Gonorrhoea, caused by Neisseria gonorrhoeae (N. gonorrhoeae), remains one of the most common sexually transmitted infections (STIs) and contributes to a substantial burden of morbidity, mortality, and infertility worldwide.1 Penicillins, tetracyclines, and fluoroquinolones were commonly used for the treatment of gonococcal infections until the emergence of resistance to these antimicrobials.2–7 Third-generation cephalosporins, both injectable and oral, are being used globally as first-line therapy. Resistance to oral third-generation cephalosporins such as cefixime and ceftibuten was reported from Japan, Hong Kong, and Taiwan.8–12 Although, treatment failures with the injectable cephalosporins like ceftriaxone have not been reported,10,13,14 decrease in susceptibility to these agents has been documented in many countries like in India, United states, counties in the World Health Organization (WHO) Western Pacific Region (Australia, Brunei, China, and Papua New Guinea), Vietnam, and Greece.13,15–18 Some of these isolates are already resistant to multiple drug classes, such as quinolones, macrolides, penicillins, and tetracyclines.13,15,18–21
The term multiresistant or “multidrug-resistant N. gonorrhoeae” (MDR-NG) has been used in many studies, but without precise definitions.13,15,18–21 Multiresistant isolates were earlier defined as quinolone-resistant N. gonorrhoeae (QRNG) and penicillinase-producing N. gonorrhoeae (PPNG); QRNG- and tetracycline-resistant N. gonorrhoeae (TRNG); QRNG, PPNG, and TRNG; QRNG and azithro resistant (AzR).3,13 Most of these antibiotics such as penicillins, tetracyclines, and fluoroquinolones are no longer used for treatment of gonorrhoea. Therefore, Tapsall et al.,22 have revised concepts for defining MDR-NG and for the first time devised the term extensively drug-resistant N. gonorrhoeae (XDR-NG). For the current definition, antibiotics have been classified into 3 categories depending on their use: category I contains antibiotics currently recommended for the treatment of gonorrhoea (injectable cephalosporins/oral cephalosporins/spectinomycin). Category II has less frequently used or else proposed for more extensive use (penicillins/fluoroquinolones/azithromycin/aminoglycosides/carbapenems) and antibiotics now superseded or else regarded as inappropriate are included in category III (chloramphenicol, thiamphenicol, tetracyclines, rifampicin, cotrimoxazole, erythromycin). MDR-NG by the revised criteria,22 are defined as those resistant to one of the antibiotic classes listed in category I, plus 2 or more in category II. XDR-NG are defined as those resistant to 2 or more of the antibiotic classes in category I and 3 or more in category II.22 An important distinction is drawn in category I between the oral and injectable forms of the cephalosporins for defining MDR-NG and XDR-NG because treatment failure due to antimicrobial resistance has been documented for the oral, but not the injectable forms of these antibiotics. These definitions with the stricter criteria applied, were derived in response to the multiple and varied definitions currently used loosely. The current study was aimed to determine the trend of MDR-NG and characterize their profile in India using the previous and current definitions of MDR-NG during the period 2000 to 2009.
A total of 764 N. gonorrhoeae consecutive isolates collected from January 2000 to December 2009 were included in this study. Methods used for isolation, identification of N. gonorrhoeae and β lactamase testing have been described previously.4,13,23 All gonococcal isolates were tested for susceptibility to ciprofloxacin, ceftriaxone, spectinomycin, penicillin, and tetracycline by the Etest ([AB Biodisk, Solna, Sweden), (media used—as recommended by manufacturer, using GC agar base with supplement]) and by the disc diffusion method using Calibrated Dichotomous Sensitivity technique (media used—Chocolate agar with Columbia agar base).24 Cefixime testing was performed by the Clinical and Laboratory Standards Institute (CLSI) technique using GC agar base with 1% isovitalex or vitamino growth supplement (Hi Media, India). Susceptibility testing for azithromycin started in November 2004 and was carried out by Etest and the CLSI disc diffusion method.25
The criteria used for phenotypic characterization of N. gonorrhoeae isolates were as follows: PPNG = β lactamase positive and non-TRNG with tetracycline minimum inhibitory concentration (MIC) <16 μg/mL; TRNG = β lactamase negative with tetracycline MIC ≥16 μg/mL; PPNG-TRNG = β lactamase positive with tetracycline MIC ≥16 μg/mL; QRNG = MIC ≥1 mg/L; high-level resistance (HLR) to ciprofloxacin = MIC ≥4 μg/mL. Calibrated Dichotomous Sensitivity breakpoints were followed for interpreting strains as susceptible and less susceptible to ceftriaxone.24,26 CLSI criteria were used to define isolates sensitive to cefixime as it does not define resistant breakpoints for it.25 Criteria used for interpretation of azithromycin resistant (AzR) was as recommended by the Neisseria Reference Laboratory at CDC27 = MIC ≥1 μg/mL and zone diameter ≤30 mm. The data from 2000 to 2009 were analyzed for trends of MDR in N. gonorrhoeae using the previous and current definitions as described earlier in the text.3,13,22
WHO reference strains A-E, G, K, L, O, and Q were used as control strains during antimicrobial susceptibility testing.26 From 2001 to 2009, this center participated in the External Quality Assurance Scheme (EQAS) of gonococcal antimicrobial susceptibility testing conducted by the Neisseria Reference Laboratory, WHO Collaborating Centre for STD, Sydney, Australia. EQAS results every year showed almost 100% agreement with the reference laboratory expected results, except some disagreement of results was observed for 1 strain for ceftriaxone in 2002, 2007, 2008, and for 1 strain for penicillin in 2004. On repeat testing, 100% agreement was observed.
The differences in different categories of MDR strains were statistically compared by determining standard error of 2 proportions, tested for significance by using Z test, and P values were determined.
Trend of MDR-NG using the previous definition is shown in Table 1. A total of 26% strains were observed to be MDR-NG and out of these, QRNG + PPNG category was maximum (16.6%), followed by QRNG + TRNG (5.4%), QRNG + PPNG + TRNG (3.9%), and QRNG + AzR (0.4%). QRNG + PPNG strains were significantly higher than QRNG + TRNG (P < 0.001), QRNG + PPNG + TRNG (P < 0.001), and QRNG + AzR (P < 0.001). Of 199 MDR strains, almost one-third that is 67 were having HLR to ciprofloxacin. HLR to ciprofloxacin in QRNG + PPNG + TRNG was significantly higher (P < 0.001) than in the QRNG + PPNG category. However, it was insignificantly higher when compared with that of QRNG + TRNG category. MDR strains increased insignificantly from 14.4% in 2000 to 36.1% in 2005 with insignificant decrease to 23.6% in 2006. Thereafter, there was significant rise (P < 0.05) to 47.1% in 2009. Even the percentage of MDR strains with HLR to ciprofloxacin increased significantly (P < 0.05) from 1.6% in 2000 to 15.7% in 2009. All the 274 strains tested for azithromycin were susceptible except one which was resistant to azithromycin in 2009, and this strain was QRNG + AzR. This strain was found to be PPNG also.
On analyzing the resistance pattern according to current definition, no strain was observed to be MDR or XDR. Only one strain resistant to spectinomycin was susceptible to all other antimicrobials. Twelve strains were less susceptible to ceftriaxone and cefixime. Out of these 12 strains, 4 were MDR by old definition, i.e., 2 strains were QRNG + PPNG, 1 was QRNG + TRNG, and 1 was QRNG + PPNG + TRNG.
The current study is the first report assessing the burden of MDR-NG using the previous and current definitions and for the first time, the current proposed WHO definitions of MDR-NG and XRD-NG have been applied to a longitudinal sample of gonococcal isolates examined in Delhi, India over a decade.
According to the old definition, 26% strains were observed to be MDR-NG. Our results are comparable with the data from Western Europe,3 where multiresistance was determined to be 21.6%.
In the current study over a period of 10 years, out of 12 isolates less susceptible to ceftriaxone, 4 (33.3%) were observed to be MDR. Similarly, in a study from Netherlands,28 30.3% strains with reduced susceptibility to cefotaxime were found resistant to multiple antibiotics such as penicillin, tetracycline, and ciprofloxacin. However, in China,29 89.9% (413 of 459) of isolates with decreased susceptibility to ceftriaxone, exhibited resistance to both penicillin and ciprofloxacin. In Australia,6 from 2001 to 2006, 134 isolates were reported with ceftriaxone MICs in the less susceptible range and most of these isolates were also multiresistant (to penicillins and quinolones).
In the current study, no strain was observed to be MDR by current WHO criteria. However, this situation of a lack of MDR is not universal. A recent study from Hong Kong described treatment failure with oral ceftibuten was of around 3.7%.11 Further, the involved N. gonorrhoeae strains were MDR-NG according to new definition, as they also manifested resistance to quinolones and penicillins. Cases with cephalosporin treatment failure were reported from Japan and were also resistant to quinolone and penicillin.8–10 These findings suggest the appearance of MDR-NG by current definition in Japan too, with the threat of emergence of these strains worldwide especially in India, Netherlands, China, Australia, United states, counties in the WHO Western Pacific Region, Vietnam, and Greece, where less susceptible ceftriaxone strains have been reported.13,15–18 The emergence and dissemination of such strains are of particular concern.
To conclude, evolution of true resistance to third-generation cephalosporins would lead to spread of MDR-NG and XDR-NG according to the current WHO definitions, as has already happened in Hong Kong and Japan and seriously hamper effective control of gonorrhoea. Current definitions seems to be a practical way forward as these are based on antibiotics currently recommended for the treatment of gonorrhoea. These should be universally applicable so that we can all make sense of the data emanating from different sources.
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