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Journal of Clinical Microbiology, December 2008, p. 3931-3934, Vol. 46, No. 12
0095-1137/08/$08.00+0     doi:10.1128/JCM.00577-08
Copyright © 2008, American Society for Microbiology. All Rights Reserved.

Molecular Epidemiological Identification of Neisseria gonorrhoeae Clonal Clusters with Distinct Susceptibility Profiles Associated with Specific Groups at High Risk of Contracting Human Immunodeficiency Virus and Syphilis

Wing-Wai Wong,² Chung-Ter Huang,¹ Lan-Hui Li,² Chien-Chou Chiang,² Bor-Dong Chen,¹ and Shu-Ying Li^1*

Research and Diagnostic Center, Centers for Disease Control, Taipei, Taiwan,¹ Branch for Disease Control and Prevention, Taipei City Hospital, Taipei, Taiwan²

Received 26 March 2008/ Returned for modification 13 June 2008/ Accepted 1 October 2008

Top ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

 
From April 2006 to August 2007, a total of 146 Neisseria gonorrhoeae isolates collected from 139 male patients in Taipei, Taiwan, were analyzed by N. gonorrhoeae multiantigen sequence typing (NG-MAST) and antibiotic susceptibility testing. The resistance rates of all isolates to ciprofloxacin, cefpodoxime, and cefixime were 76.7 (112/146), 21.2 (31/146), and 16.4% (24/146), respectively. NG-MAST identified 71 sequence types (STs), of which 21 STs contained 2 to 21 isolates. The isolates that belonged to the three major ST clusters typically were from patients who had specific epidemiological characteristics (such as sexual orientation and human immunodeficiency virus status). The major ST clones exhibited distinct resistance profiles and are associated with specific groups at high risk of human immunodeficiency virus and syphilis infections.

Top ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

 
The incidence of gonorrhea has increased in Taiwan in the last decade, as it has in many other developed countries (5, 6). The number of confirmed cases of gonorrhea reported to the Center for Disease Control of Taiwan increased from 361 (1.62 cases per 100,000 population) in 2000 to 1,978 (8.70 cases per 100,000 population) in 2004 and to 1,515 (6.67 cases per 100,000 population) in 2005 (2).

The rapid emergence and spread of gonococcal resistance to ciprofloxacin has reduced the options of effective treatment for gonococcal infections and has become a concern worldwide (10, 14). In Taiwan, the prevalence of gonococcal isolates with resistance to ciprofloxacin has increased from 25% in 1999 to 2000 to 93.1% in 2003 (9). Therefore, cephalosporin antibiotics are now the preferred choice for treatment in many clinics in Taiwan and elsewhere. Recently, however, reduced susceptibility to cephalosporin antibiotics has gradually increased (1, 13, 16).

Molecular epidemiology has been applied to identify the transmission chain and sexual network of sexually transmitted diseases (STDs) (3, 4, 7, 8). Molecular typing methods such as pulsed-field gel electrophoresis-based methods, antimicrobial susceptibility patterns, porB gene sequencing, and opa typing have been used to assess the clonal relatedness of N. gonorrhoeae strains (9, 15, 16). Recently, N. gonorrhoeae multiantigen sequence typing (NG-MAST) of two genes (por and tbpB) has been developed to assess the genetic diversity of N. gonorrhoeae (4). NG-MAST is simple, fast, and highly discriminatory and is accessible via a central database on the Internet. The digital and interlaboratory-compatible nature of NG-MAST typing is helpful for global surveillance.

In this study, we used NG-MAST to assess the genotype distribution of N. gonorrhoeae in Taiwan and to analyze the association of certain genotypes with groups at high risk of infections as well as antibiotic resistance characteristics. The data obtained in this study will contribute to identifying core groups for intervention as well as elucidating the domestic and international transmission of resistant clones within specific sexual networks.

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Patients. During the study period (April 2006 to August 2007), a total of 149 patients, including 139 males and 10 females, were diagnosed with gonorrhea at the Branch for Disease Control and Prevention, Taipei City Hospital, Taiwan. Due to the low percentage of female patients in the population, this study focused mainly on the subset of the male population with gonorrhea. The hospital is oriented to the treatment and control of STDs and the prevention of the transmission of human immunodeficiency virus (HIV). In addition, this hospital offers counseling and a nursing clinic for STD patients. Epidemiological data, including age, sexual orientation, and coinfections with other STDs (HIV and syphilis), were recorded.

Bacterial strains. N. gonorrhoeae isolates were recovered from the urethral discharge specimens of patients with gonorrhea. The urethral discharge was plated onto Thayer-Martin agar (Creative Microbiologicals, Taipei County, Taiwan) in the clinic and incubated for 36 to 48 h at 37°C in a 5% CO₂ atmosphere. N. gonorrhoeae isolates were identified by colony morphology, a positive oxidase test, a positive superoxol test, and a Gram stain smear with gram-negative diplococci. These clinical isolates were suspended in tryptic soy broth (Becton Dickinson, Cockeysville, MD) medium containing 20% glycerol and were stored at –80°C until tested.

Antimicrobial susceptibility tests. Antimicrobial susceptibility testing was performed and defined by the disk diffusion test as recommended by the Clinical and Laboratory Standards Institute (CLSI). Resistance profiles of all 146 N. gonorrhoeae isolates to seven antimicrobial agents (penicillin, cefixime, cefpodoxime, ceftriaxone, tetracycline, ciprofloxacin, and spectinomycin) were surveyed by the disk diffusion test (BBL Sensi-Disc; BD).

Molecular typing. Genomic DNA of N. gonorrhoeae clinical isolates was extracted using the QIAamp DNA mini kit (Qiagen, Hilden, Germany). The NG-MAST molecular typing for N. gonorrhoeae genotyping was conducted by the sequencing of internal fragments of two highly polymorphic loci, por and tbpB (11). The sequence data of por and tbpB were uploaded onto the database of the MAST website (www.ng-mast.net) to obtain the allele number and the sequence type (ST).

Statistical analysis. We used the chi-square tests of SPSS version 10.0 to establish relationships between STs and clinical features (sexual orientation, HIV status, and antibiotic susceptibility). Differences for which P < 0.05 were considered statistically significant.

Top ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

 
From April 2006 to August 2007, a total of 146 N. gonorrhoeae isolates were collected from 139 male patients who were diagnosed with gonorrhea. The median age of the patients infected with N. gonorrhoeae was 34 years (range, 18 to 81 years); 64.0% of the patients were adults 21 to 40 years old. Of the 139 patients, 132 (95%) provided information of their sexual orientation. It was shown that 65.2 (86/132) and 43.8% (46/132) of these patients identified as heterosexual and men who have sex with men (MSM)/bisexual, respectively. However, the numbers of MSM/bisexual patients could be underestimated. The percentage of patients coinfected with HIV, syphilis, or both were 15.8 (22/139), 12.2 (17/139), and 5% (7/139), respectively.

In this study, the isolates were divided by NG-MAST into 71 STs, of which 21 STs contained 2 to 21 isolates and the remaining 50 (34.2%) STs had only one isolate. The most common STs were ST547 (14.4%), ST835 (7.5%), ST2180 (4.8%), and ST2148 (4.1%), which constituted 30.8% of the isolates (Fig. 1). The isolates belonging to ST547, ST835, and ST2180 were significantly more likely to be from the MSM population and HIV-positive patients than the isolates that belonged to other STs (P < 0.001) (Table 1). In contrast, 73.2% of STs with fewer than four isolates and 100% of the isolates that belonged to ST2148, ST2149, and ST421 were recovered from heterosexuals (Fig. 1).

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FIG. 1. Distribution of NG-MAST STs of the N. gonorrhoeae isolates (n = 146) and the sexual orientation of the patients. Other STs were represented by fewer than four isolates.

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TABLE 1. Characteristics of patients infected with isolates that belonged to the three most prevalent STs and other STs

Seven paired isolates were collected from seven patients who were reinfected with gonorrhea within 2 weeks to 11 months. NG-MAST analysis showed that three patients were reinfected with the same ST, and the remaining four patients were reinfected with a different ST. According to the medical records, clinical treatments were successful.

The antibiotic susceptibility testing of 146 isolates was performed by disk susceptibility tests. Overall, 61.6% of isolates were resistant to penicillin, 90.4% were resistant to tetracycline, and 76.7% were resistant to ciprofloxacin. Resistance to cefixime and cefpodoxime was 16.4 and 21.2%, respectively. All isolates were susceptible to ceftriaxone and spectinomycin.

The antibiotic susceptibility profiles of the isolates were further analyzed by NG-MAST (Table 1). Of the 21 isolates belonging to ST547, 20 (95.2%) were susceptible or intermediate to all of the antibiotics except tetracycline. Furthermore, only 11.6% (17/146) of isolates were susceptible to ciprofloxacin, and 64.7% (11/17) of these isolates belonged to ST547. In contrast, all isolates of ST835 and ST2180 were resistant to ciprofloxacin and tetracycline and significantly more likely to be resistant to cefixime and cefpodoxime than the isolates that belonged to the other STs (P < 0.001). Isolates of both ST835 and ST2180 comprised 54.2 (13/24) and 51.6% (16/31) of the isolates resistant to cefixime and cefpodoxime, respectively.

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The increase of the incidence of gonorrhea and the emergence of resistant strains have become major concerns (2, 5, 6). Molecular epidemiology studies are useful in the identification of the groups at high risk of infection that are responsible for such an increase and in the identification of the transmission network. In this study, a gene sequence-based typing method, NG-MAST, with high discriminatory power was applied to determine the genotypes of N. gonorrhoeae isolates recovered from patients in Taiwan. NG-MAST identified 71 STs, of which 21 STs contained more than two isolates, with ST547, ST835, ST2180, and ST2148 being the predominant genotypes. The large ST clusters suggested that multiple clonal transmissions existed in Taiwan. The remaining 50 STs had only one isolate each, which may be due to the limited strain collection, the local emergence of new STs, or the recent introduction of foreign STs. For the seven patients with paired isolates, three were reinfected with the same ST, which may indicate reinfection from the same sexual contact, while four had different STs, implying additional infection from another sexual contact. There was no correlation between the time interval and the likelihood of acquiring a different ST.

NG-MAST typing allows the tracing of the international transmission of N. gonorrhoeae clones via groups at high risk of infection. Genotypes in this study, including ST547, ST835, and others (ST225, ST304, ST340, ST419, ST421, ST437, ST566, ST766, and ST1412), have been reported in other countries or regions, e.g., England, Hong Kong, and Australia (http://www.ng-mast.net) (4, 12). The most prevalent ST, ST547, has been reported to be one of the seven major genotypes in MSM in the United Kingdom (4). Our data for the genotyping surveillance of N. gonorrhoeae also indicate that the isolates that belonged to ST547, ST835, and ST2180 were recovered mainly from MSM patients and patients coinfected with HIV. The results in this study could help to alert patients of their possible involvement in a high-risk sexual network and to identify core groups for future intervention.

The emergence and spread of quinolone-resistant gonococci in recent years in many countries has become a therapeutic challenge. In 2005, a 95.2% rate of ciprofloxacin resistance of N. gonorrhoeae was reported in Taiwan (9). A similar trend was observed in this study, with a ciprofloxacin resistance rate of 76.7%. Recently, the expanded-spectrum cephalosporins were increasingly used as alternative antibiotics for the treatment of N. gonorrhoeae and have retained high efficacy. However, increased resistance to these antibiotics has been increasingly reported (14). In this study, a higher rate of clinical isolates with resistance to the oral cephalosporin, cefixime, has been observed (16.4% in 2007 and 9% in 2003) compared to the rate in a previous report in Taiwan (9). Although the susceptibility of these isolates to injectable ceftriaxone was still 100% in Taiwan and worldwide, the increasing frequency of gonococci with reduced susceptibility to this expanded-spectrum cephalosporin should be closely monitored.

The major ST clones exhibited distinct resistance profiles. It was found that 95.2% of the isolates belonging to ST547 were susceptible or intermediate to all of the antibiotics except tetracycline. Isolates belonging to ST547 also constitute 64.7% (11/17) of the isolates with susceptibility to ciprofloxacin. In light of the high ciprofloxacin resistance rate of clinical isolates in Taiwan, it is probable that ST547 was introduced to Taiwan through MSM groups. Our data also showed that all clinical isolates that belonged to two major genotypes, ST835 and ST2180, were resistant to tetracycline and ciprofloxacin and also showed high resistance to penicillin, cefpodoxime, and cefixime. There were significant differences between the two STs (ST835 and ST2180) and other STs with respect to the prevalence of resistance to cefpodoxime and cefixime.

This study is the first report on the genotype distribution in relation to the antimicrobial susceptibility of N. gonorrhoeae in Taiwan. The propensity of groups at high risk of infection to serve as spatial bridges to import or export specific clones should be continuously monitored. The emergence and transmission of N. gonorrhoeae isolates resistant to expanded-spectrum cephalosporins, especially in groups at high risk of infection, deserve further attention. Such data will be helpful in identifying groups at high risk of infection as well as the international transmission routes of resistant strains among such groups.

 
This work was supported by grants DOH96-DC-2017 and DOH97-DC-2008 from the Centers for Disease Control, Department of Health, Taiwan.

 
* Corresponding author. Mailing address: Research and Diagnostic Center, Centers for Disease Control, No. 161, Kun-Yang Street, Taipei, Taiwan. Phone: 886-2-26531388. Fax: 886-2-26513572. E-mail: syl{at}cdc.gov.tw

Published ahead of print on 8 October 2008.

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1
1. Anonymous. 2006. Surveillance of antibiotic resistance in Neisseria gonorrhoeae in the WHO Western Pacific Region, 2005. Commun. Dis. Intell. 30:430-433.[Medline]
2
2. Centers for Disease Control, Department of Health, Taiwan. 2006. Statistics of communicable diseases and surveillance report Republic of China, 2005. http://www.cdc.gov.tw.
3
3. Chen, Y. M., Y. C. Lan, S. F. Lai, J. Y. Yang, S. F. Tsai, and S. H. Kuo. 2006. HIV-1 CRF07_BC infections, injecting drug users, Taiwan. Emerg. Infect. Dis. 12:703-705.[Medline]
4
4. Choudhury, B., C. L. Risley, A. C. Ghani, C. J. Bishop, H. Ward, K. A. Fenton, C. A. Ison, and B. G. Spratt. 2006. Identification of individuals with gonorrhoea within sexual networks: a population-based study. Lancet 368:139-146.[CrossRef][Medline]
5
5. Fenton, K. A., and J. Imrie. 2005. Increasing rates of sexually transmitted diseases in homosexual men in Western Europe and the United States: why? Infect. Dis. Clin. N. Am. 19:311-331.[CrossRef][Medline]
6
6. Fenton, K. A., and C. M. Lowndes. 2004. Recent trends in the epidemiology of sexually transmitted infections in the European Union. Sex Transm. Infect. 80:255-263.[Abstract/Free Full Text]
7
7. Hjorth, S. V., E. Bjornelius, P. Lidbrink, L. Falk, B. Dohn, L. Berthelsen, L. Ma, D. H. Martin, and J. S. Jensen. 2006. Sequence-based typing of Mycoplasma genitalium reveals sexual transmission. J. Clin. Microbiol. 44:2078-2083.[Abstract/Free Full Text]
8
8. Hsu, M. C., P. Y. Tsai, K. T. Chen, L. H. Li, C. C. Chiang, J. J. Tsai, L. Y. Ke, H. Y. Chen, and S. Y. Li. 2006. Genotyping of Chlamydia trachomatis from clinical specimens in Taiwan. J. Med. Microbiol. 55:301-308.[Abstract/Free Full Text]
9
9. Hsueh, P. R., S. P. Tseng, L. J. Teng, and S. W. Ho. 2005. High prevalence of ciprofloxacin-resistant Neisseria gonorrhoeae in Northern Taiwan. Clin. Infect. Dis. 40:188-192.[CrossRef][Medline]
10
10. Martin, I. M., S. Hoffmann, and C. A. Ison. 2006. European surveillance of sexually transmitted infections (ESSTI): the first combined antimicrobial susceptibility data for Neisseria gonorrhoeae in Western Europe. J. Antimicrob. Chemother. 58:587-593.[Abstract/Free Full Text]
11
11. Martin, I. M., C. A. Ison, D. M. Aanensen, K. A. Fenton, and B. G. Spratt. 2004. Rapid sequence-based identification of gonococcal transmission clusters in a large metropolitan area. J. Infect. Dis. 189:1497-1505.[CrossRef][Medline]
12
12. Palmer, H. M., H. Young, I. M. Martin, C. A. Ison, and B. G. Spratt. 2005. The epidemiology of ciprofloxacin resistant isolates of Neisseria gonorrhoeae in Scotland 2002: a comparison of phenotypic and genotypic analysis. Sex Transm. Infect. 81:403-407.[Abstract/Free Full Text]
13
13. Tanaka, M., H. Nakayama, K. Huruya, I. Konomi, S. Irie, A. Kanayama, T. Saika, and I. Kobayashi. 2006. Analysis of mutations within multiple genes associated with resistance in a clinical isolate of Neisseria gonorrhoeae with reduced ceftriaxone susceptibility that shows a multidrug-resistant phenotype. Int. J. Antimicrob. Agents 27:20-26.[CrossRef][Medline]
14
14. Tapsall, J. W. 2005. Antibiotic resistance in Neisseria gonorrhoeae. Clin. Infect. Dis. 41(Suppl. 4):S263-S268.[CrossRef][Medline]
15
15. Unemo, M., H. M. Palmer, T. Blackmore, G. Herrera, H. Fredlund, A. Limnios, N. Nguyen, and J. Tapsall. 2006. Global transmission of prolyliminopeptidase-negative Neisseria gonorrhoeae strains: implications for changes in diagnostic strategies. Sex Transm. Infect. 83:47-51.[CrossRef][Medline]
16
16. Whiley, D. M., E. A. Limnios, S. Ray, T. P. Sloots, and J. W. Tapsall. 2007. Diversity of penA alterations and subtypes in Neisseria gonorrhoeae strains from Sydney, Australia, that are less susceptible to ceftriaxone. Antimicrob. Agents Chemother. 51:3111-3116.[Abstract/Free Full Text]

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Journal of Clinical Microbiology, December 2008, p. 3931-3934, Vol. 46, No. 12
0095-1137/08/$08.00+0     doi:10.1128/JCM.00577-08
Copyright © 2008, American Society for Microbiology. All Rights Reserved.

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• Pandori, M., Barry, P. M., Wu, A., Ren, A., Whittington, W. L. H., Liska, S., Klausner, J. D. (2009). Mosaic Penicillin-Binding Protein 2 in Neisseria gonorrhoeae Isolates Collected in 2008 in San Francisco, California. Antimicrob. Agents Chemother. 53: 4032-4034 [Abstract] [Full Text]  
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This Article Abstract Full Text (PDF) Alert me when this article is cited Alert me if a correction is posted Services Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Reprints and Permissions Copyright Information Books from ASM Press MicrobeWorld Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Wong, W.-W. Articles by Li, S.-Y. Search for Related Content PubMed PubMed Citation Articles by Wong, W.-W. Articles by Li, S.-Y.