Objective To establish a molecular typing method (opa-typing) for Neisseria gonorrhoeae, and to evaluate its performance based on epidemiological data. Methods Twenty-six gonorrhea patients were recruited from March to April 2006 at two sites, including 17 cases from the STD Clinic of Institute of Dermatology, Chinese Academy of Medical Sciences and Peking Union Medical College and 9 cases from the STD Clinic of Shandong Provincial Institute of Dermatology. Of the 26 patients, 6 were from three known sexual links, while the remaining 20 patients did not have any sexual contact with each other. The opa gene was amplified by using PCR from Neisseria gonorrhoeae isolates from these patients followed by overnight digestion with restriction enzymes Taq Ⅰ and Hpa Ⅱ. The enzyme digestion band patterns were analyzed using the Cel-Compar program. Results The opa gene was successfully amplified from all the 26 Neisseria gonorrhoeae strains, and restrictedly digested by endonucleases Taq Ⅰ and Hpa Ⅱ . Identical band patterns were observed between patients with sexual links, but not among the remaining 20 patients. Conclusions The results of opatyping of Neisseria gonorrhoeae coincide with the information on sexual behaviour provided by patients. Opatyping may serve as a reliable tool in sexual network analysis.

The chemical composition, separation and identification of the components as well as the tumor photobiological activities of the major components of the new tumor-photochemodiagnostic and photochemotherapeutic agent photocarcinorin (PsD-007) were represented in this paper. It has been shown by the results of HPLC analysis in combination with spectroscopic determinations that PsD-007 is composed of 7 different porphyrins: MHD, DMD, MVD, AHD, HVD, Hp and Pp. The experimental results show that MHD, DMD and MVD are the major tumor photobiologically active components of PsD-007.

To investigate the clinical improvement of limping gait in patients with ankylotic hips and limb length discrepancy.Methods:From 1996 to 2005,12 patients with ankylotic hips and limb length discrepancy were treated by distraction osteogenesis with a mono-lateral external fixator and an intramedullary nail.The limb length discrepancy was 6.20-12.50 (median 8.45) cm.Limping gait was classified according to the recommendations of the American Academy of Orthopedic Surgeons/Hip Society and scored according to Harris:no limping scored 11 points,mild limping scored 8 points,moderate limping scored 5 points,while severe limping scored 0 points.Limping gait was severe in all patients pre-operatively and the total score was 0.Results:All patients were followed up for 30.00-46.00 (median 38.55) months,and all reported improvement in limping gait.The gain in length was 6.00-12.50 (median 8.20) cm,and the mean residual limb length discrepancy was 0-0.50 (median 0.20) cm.The total treatment time was 41.00-82.00 (median 61.50) weeks,the lengthening time was 14.00-38.00 (median 29.55) weeks.At the last follow-up,10patients had mild limping gait and 2 had moderate limping gait; the total score was 90.00.The median score was 7.50 (P25 was 8.00,P75 was 8.00).According to Wilcoxon signed rank test,the post-operative limping gait scores were significantly higher than pre-operative (P=0.001 ).Conclusion:Femoral lengthening can improve the limping gait significantly in ankylotic hips and limb length discrepancy.

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 study the current status of antimicrobial resistance of clinical Neisseria gonorrhoeae isolates in China by analyzing the surveillance results in 2008.Methods N. gonorrhoeae strains were collected from 951 eligible patients at national monitoring sites for resistance of N. gonorrheae,including 156 patients from Jiangsu province,71 from Zhejiang province,102 from Fujian province,207 from Guangdong province,77 from Guangxi province,43 from Hainan province,80 from Sichuan province,44 from Chongqing,45 from Xinjiang Uygur Autonomous Region,72 from Shaanxi province,and 54 from Tianjin.The production of β-lactamase was detected by paper acidometric testing,and minimum inhibitory concentrations(MICs)were determined by agar dilution method for spectinomycin,ciprofloxacin,cefiriaxone,tetracycline,respectively.Results Among the 951 N. gonorrhoeae isolates,2(0.21%)were resistant to spectinomycin,451(47.42%)showed reduced sensitivity to ceflriaxone,928(97.58%)were resistant to ciprofloxacin.Penicillinase-producing N.gonorrhoeae (PPNG) and plasmid mediated tetracycline-resistant N. gonorrhoeae (TRNG) accounted for 34.91%(332/951)and 51.21%(487/951) of these isolates respectively.Kendall rank correlation analysis showed that there was a positive correlation between the positivity rate of TRNG and PPNG(r=0.20,P<0.01),but a negative correlation between the susceptibility to cefiriaxone in N.gonorrhoeae and positivity rate of PPNG(r=-0.09,P<0.01).No correlation was observed between the susceptibility to cefiriaxone and susceptibility to ciprofloxacin or the positivity rate of TRNG,or between the susceptibility to ciprofloxacin and positivity rate of PPNG or TRNG.Chi-square analysis showed a marked increase in the percentage of N.gonorrhoeae isolates with reduced susceptibility to ceftriaxone in Guangxi province,Hainan province,Xinjiang Uygur Autonomous Region and Shaanxi province,the percentage of N.gonorrhoeae isolates with resisitance to spectinomycin in Shaanxi province,prevelance of TRNG in Guangdong province,and prevelance of PPNG in Sichuan and Zhejiang provinces compared with the average level (all P<0.05).Conclusions Thero is a significant diffefence in antimicrobial susceptibility of N.gonorrhoeae from difierent areas of China.A significant elevation is observed in the percentage of N.gonorrhoeae with reduced susceptibihty to cefifiaxone and resistance to spectinomycin in Shaanxi province.to which close attention should be paid.

ObjectiveTo evaluate the performance of a three-gene typing system in the determination of Treponema pallidum (Tp) genotypes.MethodsTo determine the genotypes of Tp,three targets were assessed,including the number of 60 base-pair repeats,restriction fragment length polymorphism(RFLP) pattem of tprEGJ gene after MseI digestion and the sequence of tp0548 gene.The DNA extracted from the Nichols strain of Tp served as the positive control,and that from the moist ulcer of patients with genital herpes and negative RPR or TPPA test results served as the negative control.To validate the typing method,clinical specimens were collected from the moist skin lesions of patients with primary or secondary syphilis,and subjected to the amplification of polA gene by PCR.The enhanced molecular typing system was used to determine the genotypes of Tp in Tp DNA-positive specimens.ResultsThe Nichols strain harbored a genotype of 14a/a.No amplification of any of the three target genes was found in the negative control.The arp gene,tprEGJ gene and tp0548 gene were amplified from 94.1％,91.2％ and 94.1％ of the 40 clinical specimens,and the genotype was successfully determined by the three-gene typing system for 91.2％ of the clinical Tp strains.The predominant type of arp,tprEGJ and tp0548 genes was 14 repeats,d and f,respectively in these clinical Tp isolates.ConclusionThe enhanced molecular tying method for Tp exhibits high sensitivity,specificity and discrimination potential.

ObjectiveTo investigate the prevalence of syphilis among outpatients in sexually transmitted disease (STD) clinics in Guangxi Zhnang Autonomous Region,and to assess the socioeconomic and behavioral factors associated with the infection.MethodsThe outpatients to 14 STD clinics in 8 cities of Guangxi Zhnang Autonomous Region were investigated with questionnaires by their doctors at the first visit.Venous blood samples were obtained from these outpatients and subjected to toludine red unheated serum test (TRUST) to screen for syphilis.Treponema pollidum particle agglutination (TPPA) was performed for TRUSTpositive samples.The epidemiological data were collected by using EpiData software,statistically analyzed by using SPSS13.0 software package.ResultsA total of 10 930 STD outpatients were recruited in the study,and 1297 samples were confirmed to be both TRUST and TPPA positive.The prevalence of syphilis was 11.9％ in all of the outpatients,14.3％ in female outpatients and 10.3％ in male outpatients,13.3％ in the outpatients of Zhuang nationality,and 11.4％ in those of Han nationalily.Multivariate analysis showed that syphilis was independently related to female sex[odds ratio(OR) 2.23,95％ confidence interval(CI) 1.69 - 3.00,P＜0.01 ],low educaiion level (middle school:OR 1.70,95％ CI 1.11 - 2.62,P ＜ 0.05; primary school or illiteracy,OR 1.98,95％ CI 1.13 - 3.46,P＜0.05),annual income of more than 30000 Yuan (OR 1.91,95％ CI 1.18 -3.10,P ＜ 0.01 ),commercial sex workers or having multiple sexual partners(OR 1.54,95％ CI 1.16 - 2.06,P ＜0.01 ).ConclusionsSyphilis serology should be the routine test in STD clinical settings in Guangxi region,and the intervention should be enhanced to control the prevalence of syphilis in high-risk populations.

Objective To estimate the prevalence of hepatitis C virus (HCV) infection among sexually transmitted disease (STD) clinic attendees infected with human immunodeficiency virus type 1 (HIV-1) in Guangxi Zhuang Autonomous Region.Methods Totally,11 553 blood plasma samples were collected from STD clinic attendees in Guangxi Zhuang Autonomous Region,and subjected to HIV-1 antibody screening and confirmatory testing.Enzyme linked immunosorbent assay (ELISA) was performed to detect anti-HCV antibodies in 140 anti-HIV-1 antibody-positive samples and 282 anti-HIV-1 antibody-negative samples from age-and marital status-matched attendees.Chi-square test was performed to assess the differences in the prevalence rate of HCV infection between anti-HIV-1-negative and-positive samples,and Logistic regression analysis to evaluate the risk factors for HCV and HIV co-infection.Results The positivity rate of anti-HCV antibodies was 33.57％ (47/140)among anti-HIV-1-positive samples,significantly higher than that in anti-HIV-1-negative samples (1.06％ (3/282),x2 =94.66,P ＜ 0.05).Logistic regression analysis showed a statistical increase in the prevalence of HCV/HIV co-infection in individuals reporting more than one sexual partners compared with those reporting only one sexual partner (OR =2.4,95％ CI (1.0-5.6),P =0.05),and in intravenous drug users compared with non-intravenous drug users (OR =20.8,95％ CI(5.7-76.5),P ＜ 0.05).Conclusions HCV infection appears to be associated with HIV-1 infection,and comprehensive intervention on HIV-1-infected patients may slow down HCV transmission.

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.

ObjectiveTo establish a rapid,sensitive and accurate method to detect Mycoplasma genitalium,and to evaluate the prevalence of M.genitalium among unlicensed prostitutes from Hezhou city in Guangxi Zhuang Autonomous Region.MethodsA pair of primers and Taqman MGB probe were designed and synthesized for the Pa gene of M.genitalium.Standard samples were prepared with the M.genitalium type strain G37.The established Taqman MGB real time fluorescence-based PCR assay was used to detect M.genitalium in the standard samples and cervical swab specimens collected from unlicensed prostitutes in Hezhou city of Guangxi Zhuang Autonomous Region.ResultsThe established Taqman MGB real time PCR exhibited a wide linear range( 1 × 10 copies/μl to 1 × 106 copies/μl,R2 =0.993),good repeatability(intra-assay variation;0.7％,inter-assay variation:1.09％) and hign sensitivity with the limit of detection being 10 copies/μl and limit of quantification being 50 copies/μl.As the assay showed,12.1％ of the 404 cervical swab samples were positive for M.genitalium.ConculsionThe Taqman MGB real time fluorescence-based PCR is a rapid and sensitive method for the quantitative and qualitative detection of M.genitalium.