METHODS

Bacterial isolates

In all, 793 consecutive isolates of N gonorrhoeae were collected from outpatients with gonorrhoea attending STD clinics of Guangdong Provincial Centers for STIs and Skin Diseases Control and Prevention, and Guangzhou Center for STD Control and Prevention from 1996 to 2001 in Guangzhou, China.

Isolation of N gonorrhoeae

Urethral and endocervical specimens were directly inoculated on Thayer-Martin medium (GC agar base + haemoglobin + growth supplement + VCNT) and GC agar supplemented with 10% defibrinated sheep blood. The plates were incubated at 35°C in a humid candle extinction jar for 36–48 hours. The isolates were identified as N gonorrhoeae on the basis of colony morphology, Gram staining, oxidase test, and carbohydrate degradation tests. All stains were preserved in skimmed milk and stored at −70°C before use.

Antibiotic susceptibility

All the isolates were examined for susceptibility to penicillin G, ciprofloxacin, ceftriaxone, and spectinomycin by the agar plate dilution method for minimum inhibitory concentrations (MICs).⁵ The ranges of concentration of antibiotics were as follows: penicillin G, 0.31–32 μg/ml; tetracycline, 0.5–32 μg/ml; spectinomycin, 4–128 μg/ml; ciprofloxacin, 0.15–32 μg/ml and ceftriaxone, 0.002–1 μg/ml. The sensitivity of isolates was determined according to agar plate dilution methods similar to those in use by the Western Pacific WHO GASP programme as follows. The medium used was GC agar (Oxoid) supplemented with 10% defibrinated sheep blood. Pure subculture from 18 to 24 hours’ growth on GC agar was suspended in saline and adjusted to contain approximately 10⁸ cfu/ml as inoculum. A multipoint inoculator was used to inoculate the suspension onto antibiotic containing media. After overnight incubation at 35°C in a carbon dioxide incubator, the MICs were read.

Interpretation of susceptibility

Two methods were used in AHPWPR, one is the American method and other the non-American method. We apply the latter method, modified by using sheep blood instead of lysed horse blood. The laboratory obtained satisfactory results in the China GASP QA programme external quality assurance programme conducted by the Nanjing National Reference STD centre. The American method uses MIC ⩾2 mg/l as penicillin resistance, while the non-American method standard is 1 mg/l. The criteria for resistance in this test system are shown in table 1.

WHO N gonorrhoeae reference strains A–E, kindly provided by Dr Ye Shunzhang, the National Center for STD control, Nanjing, were used as controls in each antimicrobial assay. Because these reference strains are all quinolone sensitive, we also reference the external QA results as control.

A β lactamase assay was performed by the paper acidometric method.⁶ WHO reference strains A and E were used as negative and positive control, respectively.

Statistics

Geometric MICs were determined using the base₂logarithms. The Mann-Whitney U test was used for evaluating the trends of the resistant rates.

RESULTS

Among the 793 gonococcal isolates examined, 55 PPNG strains were identified by the paper acidometric method. The prevalence dramatically increased from 2% to 21.8% in 1996–2001 (U=6.38, p<0.01) (table 2).

The distribution of MIC₅₀, MIC₉₀, and MICmean of the four antibiotics to 793 consecutive strains collected in Guangzhou during these 6 years is summarised in table 3. Out of the four antibiotics tested, ceftriaxone and spectinomycin appeared to be the most effective agents and the MICs remained stable over the years, while MICs of penicillin G and ciprofloxacin enlarged dramatically. MIC₅₀, MIC_90,and MICmean of penicillin G multiplied by 2, 8, and 3.5 times from 1996 to 2001, respectively; those of ciprofloxacin by 33, 16, and 18.7 times higher in 1996–9, respectively, and declined slightly in 2000–1.

Antimicrobial susceptibilities of 793 strains, interpreted according to WHO standards, were shown in table 2. It appeared that the strains were more sensitive to ceftriaxone and spectinomycin. Only two spectinomycin resistant gonococcal strains were isolates during the 6 years. The proportion of sensitive strains to penicillin decreased from 8.4% to 0.9%, while the resistant rate increased from 57.2% to 81.8% (U=4.39, p<0.01). A significant trend for increasing resistance to ciprofloxacin developed dramatically from 1996 to 2001. The proportion of sensitive organisms dropped from 18.6% to 1.8%, while the resistant rate rose steeply from 17.6% to 72.7% (U=9.67, p<0.01).

DISCUSSION

The antibiotic resistant profile changes constantly over time. Since the emergence of PPNG in 1976 and the chromosomally mediated penicillin resistant isolate in 1983, the resistant gonococci have spread worldwide and the prevalence increased rapidly. In south east Asian, very high proportions of penicillin resistance were recorded—Korea (91%), the Philippines (89%), China (80%), Singapore (58%), Hong Kong (54%), and Vietnam (48%) in 2000.⁷ We identified 546 penicillin resistant strains out of 793 consecutive isolates in Guangzhou, in which 55 were PPNG and 491 chromosomally mediated penicillin resistant. The prevalence of PPNG were lower during the first 4 years but rose steeply from 5.4% to 21.8% (U=4.2, p <0.01) in the past 2 years, indicating that Guangzhou had become a highly prevalent region. The penicillin resistant rates increased from 57.2% to 72.7% in 1996–2001 and were higher than the report (66.7%) by the National Center for STD Control.³ The resistant trend was statistically significant (U=4.39, p <0.01). The MIC₅₀, MIC₉₀and MICmean in 2001 were 2, 8, and 3.5 times higher than those in 1996, respectively. A high rate (21.8%) of PPNGs in 2001 might cause the MIC increase. The high prevalence of PPNGs may cause an increase of penicillin MIC and, in addition, the widespread use of penicillin for treatment might also have contributed to the findings of a steep increase in syphilis infection in Guangzhou in recent years.

Quinolones had been recommended by the Centers For Disease Control and Prevention (CDC) for treatment of gonorrhoea since 1989.⁸ With widely use of these agents, resistance has sharply increased in many districts of south east Asia and the resistant strains have spread recently. In Hong Kong, quinolone resistant N gonorrhoeae (QRNG) increased from 0.5% in 1992 to 10.4% in 1994⁹ and from 60% in 1998 to 80% in 2000.⁷ Other information identified an increasing number of QRNG in Japan.¹⁰ A report from Sydney, Australia, represented 3% QRNG from 1991 to 1994, and in the first 6 months of 1995, the proportion (8%) of QRNG rose substantially.¹¹ It was considered that the appearance of QRNG in Sydney has been caused by the continuing importation of resistant strains by travellers entering or returning to Australia from Asian destinations. In Guangzhou, our finding was similar. There had been a significant increase in the rate of ciprofloxacin resistant N gonorrhoeae from 17.6% in 1996 to 72.7% (U=9.67, p <0.01) in 2001. A WHO document stated that an agent should be withdrawn from treatment regimens if the rate of 5% resistance to the agent occurs.¹² The MIC₅₀, MIC_90,and MICmean multiplied by 33, 16, and 18.7 times in 1996–9, respectively, and then declined slightly in 2000–1. A recent study on the correlation between the in vitro susceptibility to ciprofloxacin and treatment outcome showed that 96% isolates from patients who had treatment failure were MICs 1–32 μg/ml.¹³ The emergence and wide spread of the highly ciprofloxacin resistant strains (MIC ⩾32 μg/ml) might mean no dose of this agent (or other quinolones) would effect a cure.

Spectinomycin and ceftriaxone are highly effective as single dose treatment for gonorrhoea. Susceptibility to ceftriaxone was proved to have risen slightly in recent years.¹⁴ In Guangzhou, the susceptibilities to both of the antibiotics remained unchanged from 1996 to 2001. The MIC₅₀, MIC₉₀, and MICmean all fell in the sensitive range of the interpretative criteria although two spectinomycin resistant strains were isolated in 1996. The results showed that spectinomycin and ceftriaxone should be the first line treatment for gonorrhoea in Guangzhou, China.

In summary, the results of this study clearly demonstrate that penicillin/ciprofloxacin resistant gonococcal strains are frequently found in Guangzhou. Local susceptibility data of periodic surveillance are useful in guiding clinicians in the use of appropriate antibiotics for the treatment of gonorrhoea.