Study On The Opa-typing Of Neisseria Gonorrhoeae

Prevalence of sexually transmitted diseases (STDs) has been increasing in Chinasince the 1980s. Currently, gonorrhea is one of the most frequently reported STDs inChina. Antimicrobial resistance of Neisseria gonorrhoeae has been a challenge to thetreatment of gonococcal infections in the past 30 years. PPNG and TRNG were firstidentified in 1976 and 1985 respectively. Since then, they have been spreadingworldwide, and the prevalence of PPNG and TRNG reached to a level in manycountries around the world, especially in developing countries in Asia and Africa.While gonorrhoeae strains with decreased susceptibility to fluoroquinolone arepopular in Southeast Asia and Pacific region, they were emerged in some Europeancountries. Cases of decreased susceptibility to spectinomycin and ceftriaxone werealso reported sporadically. In order to monitor the epidemic of antibiotic susceptibilityto gonorrhoeae and to find out its characterizations in China, the gonococcalantimicrobial susceptibility program (GASP) was established in 1987 as onecomponent of National STD Surveillance Program.Typing of N. gonorrhoeae strains not only can provide valuable insight into N.gonorrhoeae strains circulating within a given community but also can enhancemonitoring of their susceptibility to antibiotics, which will be helpful for improvingstrategies for STDs prevention and control. Traditional methods used for N.gonorrhoeae typing are often based on phenotypic characteristics, such as auxotypingand serotyping. However, these two typing methods have a number of limitations. Inthe recent decade, typing methods based on DNA analysis such as opa-typing havebeen developed and used for analysis of N. gonorrhoeae strains. Previous studies haveshown that opa-typing has greater discriminatory power than the traditionalphenotypic and other genotypic techniques.ChapterⅠ. Surveillance of antimicrobial susceptibilities of Neisseria gonorrhoeaein Jinan, Fuzhou and Nanning. To monitor antibiotic resistance of Neisseria gonorrheae in Jinan, Fuzhou andNanning based on analysis of the results from 2005. The production ofβ-lactamasewas determined by paper acidometric method. Minimum inhibitory concentrations(MICs) of ceftriaxone, tetracycline, ciprofloxacin and spectinomycin were determinedby an agar plate dilution method. A total of 379 clinical isolates of Neisseriagonorrhoeae were examined. During the period of 2005, positivity rate of PPNG was26.6％.Prevalenco of TRNG was 28.2％.Positivity rate of chromosomal resistance tociprofloxacin was 98.4％.There was no gonococcal isolate resistant to ceftriaxone, butthe prevalence of strains less susceptible to ceftriaxone was 32.5％. No Neisseriagonorrhoeae strain resistant to spectinomycin in 2005.Among Neisseria gonorrhoeaestrains isolated in the three cities, plasmid mediated resistance(including PPNG andTRNG)was high, cefriaxone and spectinomycin are considered as effective drugs forthe treatment of gonorrhea.ChapterⅡ. Establishment and evaluation of Opa-typing method.Opa-typing method was established in our study, the main procedure as follows:1) Preparation of DNA by using the AMPLICOR CT/NG Specimen Preparation kit;2) PCR amplification of opa gene and determination of PCR products using 1.5％agarose gel electrophoresis; 3) PCR products were then digested overnight at 65℃with 5U of the restriction enzyme TaqⅠ. Isolates that gave identical band patternswhen digested with TaqⅠwere subsequently digested overnight at 37℃with 5U ofHpaⅡ. 4) The resulting fragments were separated on a 10％polyacrylamide gel withan electrophoresis unit. 5) Bands were visualized by BioRad Electrophoresis ImageAnalysis System after silver staining. 6) Images of band patterns produced by TaqⅠand HpaⅡwere saved in TIFF format and the pattern profiles were analyzed using theGelComparⅡprogram. Twenty-six Neisseria gonorrhoeae strains were collected fromNanjing and Jinan, 6 of them were from three known sexual links, while theremaining 20 isolates without any sexual links among them. Isolates that gaveidentical band patterns by TaqⅠor HpaⅡwere from known sexual links, while isolates that gave different band patterns by TaqⅠor HpaⅡwere from the 20 isolateswhich without any sexual links each other.ChapterⅢ. Opa-typing of Neisseria gonorrhoeae strains isolated from patientsattending at STD clinics in ChinaGonorrhea has been one of the most common sexually transmitted diseases(STDs) in China, a clear understanding of its transmission dynamics is important informulating prevention and control measures. The goal of the study is to investigatethe distribution of opa-types in patients attending at STDs clinics in China and toevaluate the concordance between epidemiologic data and opa-typing results.Opa-typing was conducted for 330 Neisseria gonorrhoeae strains isolated from thepatients at 4 STDs clinics in China, and the epidemiologic data was collected as well.A total of 309 opa types were detected from the 330 isolates. Two hundred andninety-two opa types were unique, and 17 opa types were found in more than onepatient. Opa-typing confirmed all 9 sexual links which were revealed byepidemiologic information, and further identified 9 opa clusters and 8 similar pairs.Opa-typing is a discriminatory tool which can be used in epidemiologic studies ongonococcal infections. This technique is more powerful than epidemiologic data toidentify sexual links and improve our understanding of the transmission dynamics ofgonorrhea.