Neisseria gonorrhoeae is one of the important pathogens of sexually transmitted infections. Representative infections caused by N. gonorrhoeae include male urethritis, female cervicitis, pharyngeal infection, rectal infection, and conjunctivitis. After clinical strains of N. gonorrhoeae became resistant to penicillin, tetracyclines, fluoroquinolones, and oral third-generation cephalosporins were no longer recommended for the treatment of gonorrhea because of decreased susceptibilities to these agents.1–5 Furthermore, in 2009, the high-level ceftriaxone-resistant strain H041 was isolated in Japan.6 Thus, gonococcal infections are difficult diseases to treat because of their multidrug antimicrobial resistance. In this situation, monitoring of the antimicrobial resistance of N. gonorrhoeae for disease management and control or to establish treatment guidelines is important.7 The outbreak of antimicrobial-resistant N. gonorrhoeae, for example, that resistant to oral quinolone or third-generation cephalosporin, has begun in Asia and in particular in Japan. Therefore, it is very important that we understand the trend of antimicrobial resistance of N. gonorrhoeae in both Asia and Japan. Nationwide surveillance conducted by the surveillance committee of 3 Japanese societies, the Japanese Association of Infectious Diseases, the Japanese Society of Chemotherapy, and the Japanese Society of Clinical Microbiology, has been performed only twice; however, the number of tested N. gonorrhoeae isolates was small, 83 in 2009–2010 and103 in 2012–2013. Our large-scale continuous surveillance of the antimicrobial susceptibility of N. gonorrhoeae began in 2000 under the guidance of the Department of Urology, Gifu University. We report our surveillance data from 2000 to 2015.
MATERIALS AND METHODS
We collected N. gonorrhoeae strains isolated from patients with gonococcal infections who visited our cooperating medical institutions in the regions of Hokkaido, Miyagi, Tokyo, Saitama, Shizuoka, Aichi, Gifu, Ishikawa, and Shiga in Japan from 2000 to 2015. Neisseria gonorrhoeae was identified and confirmed by the Gonochek-II Reagent Tubes (EY Laboratories Inc., San Mateo, CA). If the result of the Gonochek-II kit was in doubt, we performed the NH-20 Rapid “Nissui” ID test (NISSUI Pharmaceutical Co., Ltd., Tokyo, Japan) to confirm N. gonorrhoeae. In the very few cases in which N. gonorrhoeae still could not be determined, we used in-house PCR for identification (details not shown). MICs of penicillin G (Wako Pure Chemical Industries, Ltd., Osaka, Japan), cefixime (Astellas Pharma, Tokyo, Japan), ceftriaxone (Chugai Pharmaceutical Co., Ltd., Tokyo, Japan), tetracycline (Wako Pure Chemical Industries, Ltd.), spectinomycin (LKT Laboratories, Inc., St. Paul, MN), azithromycin (Sigma-Aldrich, St. Louis, MO), and levofloxacin (Daiichi Sankyo Company Ltd., Tokyo, Japan) were determined by the agar dilution method approved by the Clinical and Laboratory Standards Institute (CLSI).8 We tested the antimicrobial susceptibility ranging from 0.001 to 512 mg/L for each drug. The N. gonorrhoeae American Type Culture Collection 49226 strain was used as the quality control strain in each test. The breakpoints defined by the CLSI were used in this study.9 Because the breakpoint of levofloxacin is not defined by the CLSI, we used one half of the ofloxacin breakpoint as the levofloxacin breakpoint. The breakpoint of azithromycin is also not defined by the CLSI but is defined in European Committee on Antimicrobial Susceptibility Testing (EUCAST).10 In general, if a testing method is different, another breakpoint cannot be indicated. However, EUCAST has not yet recommended a specific method or medium for antimicrobial susceptibility testing of N. gonorrhoeae. Therefore, EUCAST recommends that the method or medium for testing the antimicrobial susceptibility of N. gonorrhoeae follows existing national or international guidelines, such as those of the CLSI. Therefore, we used the EUCAST breakpoints of azithromycin in this study. Because there are only a few cases of treatment failure using ceftriaxone for gonococcal infection at present, the true breakpoint for ceftriaxone against N. gonorrhoeae is unknown and remains controversial. Therefore, we chose to use both the CLSI and EUCAST breakpoints in this study.
This study was approved by the Institutional Review Board of the Graduate School of Medicine, Gifu University, Gifu, Japan (no. 28-81).
Susceptibilities of 2471 isolates of N. gonorrhoeae to each drug were evaluated and are listed in Table 1. Strains were isolated from the urethra (n = 2317), pharynx (n = 79), and cervix (n = 75). Results of antimicrobial susceptibility against each antimicrobial agent are listed in the supplemental data contents, https://links.lww.com/OLQ/A144. Most of the strains isolated throughout the study period were not susceptible to penicillin G. The highest rate of isolation of strains susceptible to penicillin G was 2.2% in 2003. The strains not susceptible to cefixime were isolated at a rate of around 20% to 40% (Fig. 1). In 2015, the rate of the strains not susceptible to cefixime was 27.9%. The rates of nonsusceptible strains against ceftriaxone by EUCAST and CLSI are shown in Figure 2. None of isolates had a MIC >1 mg/L against ceftriaxone throughout the study period. However, 6 strains with a MIC of 0.5 mg/L against ceftriaxone were isolated: 1 strain from a male urethra in 2003, 3 strains (2 strains from male urethras and 1 strain from a male pharynx) in 2011, 1 strain each from a male urethra in 2014 and in 2015. Most of the strains showed nonsusceptibility to tetracycline. From 2000 to 2013, the rate of tetracycline-susceptible strains remained around 10% to 20%. However, these strains began to increase from 2014, and the rate for such strains had risen to 28.3% in 2015. Throughout the study period, most strains were susceptible to spectinomycin (Fig. 3). Only 2 strains, 1 strain each in 2001 and 2002, showed resistance to spectinomycin. Antimicrobial susceptibility testing of azithromycin for N. gonorrhoeae was started from 2002, and in 2002 and 2003, the rates of azithromycin-susceptible strains were both >90%. However, the rate of such strains suddenly decreased to 40% in 2004 and remained at around 40% to 60% thereafter. In 2015, the rate was 47.4%. Furthermore, N. gonorrhoeae strains showing a MIC against azithromycin of >1 mg/L have been isolated continuously at a rate of around 10% to 40% since 2004, and 51 strains (18.8%) were isolated in 2015 (Fig. 4). Strains of N. gonorrhoeae not susceptible to levofloxacin were isolated at a rate of around 70% to 90% over the study period.
The rates of multidrug-resistant strains against ceftriaxone, azithromycin, and levofloxacin are shown in Table 2. None of the strains had resistance only to both ceftriaxone and azithromycin. The number of strains resistant to all 3 drugs by the EUCAST breakpoint was low and did not increase significantly. In addition, most of the strains were not resistant to all 3 drugs by the CLSI breakpoint.
The rates of resistance according to the isolation site are shown in Table 3. The rates of resistance of the strains isolated from the pharynx were not higher than those of the strains isolated from the urethra or cervix.
Because of the increased antimicrobial resistance to penicillin G, tetracycline, and levofloxacin, these drugs cannot be used in the treatment of gonorrhea in Japan, as reported elsewhere.11,12 Furthermore, about 30% of the strains were not susceptible to cefixime. Therefore, cefixime also cannot be used to treat gonorrhea because a cure rate >95% is needed for sexually transmitted infections.13 In this situation, the recommendation of oral third-generation cephalosporins has already been halted in each country or region's guideline for gonorrhea.14,15 Spectinomycin still maintains good antimicrobial activity against N. gonorrhoeae. However, spectinomycin is not recommended as first-line therapy for gonorrhea because the clinical efficacy of spectinomycin is low for N. gonorrhoeae pharyngeal infection due to lower tissue penetration.16 Therefore, therapies based on ceftriaxone and/or azithromycin as the recommended drugs are considered for gonorrhea treatment in each country or region’s guidelines.15 Unfortunately, the emergence of N. gonorrhoeae strains with resistance to these drugs has been reported throughout the world and is now becoming a serious public health problem.15
Azithromycin has antimicrobial activity for N. gonorrhoeae. A 2-g dose of extended-release azithromycin can be used to treat gonococcal infections because it has an indication for gonococcal infection in Japan, but it is not recommended in the Japanese guideline. However, clinical strains with decreased susceptibility to azithromycin have emerged in many countries worldwide, and clinical strains with high-level resistance to azithromycin have been reported in several countries.17–20 In this study, the rate of strains resistant to azithromycin suddenly increased to 60% in 2004. Azithromycin has been available in Japan for chlamydial urethritis and cervicitis since 2004; however, we do not know whether this event was associated with the decreased susceptibility to azithromycin. The breakpoint of azithromycin for N. gonorrhoeae has not yet been defined clearly and is not described by the CLSI. When a 2-g dose of azithromycin is used in monotherapy, susceptibility is defined as a MIC of ≤0.25 mg/L, and resistance is defined as a MIC of ≥0.5 mg/L in EUCAST. However, there is little evidence for these values. We previously reported the efficacy of a 2-g dose of extended-release azithromycin with pharmacokinetics equal to a 1.5-g dose of the immediate-release formation for gonococcal urethritis.21 In that report, about half of the N. gonorrhoeae strains with a MIC of 1 mg/L were not eradicated. In the present study, around 10% to 40% of the strains with a MIC of 1 mg/L were isolated since 2004, and 18.8% were isolated in 2015. Therefore, monotherapy with a 2-g dose of either immediate-release or extended-release azithromycin is not recommended by our antimicrobial susceptibility surveillance.
Ceftriaxone is effective for gonococcal infections including urethritis and pharyngeal infection. High-level ceftriaxone-resistant strain H041 was isolated from the pharynx of a female commercial sex worker in Japan.6 Afterward, F89 ceftriaxone-resistant strains similar to the H041 strain were reported from France and Spain.22,23 Therefore, isolates having a ceftriaxone MIC of 0.5 mg/L were reported.24,25 In our study, 6 strains with a ceftriaxone MIC of 0.5 mg/L against N. gonorrhoeae were isolated. We reported the characteristics of 5 of these strains previously.26,27 Fortunately, they were isolated sporadically, and genotypes and alterations of PBP2 were each different again. The rate of strains with a ceftriaxone MIC of 0.25 mg/L was 12.9% in 2015. The resistance criteria are different between the CLSI (MIC ≥0.5 mg/L) and EUCAST (MIC ≥0.25 mg/L) guidelines. A ceftriaxone dose of 250–500 mg is recommended to treat gonorrhea by many guidelines around the world. Pharmacodynamic studies have predicted that treatment failures with 250-mg or 500-mg doses of ceftriaxone in monotherapy are likely for strains with MICs of 0.5 mg/L.28 However, there was 1 report of treatment failure with 250-mg and 500-mg ceftriaxone therapy against a N. gonorrhoeae pharyngeal infection with a ceftriaxone MIC of 0.25 mg/L. Therefore, a MIC ≥0.25 mg/L should be used as the resistance criterion when considering the ceftriaxone dose recommended for worldwide use and for pharyngeal infection. The increase of strains with a ceftriaxone MIC of 0.25 mg/L in this study is a serious problem. Fortunately, a single 1-g dose of ceftriaxone intravenously or a single 2-g dose of spectinomycin intramuscularly is recommended in the Japanese guideline to treat gonorrhea.14 Dual therapy as performed in the United States and Western Europe is not recommended in Japan. Pharmacodynamic analyses showed that strains exhibiting MICs up to 0.5 mg/L were eradicated by the single-dose regimen of ceftriaxone 1 g.28 In a previous study, a single 1-g dose of ceftriaxone was effective against gonococcal infection,29,30 and we experienced eradication of strains with a MIC of 0.5 mg/L against N. gonorrhoeae treated with a single 1-g dose of ceftriaxone (data not shown).
This study includes some limitations. This surveillance may include sampling bias according to the local bias of the cooperating medical institutions in Japan, but the main medical institutions cooperating in this surveillance did not change throughout the study period. We did not perform statistical analysis during the entire study period to evaluate such bias. However, the participating medical institutions showed regional disparities in the nationwide surveillance performed twice before.11,12 Nevertheless, this surveillance has been continued on a large scale for many years, and we believe its quality to be as high as that of the national surveillance. Recently, molecular typing was carried out by one such surveillance. Although molecular typing of N. gonorrhoeae is not currently performed in the present surveillance, we expect that it will be included in the future.
In conclusion, the results of this surveillance showed that strains with a 0.25-mg/L MIC of ceftriaxone were isolated at rates of around 5% to 10% throughout the study period, and a 0.5-mg/L MIC of ceftriaxone was isolated in 6 strains. Pharmacodynamic analyses indicated that a single 1-g dose of ceftriaxone is recommended to treat gonorrhea. However, the trend of antimicrobial-resistant N. gonorrhoeae, especially that for ceftriaxone, will require further investigation in the future. As strains with high-level ceftriaxone resistance continue to spread, higher doses of ceftriaxone in monotherapy or multiple doses of ceftriaxone should be considered.
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