STRATEGIES FOR CONTROL OF GONORRHEA have relied on the use of highly effective and single-dose therapy administered at the time of diagnosis. Because of the appearance and the subsequent increase in the prevalence of penicillinase-producing Neisseria gonorrhoeae (PPNG) and N gonorrhoeae isolates with chromosomally mediated resistance to penicillin and tetracycline (CMRNG-PT), in 1993 the Centers for Disease Control and Prevention advocated the use of broad-spectrum cephalosporins or fluoroquinolones as first-line therapy for uncomplicated gonorrhea. 1,2 However, ciprofloxacin resistance has been reported in the USA, UK, World Health Organization (WHO) Western Pacific Region, and Asia, 3–5 including India. 6–8 In India, including New Delhi, ciprofloxacin (500 mg) is still being used as single-dose treatment for gonorrhea, as recommended by the National AIDS Control Organization [NACO] for a syndromic approach. 9
According to the WHO's suggestion of global monitoring of resistant strains of N gonorrhoeae, a Gonococcal Antimicrobial Susceptibility Program (GASP) was established in its Southeast Asia Region (SEAR), with a coordinating laboratory at the Regional STD Teaching, Training, and Research Center of VMMC and Safdarjang Hospital, in New Delhi, India. In that center, gonorrhea accounted for 19.4% of all STD cases from 1999 to 2001. Since 1995, this center has been continuing the monitoring of antimicrobial susceptibility of isolates from patients from different parts of Delhi who have acute gonococcal urethritis. 10
The aim of the current study was to determine the antimicrobial susceptibility pattern of N gonorrhoeae isolates to different antibiotics by disc diffusion and to determine the MICs of ciprofloxacin, penicillin, and ceftriaxone against these isolates.
A total of 448 male patients with acute gonococcal urethritis attending the aforementioned Regional STD Teaching, Training, and Research Center in New Delhi during the period of January 2000 through December 2001 were included in the study. Of these patients, 321 were direct smear–positive. Culture was performed in 273 smear-positive cases, and N gonorrhoeae was isolated in 257 (94.1%).
For isolation of N gonorrhoeae, urethral swabs were inoculated directly onto chocolate agar (Columbia agar base and sheep blood) and saponin lysed blood agar with VCNT inhibitors. 11 The inoculated culture plates were incubated at 36°C in a moist atmosphere containing 5% CO2 (candle extinction jar) for 24 to 48 hours. Isolates were identified on the basis of colony morphology, Gram staining, an oxidase test, and superoxol and rapid carbohydrate utilization tests. 11
Susceptibility testing of 241 consecutive isolates (125 isolates in 2000 and 116 in 2001) was done by the Australian Gonococcal Surveillance Program (AGSP) method, 11 based on the Calibrated Dichotomous Sensitivity (CDS) test on chocolate agar plates with low-potency antibiotic discs (Oxoid, Basingstoke, UK). The discs were supplied by Dr. J. Tapsall, of the Neisseria Reference Laboratory, at the Prince of Wales Hospital (Sydney, Australia), and included penicillin (0.5 IU), tetracycline (10 μg), ceftriaxone (0.5 μg), ciprofloxacin (1 μg), spectinomycin (100 μg), and nalidixic acid (30 μg). The strains were defined as susceptible, less sensitive, and resistant (Table 1). Nalidixic acid was used only to identify strains less sensitive to ciprofloxacin, and results of susceptibility to this antibiotic are not included.
MICs of ciprofloxacin, penicillin, and ceftriaxone were determined by Etest (AB Biodisk, Solna, Sweden) on chocolate agar plates for 110, 68, and 17 consecutive isolates, respectively, according to the steps suggested by the manufacturer. The strains were labeled as susceptible (MIC, ≤0.03 μg/mL), less sensitive (MIC, 0.06–0.5 μg/mL), or resistant (MIC, ≥1.0 μg/mL) on the basis of WHO criteria. In this system, unlike others, the breakpoints are the same for all the antibiotics.
Five WHO standard strains of N gonorrhoeae A to E and provisional strains F to J were used as controls for disc diffusion and MIC testing. β-Lactamase production was detected by the chromogenic cephalosporin method with use of nitrocefin freeze-dried powder (Oxoid). 11
The antimicrobial susceptibility testing of 241 N gonorrhoeae isolates by disc diffusion in 2000 to 2001 revealed that 100% of the strains were susceptible to ceftriaxone and spectinomycin. Resistance to penicillin, tetracycline, and ciprofloxacin was observed in 125 (51.9%), 22 (9.1%), and 193 (80.1%), respectively; 51 isolates (21.2%) were found to be PPNG, and 62 (25.7%) and 3 (1.2%) were less sensitive to penicillin and ciprofloxacin, respectively. Of the 193 ciprofloxacin-resistant strains, 47 (24.4%) were PPNG, 112 (58.0%) were penicillin-resistant non-PPNG, and 34 (17.6%) were penicillin-susceptible.
On the basis of MIC values, 35.3% and 52.9% of strains were found to be resistant and less sensitive, respectively, to penicillin. In testing of ciprofloxacin, 67.3% and 28.2% of strains were observed to be resistant and less sensitive, respectively. Only one isolate (5.9%) was found to be less sensitive to ceftriaxone. The MIC values for resistant strains varied between 0.06 and 2 μg/mL (penicillin), 1 and 32 μg/mL (PPNG), and 0.06 and 32 μg/mL (ciprofloxacin).
The antimicrobial susceptibility pattern of N gonorrhoeae, based on disc diffusion with the various antibiotics in 2000 and 2001, was compared with data from previous years (i.e., 1995 to 1999). 10 All the strains were found to be susceptible to ceftriaxone and spectinomycin throughout the period. Penicillin resistance increased significantly (P < 0.001), from 6.8% in 1996 to 66.7% in 2001 (Figure 1). Even the percentage of less sensitive isolates increased from 0% in 1996 to 25% in 2001, and that of PPNG isolates increased from 3.4% in 1996 to 26.8% in 2001 (Figure 1). PPNG accounted for 43.5% of penicillin-resistant isolates between 1996 and 2001.
Tetracycline resistance remained constant at 12% from 1998 to 2001 but decreased to 7.4% in 2001. The ciprofloxacin resistance showed a significant increase (P < 0.001), from 3.4% in 1996 to 90.7% in 2001 (Figure 1).
MICs of ciprofloxacin, penicillin, and ceftriaxone against the isolates studied in the years 1996 to 1999 and 2000 to 2001 are shown in Figure 2. It was observed that there was a significant increase (P < 0.001) in ciprofloxacin-resistant strains having MICs ≥1 μg/mL, from 21.8% in the period of 1996 to 1999 to 67.3% in 2000 to 2001. For a total of 20.9%, MICs were ≥4 μg/mL. An insignificant increase (P > 0.1) in penicillin-resistant strains (MIC, ≥1 μg/mL) was also observed, from 10.9% in 1996 to 1999 to 35.3% in 2000 to 2001. For 100% of strains, ceftriaxone MICs were ≤0.03 μg/mL in 1996 to 1999, but in 2001, for 1 strain (5.9%) the MIC was ≥0.06 μg/mL.
There has been a progressive increase in the antimicrobial resistance of N gonorrhoeae worldwide, and a few reports from India based on disc diffusion data are available. 6–8,10,12 Assessment of the antimicrobial resistance is of great importance in monitoring the emergence and spread of resistance and in planning appropriate treatment regimens.
In the current study, the incidence of PPNG strains was observed to be 26.8% in 2001. In contrast, Bhalla et al in Delhi 6 and Divenkar and Gogate in Mumbai 8 reported 8% and 94% PPNG, respectively, in 1998 and 1999. Results from this center compare well with recent observations in other SEAR countries such as Bangladesh 13 and Thailand. 14 The high-level penicillin resistance observed in the current study may be due to a consequence of therapy with penicillin and/or its derivatives for unrelated illnesses, e.g., syphilis and upper respiratory tract infections.
In the WHO SEAR, tetracycline-resistant N gonorrhoeae (TRNG) poses a significant problem, particularly in Thailand 14 and Indonesia. 15 In the current study, tetracycline resistance, as determined by disc diffusion test, remained constant at 12% during 1998 to 2000 but decreased to 7.4% in 2001. This remains unexplained in spite of the drug's regular use in syndromic management of urethral discharge, as per treatment guidelines for the country. In Bangladesh, 10% of 94 isolates were TRNG in 1997, 13 and these results are comparable with the findings in our study. In contrast, Bhalla et al 6 found that 28% of isolates in New Delhi were TRNG.
Fluoroquinolones have been recommended by the WHO for use in Southeast Asia and Central Africa for the primary treatment of urethral discharge in a syndromic approach. Fluoroquinolones also have been broadly used in the treatment of other infectious diseases since the late 1980s. As a consequence of the large-scale use of this group of antimicrobials in areas where over-the-counter availability of drugs without prescription is common, a substantial increase in resistant strains may occur. The significant increase in ciprofloxacin resistance in the current study indicates that resistance has developed under selective antibiotic pressure. Level of resistance has also risen substantially, with 20.9% of strains having MICs of ≥4 μg/mL. Reports from other centers also have highlighted the dramatic increase in quinolone resistance demonstrated by the disc diffusion method as well as the increase in isolates against which the MIC of ciprofloxacin is unusually high. 13,16–22 The resistance has gradually developed, and noncompliance and prescription of dosages higher than needed probably have contributed further to the high-level resistance observed in this study. The current study revealed increases in resistance to both penicillin and ciprofloxacin. In contrast, Kam et al, in Hong Kong, 23 reported that as the proportion of ciprofloxacin-resistant isolates increased, the proportion of PPNG isolates decreased.
The detection of increasing MICs and less sensitivity (5.9%) of isolates to ceftriaxone, as noted in this study, cannot be ignored; there have been similar reports from other countries, 13,22 and this antibiotic has been used in first-line drug therapy at this center for treatment of gonorrhea for the last 5 years. If N gonorrhoeae isolates in this country develop resistance against this antimicrobial—as per a recent report from Zhanjiang, China, 24 where 16.48% of gonococcal isolates were observed to be resistant to ceftriaxone—then a decision regarding the use of an alternative antimicrobial is urgently needed.
In the current study, the rate of sensitivity to spectinomycin was 100%, and the results are in accordance with reports from India and other SEAR countries such as Bangladesh, Thailand, and Indonesia. 8,13–15 Spectinomycin resistance was detected in China 24,25 and the Philippines. 26
This trend of increasing ciprofloxacin and penicillin resistance with MICs at alarming levels, as reported in this study, is a cause for concern. Periodic review of national treatment guidelines for gonorrhea and inclusion of alternative antimicrobials on the basis of antimicrobial susceptibility data from reference laboratories seem to be mandatory in view of the above findings.
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