Yaohua Xue

Association of the in vitro susceptibility of clinical isolates of chlamydia trachomatis with serovar and duration of antibiotic exposure.

Background The presence of persistent Chlamydia trachomatis infection after treatment does not always correlate with in vitro susceptibility testing. Methods The in vitro minimum inhibitory concentration (MIC) and minimal bactericidal concentration (MBC) of azithromycin, clarithromycin, roxithromycin, doxycycline, tetracycline, ofloxacin, and penicillin were tested against 61 clinical isolates of C. trachomatis on 6 serovars, and the MIC/MBC of azithromycin and ofloxacin at different points in time after antibiotic administration to infected cultures. Results Of the 7 antibiotics tested, clarithromycin showed the greatest activity against C. trachomatis isolates with MIC90 of 0.032 &mgr;g/mL and MBC90 of 0.064 &mgr;g/mL, followed by doxycycline with MIC90 0.064 &mgr;g/mL and MBC90 0.064 &mgr;g/mL, and azithromycin with MIC90 0.160 &mgr;g/mL and MBC90 0.320 &mgr;g/mL. Azithromycin had roughly the same MIC50 values (0.08 &mgr;g/mL) as the other serovars isolates tested, and other antibiotics showed a 2- to 4-fold difference in MICs50 between serovars. In addition, an increase in the azithromyin MIC was observed by 8 hours and the ofloxacin MIC by 16 hours. At 24 hours, the azithromycin MICs were greater than 40 &mgr;g/mL and ofloxacin MICs were greater than 64 &mgr;g/mL. Conclusions The current data demonstrated that the antimicrobial susceptibility of C. trachomatis was influenced by both the serovar type and the duration of exposure to antibiotics in infected cultures.

Prevalence of Chlamydia trachomatis Genotypes in Men Who Have Sex with Men and Men Who Have Sex with Women Using Multilocus VNTR Analysis-ompA Typing in Guangzhou, China.

Background Chlamydia trachomatis is one of the most prevalent bacterial sexually transmitted infection in China. Although C. trachomatis genotypes can be discriminated by outer membrane protein gene (ompA) sequencing, currently available methods have limited resolutions. This study used a high-resolution genotyping method, namely, multilocus variable number tandem-repeat analysis with ompA sequencing (MLVA)-ompA, to investigate the local epidemiology of C. trachomatis infections among men who have sex with men (MSM) and men who have sex with women (MSW) attending a sexually transmitted diseases (STD) clinic in Guangzhou, China. Methods Rectal specimens from MSM and urethral specimens from MSW were collected between January 2013 and July 2014 at the Guangdong Provincial Center STD clinic. The specimens were sent to the laboratory for analyses. All specimens that were tested positive for C. trachomatis by the commercial nucleic acid amplification tests were genotyped by MLVA-ompA. Results Fifty-one rectal specimens from MSM and 96 urethral specimens from MSW were identified with C. trachomatis. One hundred and forty-four of the 147 specimens were fully genotyped by MLVA-ompA. Rectal specimens from MSM were divided into four ompA genotypes and urethral specimens from MSW into nine genotypes. No mixed infections were found among all specimens. The most frequent genotypes were D, G, J, E and F. All specimens were further divided into 46 types after ompA genotyping was combined with MLVA. Genotypes D-8.7.1 and G-3.4a.3 were the most frequent among MSM, whereas genotypes D-3.4a.4, E-8.5.1, F-8.5.1, and J-3.4a.2 were the most frequent subtypes among MSW. The discriminatory index D was 0.90 for MLVA, 0.85 for ompA, and 0.95 for MLVA-ompA. Conclusions The most prevalent MLVA-ompA genotypes were significantly different between MSM and MSW from Guangzhou, China. Moreover, MLVA-ompA represented a more favorable degree of discrimination than ompA and could be a reliable complement for ompA for the routine subtypes of C. trachomatis.

An in vitro model of azithromycin-induced persistent Chlamydia trachomatis infection

Single-dose azithromycin is recommended for treating Chlamydia trachomatis infections. Here, we established an in vitro cell model of azithromycin-induced persistent infection. Azithromycin inhibited the replication of C. trachomatis in a dose-time-dependent manner. Electron microscopy indicated that small inclusions in the induced model contained enlarged, aberrant and non-infectious reticulate bodies. RT-PCR showed that C. trachomatis still has the ability to express the unprocessed 16S rRNA gene in the model and that C. trachomatis recovered after the removal of azithromycin with a peak recovery time of 24 h. The mutations in 23S rRNA, L4 and L22 genes were not found in persistent infection, and qRT-PCR analysis showed that the relative expression level of euo in azithromycin treated infection was upregulated while omcB was downregulated. In summary, this study provides a novel in vitro cell model to examine the characteristics of azithromycin-induced persistent infection and contribute to the development of treatments for C. trachomatis infection.

Determining the in vitro susceptibility of Neisseria gonorrhoeae isolates from 8 cities in Guangdong Province through an improved microdilution method

A microdilution method for the antibiotic susceptibility testing of Neisseria gonorrhoeae was established and improved, and the antibiotic resistance of N. gonorrhoeae samples isolated from 8 cities of Guangdong in 2016 was determined. The improved microdilution method was compared with the agar dilution method recommend by the World Health Organization (WHO) Western Pacific Region by testing the susceptibility of 100 clinical N. gonorrhoeae isolates. The essential agreement (EA), categorical agreement (CA), very major error (VME), major error (ME), and minor error (MIE) levels of the two methods were analyzed; the acceptable performance rates were measured as follows: ≥90% for EA or CA, ≤3% for VME or ME, and ≤7% for MIE. The EA, CA, VME, ME, and MIE of each method for 7 antibiotics, penicillin, tetracycline, ciprofloxacin, spectinomycin, ceftriaxone, cefixime, and azithromycin, were 96%-100%, 94%-100%, 0%-3%, 0%-2%, and 0%-6%, respectively. The Wilcoxon signed-rank test results indicated 94%-100% agreement between the 2 methods after excluding off-scale values (P > 0.05). The susceptibility of 634 N. gonorrhoeae strains to the 7 antibiotics above were tested through the microdilution method. The resistant rates of the isolates against ciprofloxacin, tetracycline, penicillin, and azithromycin were 99.8%, 88.3%, 53.8%, and 11%, and the percentages of the isolates with decreased susceptibility to ceftriaxone (minimum inhibitory concentration [MIC] ≥0.125 μg/mL) and cefixime (MIC ≥0.25 μg/mL) were 2.1% and 12%, respectively, in Guangdong. Among 8 cities, Shenzhen had the highest rates of resistance against penicillin (77.8%) and decreased susceptibility against ceftriaxone (5.6%). Zhuhai had the highest rates of decreased susceptibility against cefixime (30.1%), and Jiangmen had the highest azithromycin-resistant isolates (16.8%). The findings from this study indicated that the improved microdilution method is an alternative for testing the antimicrobial susceptibility of N. gonorrhoeae. The resistance rates of N. gonorrhoeae against penicillin, tetracycline, and ciprofloxacin were high. While ceftriaxone, cefixime, and spectinomycin remained effective against N. gonorrhoeae, their effectiveness seemed to be decreasing over time. Azithromycin therapy requires timely susceptibility test results.

P3.240 Prevalence and genotype distribution of chlamydia trachomatis in urine among males attending std clinics in guangdong province, china, 2016

Introduction Chlamydia is the most common sexually transmitted disease worldwide. Many studies have been evaluated the prevalence of Chlamydia trachomatis (CT) infection while very rare studies assessed the genotype distribution in urine among males attending sexually transmitted diseases (STD) clinics (MSCs)in China. This study aimed to investigate the prevalence and molecular epidemiology of CT infection by urine samples among MSCs from different geographic areas of Guangdong province, China. Methods A cross-sectional study was performed among MSCs from ten HIV surveillance sites of Guangdong province, China. CT DNA in male urines were extracted and detected by using the Roche cobas 4800 CT/NG. The ompA genes were amplified by nested polymerase chain reaction and sequenced. Urine leukocyte esterase test were performed. Results Of the 1926 urine specimens, 1903 urines were successfully validated for detection of CT. Of the 1903 samples, one hundred and sixty-three (8.6%, 95% CI 8.2% to 9.0%) were found to be positive for CT. One hundred and thirty CT positive specimens were successfully genotyped by nested PCR, resulting in eight genotypes. The most prevalent genotypes were D, E, F, and J with proportions of 20.8%, 20.0%, 17.7%, 16.9%, respectively. There was no significant difference between age, geographic area, leukocyte esterase test and genotype distribution. Conclusion There was a high prevalence of CT infection among males attending STD clinics in eastern area of Guangdong province, China. Promoting detection and molecular epidemiology research are needed for effective and comprehensive prevention and control programs.

Prevalence and Genotype Distribution of Chlamydia trachomatis in Urine among Men Attending STD Clinics in Guangdong Province, China, 2016

Studies have rarely assessed the genotype distribution of Chlamydia trachomatis (CT) in urine among men attending sexually transmitted disease clinics (MSCs) in China. This study was aimed at investigating the prevalence and molecular epidemiology of CT infection by examining urine samples among MSCs from different geographic areas of Guangdong Province, China. A cross-sectional study was conducted among MSCs from 10 human immunodeficiency virus sentinel surveillance sites in Guangdong Province. CT DNA was extracted from male urine samples and analyzed using a Roche cobas 4800 CT/NG. The ompA genes were amplified by nested PCR and sequenced. The leukocyte esterase test was performed by routine urine analysis at local clinics. Of the 1,903 samples, 163 (8.6%, 95% confidence interval [CI] 3.8-16.3%) tested positive for CT. The highest prevalence (10.5%) of CT infection was observed among participants aged between 21 and 30 years. A total of 130 CT-positive samples (79.8%, 130/163) were successfully genotyped by nested PCR, resulting in 8 genotypes. The most prevalent genotypes were D, E, F, and J, with proportions of 20.8%, 20.0%, 17.7%, and 16.9%, respectively. There were no significant correlations between the geographical areas, leukocyte esterase test results and genotype distribution. Promotion of detection and molecular epidemiology research is needed for effective and comprehensive prevention and control programs.

P1.003 Experimental Study on Pathogenic Diversity of Different Chlamydia Trachomatis Serovars in Mouse Genital Infection

Background Chlamydia trachomatis infection increases above gonorrhoea and syphilis, ranking first among the STDs. Molecular epidemiological researches have shown the predominant genotypes vary between regions, periods and population subgroups. However, serovars E, D and F are the most prevalent serovars. It is unclear whether the epidemiological characteristics were contributed to geography or pathogenicity. We explored the pathogenic diversity of different C. trachomatis serovars in mouse genital infection. Methods One hundred of female BALB/C mice were divided into serovar E, F, H, J and K groups. The mice in study group treated by medroxyprogesterone acetate were inoculated 107 C. trachomatis into genital tract. C. trachomatis was detected by culture, direct immunofluorescence assay (DFA) and PCR in the cervicovaginal secretion. On the days 7 and 35 after inoculation, inflammation of the cervix, uterus and oviduct were examined by HE stain, and expressions of cHSP60 and CPAF in the uterus and fallopian tube were detected by ELISA. Results The inflammatory of the cervical mucosa was more severe in serovar E group compared with J, K and H groups on day 7 post-inoculation. Accordingly, cHSP60 and CPAF expression increased significantly in E group compared with other experimental and control groups. On day 35 post-inoculation, the histo-pathological changes of the genital tract were obvious in J, K and H groups, characterised with uterine swelling, pyometra and effusion, fallopian expansion, hydrops, fibrosis and stenosis. cHSP60 and CPAF expression in H group was superior to that in other groups. Positive correlation between cHSP60 and CPAF expression was present on day 7 and 35 post-inoculation, respectively. Conclusion There existed pathogenic diversity among different C. trachomatis servoars in mouse genital infection. The expression of inflammatory cytokines of cHSP60 and CPAF during Chlamydial infection might partially explain the pathogenic mechanism and the stage of the Chlamidal infection.

P1-S1.41 Emergence of an African antimicrobial resistance genotype in Neisseria gonorrhoeae strains isolated in Guangzhou, China, 2001–2009

Background The continuing spread of drug-resistant gonococci has posed a challenge for successful treatment worldwide. Recently third-generation cephalosporins-resistant strains were isolated in Japan. Guangdong Province in South China has one of the highest gonococcal resistance rates in China and a large number of international migrants. We investigated the invitro antimicrobial susceptibility and genotypes of N gonorrhoeae strains isolated in Guangzhou, the capital city, from 2001 to 2009. Methods MICs to penicillin, ceftriaxone, tetracycline, ciprofloxacin, and spectinomycin were determined by agar plate dilution and susceptibilities were interpreted according to WHO standards. ß-lactamase production was determined by paper acidometric testing. The resistant plasmids were determined for penicillinase-producing N gonorrhoeae (PPNG) and high-level tetracycline resistant N gonorrhoeae (TRNG) by PCR and the isolates were genotyped. Results Of 1132 consecutive gonococci isolated from 2001 to 2009, no ceftriaxone and spectinomycin resistant strains were found, but the prevalence of strains less susceptible to ceftriaxone rose from 18.2% to 38.5%. The MIC90 for ceftriaxone showed intermediate sensitivity (0.06–0.125 μg/ml) and spectinomycin near the resistant level (16–32 μg/ml). The resistance to penicillin, tetracycline and ciprofloxacin increased from 81.8%, 84.5% and 72.5% in 2001 to 90.1%, 91.4% and 98.7% in 2009, respectively. A total of 313 (27.7%) PPNG and 486 (42.9%) TRNG strains were detected. 206 (18.2%) strains were both PPNG and TRNG. PPNG rose from 21.8% to 29.8% and TRNG rapidly increased from 27.2% to 56.3%. Genotyping TEM-1 gene showed 267 (99.6%) PPNGs in 2001–2008 carried the Asian-type ß-lactamase plasmids and one African-type in 2008. Genotyping of tetM gene showed that all 486 TRNGs were Dutch variants. Conclusion Gonorrhoea resistance continues to be a major public health problem in Guangzhou. The emergence of an African gonorrhoea resistance variant may be related to the large African diaspora in Guangzhou, migration of Chinese to Africa, or other migration patterns. More research is needed to determine what practices, systems, and behaviours contribute to escalating resistance patterns. Abstract P1-S1.41 Figure 1 Resistance of N gonorrhoeae isolates in Guangzhou during 2001–2009.