 ℃, and liquid nitrogen) ; no N. gonorrhoeae strains survived by day 4 when stored at 4°C; freeze-dried N. gonorrhoeae remained viable with the presence of confluent colonies for 6 months at 4 ℃. Conclusion:The self-made gonococcal selective medium demonstrated superior cultivation and isolation performance compared to commercialized gonococcal selective media; a small amount of CO 2 could promote the growth of N. gonorrhoeae; ultralow temperature and freeze-drying preservation could increase the survival time of N. gonorrhoeae, with freeze-drying at 4 ℃ being the most cost-effective long-term preservation method.

Objective To investigate in vitro combined effect of ceftriaxone and azithromycin against clinical isolates of Neisseria gonorrhoeae(NG). Methods A total of 25 NG clinical isolates were collected from the STD clinic of Dalian Dermatosis Hospital in 2012. Epsilometer test(Etest)method was used to determine the minimum inhibitory concentrations(MICs)of ceftriaxone and azithromycin against NG isolates. Fractional inhibitory concentration index (FICI) was calculated to evaluate the in vitro combined effect of ceftriaxone and azithromycin against NG isolates. Results The mean MICs of ceftriaxone and azithromycin were 0.032 mg/L (range, 0.008- 0.064 mg/L) and 0.834 mg/L (range, 0.064-4.000 mg/L), respectively. The FICI ranged from 0.724 to 2.696, and ceftriaxone and azithromycin showed an additive effect against the above NG isolates. Conclusion Ceftriaxone and azithromycin show an additive effect against NG in vitro, but further studies with large sample size are needed to confirm their effects.

Objective To observe the clinical efficacy of stage treatment withTiao He Ying Wei(regulating Ying-nutritional and Wei-defensive qi) needling in treating insomnia.Method A hundred insomnia patients presenting difficulty falling asleep were randomized into group A1 and B1, 50 cases each; 100 insomnia patients presenting difficulty maintaining sleep were randomized into group A2 and B2, 50 cases in each group; 100 insomnia patients presenting early-morning awakening were randomized into group A3 and B3, 50 cases each. Group A1, A2 and A3 were treated withTiao He Ying Wei needling, while group B1, B2 and B3 were treated with conventional medication. The Symptoms score and cerebral blood flow indicators were observed before and after the intervention.Result After the treatment, the symptoms scores were significantly changed in each group (P<0.05). The improvement of symptoms score in group A1 was superior to that in group B1 (P<0.05); the improvement of symptoms score in group A2 was superior to that in group B2 (P<0.05); the improvement of symptoms score in group A3 was superior to that in group B3 (P<0.05). The cerebral blood flow indicators (middle cerebral artery, posterior cerebral artery, anterior cerebral artery, and basilar artery) were significantly changed after the treatment in group A1, A2 and A3 (P<0.05). After the treatment, there were significant differences in comparing the cerebral blood flow indicators between group A1 and B1, A2 and B2, and A3 and B3 (P<0.05).Conclusion Stage treatment withTiao He Ying Wei needling can improve the sleep quality of insomnia patients.

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.

Objective To test the ceftriaxone susceptibility of Neisseria gonorrhoeae (NG) isolates from Nanjing city,and to assess their genotypes by using the NG multi-antigen sequence typing (NG-MAST) method.Methods A total of 204 NG strains isolated in 2007 and 81 in 2012 from Nanjing city were included in this study.The minimum inhibitory concentration (MIC) of ceftriaxone was determined for these strains using an agar dilution method.DNA was extracted by the Qiagen commercial kit from these strains followed by NG-MAST.Results All the isolates were susceptible to ceftriaxone (MIC,≤ 0.25 μg/ml).The MIC of ceftriaxone was ≥ 0.06 μg/ml for 63.2％ of all the NG strains,70.6％ of those isolated in 2007 and 44.4％ of those in 2012,and ≥ 0.125 μg/ml for 31.6 ％ of all the NG strains,39.7％ of those isolated in 2007,11.1％ of those in 2012.Totally,166 genotypes were identified among the 285 isolates,of which,73 had been reported,and 93 were previously unreported.The most prevalent genotype was ST568 (n =13) in NG strains isolated in 2007,followed by ST270 (n =9),ST421 (n =7),ST2288 (n =5),ST1731 (n =4),ST1766 (n =4),ST1866 (n =4),ST1870 (n =4),while ST2318 (n =5),ST1053 (n =4),ST5990 (n =4),ST8726 (n =4) were the common genotypes in 2012.Those isolates with identical or similar genotypes tended to display similar MICs for ceftriaxone.Conclusions The prevalent genotypes of NG are markedly different between 2007 and 2012 in Nanjing region,and there is a strong association between the genotypes and ceftriaxone susceptibility of NG.NG-MAST results may serve as a genetic marker in the surveillance of antibiotic susceptibility in NG.

Objective To discuss the feasibility of constructing the system of traditional Chinese medicine nursing diagnosis.Methods Based on the theoretical analysis and status quo analysis,the feasibility of constructing the system of TCM nursing diagnosis was discussed,and the achievements and problems waiting for settlement were also narrated.Resuts It has the foundation of constructing the system of TCM nursing diagnosis,but some problems still need to be solved.Conclusions It is feasible for building TCM nursing diagnosis system,and the TCM nursing diagnosis system does not conflict with NANDA-I.

Objective To determine Neisseria gonorrhoeae multi-antigen sequence typing (NG-MAST) sequence types in different geographical areas of China,including Changzhou and Yangzhou cities of Jiangsu province,Wuzhou and Hezhou cities in Guangxi Zhuang Autonomous region,Sanya and Qionghai cities of Hainan province,Jiangmen and Maoming cities of Guangdong province.Methods DNA was extracted using Qiagen DX extraction kits from 88 urine samples which were collected from male patients attending sexually transmitted disease (STD) clinics and positive for nucleic acid amplification test (NAAT) for N.gonorrhoeae.Two rounds of PCR were carried out to amplify the porB and tbpB genes of N.gonorrhoeae followed by gene sequencing.Sequence alignment was performed on the NG-MAST website (http://www.ng-mast.net) to determine the genotype of N.gonorrhoeae.Results The first-round PCR yielded positive results for porB and tbpB in 13.6％ (12/88) and 14.8％ (13/88),respectively,of these urine specimens,and 12 samples were successfully genotyped with the efficiency of genotyping being 13.6％.The amplification efficiency of second-round PCR was enhanced to 71.6％ and 72.7％ for porB and tbpB,respectively,and the efficiency of genotyping increased to 70.5％ (62/88).Compared with the first-round PCR,the second-round PCR showed an increase in amplification efficiency for porB and tbpB by 58.0％ and 57.9％ respectively,as well as in genotyping efficiency by 56.9％.Forty-five genotypes were identified in the 62 samples,including 40 known genotypes and 5 novel genotypes.Of these genotypes,ST1866 was the most abundant (6/62),followed by ST1972 (4/62) and ST3356 (4/62),all of which were from Jiangsu province.The ST532 genotype was identified in 3 samples from Guangdong province,ST2221 genotype in 2 samples from Guangxi Zhuang Autonomous region.Each of the remaining genotypes was identified in only 1 sample and scattered in all of these cities.The 5 novel MAST-genotypes were as follows:porB-892 and tbpB-46 (98％ similarity),porB-130 and tbpB-504 (96％ similarity),porB-2790 and tbpB-32 (99％ similarity),porB-1053 and tbpB-856 (99％ similarity).Conclusions Urine samples can be used for NG-MAST analysis,and two rounds of PCR can enhance the efficiency of genotyping.NG-MAST genotypes appear to be diverse in different geographical areas of China.

Objective To make a nationwide external quality assessment of tests for isolation and identification of N.gonorrhoeae in medical and healthcare facilities at different levels,and to analyze current problems.Methods Lyophilized quality control samples were uniformly delivered to 252 medical and healthcare facilities providing sexually transmitted disease (STD) services at different levels.Test results were analyzed by the National Center for STD Control,China Center for Disease Control and Prevention,and evaluation results were fed back to participating laboratories.Results Finally,test results were received from 203 (80.56％) facilities.The comprehensive score averaged at 87.14,and facilities achieving a comprehensive score of 80 or greater amounted to 80.30％ (163/203).The coincidence rate was 53.69％ (109/203) for all of the 5 quality control samples,82.76％ (168/203) and 87.68％ (178/203) respectively for two quality control samples containing only N.gonorrhoeae,86.21％ (175/203) and 96.06％ (195/203) respectively for a sample containing Neisseria sicca and a sample containing Enterococcus faecalis,69.46％ (141/203)for a sample containing different species of Neisseria.Conclusion The external quality assessment reveals a disparity in the capability to isolate and identify Neisseria among medical and healthcare facilities providing STD services at different levels.

Objective To make a nationwide external quality assessment for drug sensitivity testing of Neisseria gonorrhoeae, analyze the problems in and factors associated with the drug sensitivity testing, and to enhance the quality of drug sensitivity testing of N. gonorrhoeae at different monitoring sites. Methods Samples were uniformly delivered to monitoring sites by express mail service. Test results were analyzed in the National Center for STD Control, and the evaluation results were fed back to these monitoring sites. Results A total of 105 quality control samples were delivered from 2007 to 2009, with a response rate of 88.57% (93/105). Thirteen monitoring sites were enrolled in the external quality assessment, including 9 laboratories in 2007, 9 in 2008 and 13 in 2009. The total percentage amounted to 77.42% (24/31) for qualified laboratories during the 3 years, including 6 laboratories in 2007, 7 in 2008 and 11 in 2009. The coincidence rate increased for the detection of penicillinase-producing N. gonorrhoeae (PPNG), N. gonorrhoeae with chromosome-mediated ciprofloxacin resistance, and N. gonorrhoeae with chromosome-mediated spectinomycin resistance, and declined for the detection of N. gonorrhoeae with plasmid-mediated high level tetracycline-resistance (TRNG) and N. gonorrhoeae with chromosome-mediated ceftriaxone resistance. Conclusions The 3-year external quality assessment reveals an improvement in the overall quality of drug sensitivity testing of N. gonorrhoeae at national monitoring sites; the accuracy is improved markedly for the detection of PPNG, N. gonorrhoeae with resistance to spectinomycin and ciprofloxacin, but is needed to increase for the detection of ceftriaxone-resis- tant N. gonorrhoeae and TRNG.