DECREASING THE PREVALENCE OF SEXUALLY transmitted diseases (STDs) is a priority in developing countries, especially in large parts of Africa and South East Asia where the prevalence of HIV and STDs is now high.1,2 In resource-poor settings, and particularly in most developing countries, the diagnosis and control of STDs has been hampered by the lack of appropriate laboratory support and trained healthcare personnel. To overcome the lack of an appropriate point-of-care test with good sensitivity and reasonable specificity to detect the common STD pathogens, the World Health Organization (WHO) introduced treatment guidelines for the syndromic management of symptomatic patients.3 The syndromic approach uses clinical algorithms based on the patient’s symptoms and clinical signs to guide antimicrobial therapy.
Neisseria gonorrhoeae infections are one of the most widely disseminated sexually transmitted diseases worldwide. Prompt and appropriate antimicrobial treatment is important to eliminate the pathogen and restrict its transmission. However, the antibiomicrobial resistance of N. gonorrhoeae is constantly changing as a result of the extensive use and/or misuse of antibiotics for treatment of gonorrhea and importation of resistant strains.4 As a consequence, there is a need for area-specific research to underpin control programs. Periodic monitoring of the prevalence of the various pathogens and their susceptibility profiles provides essential information for updating local syndromic management guidelines. In 2001, the Pasteur Institutes International Network, supported by the French Ministry of Foreign Affairs, launched a program to conduct antimicrobial resistance surveillance studies. This program concerned parasites (Plasmodium falciparum), viruses (human immunodeficiency virus),and various bacteria. Eleven developing countries have been involved in this program.
N. gonorrhoeae infections were one of those studied in this surveillance program because the data available for the countries concerned were limited and not recent.5–8
Here, we report the prevalence of penicillinase-producing N. gonorrhoeae (PPNG) and the antimicrobial susceptibility of N. gonorrhoeae isolated in Bangui, Central African Republic; Yaoundé, Cameroon; Antananarivo, Madagascar; and Ho Chi Minh Ville and Nha Trang, Vietnam.
From March 2004 to June 2006, consecutive isolates of N. gonorrhoeae were collected from outpatients with gonorrhea attending (1) 2 health centers and the Pasteur Institute of Bangui in Bangui, Central African Republic; (2) the venereological center of Yaoundé and the Pasteur Center of Cameroon in Yaoundé, Cameroon; (3) 3 Health centers specialized in STD in Antananarivo, Madagascar; (4) the dermatological Hospital of Ho Chi Minh Ville; and (5) the Pasteur Institute of Nha Trang, the dermato-venereological hospital of Khanh Hoa province, and the HIV/AIDS provincial center in Nha Trang, Vietnam. The patients were asked to complete a questionnaire to collect data about age, gender, and previous antibiotic treatment (during the last month, including treatment for the present disease).
Isolation of N. gonorrhoeae
When patients were presented at the Pasteur Institutes, urethral and endocervical specimens were inoculated directly onto chocolate agar base supplemented with hemoglobin, 1% isovitalex, and inhibitors (vancomycin, colistin, and nystatin). The plates were incubated at 35°C in a humid candle extinction jar for 36 to 48 hours. When the recruitment occurred in various health centers, specimens were still directly inoculated onto chocolate agar plates; plates were put in a humid candle extinction jar at room temperature and collected once a day to be brought to the Pasteur Institutes within <1 hour. There, the isolates were identified as N. gonorrhoeae on the basis of colony morphology, Gram staining, oxidase tests, and carbohydrate degradation tests (Api NH; BioMérieux, Marcy L’étoile, France). All strains were stored in skimmed milk at −80°C until use.
Susceptibility to antibiotics was assessed by a disk diffusion technique on GC agar base (Oxoid) supplemented with 1% isovitalex, as recommended by the CLSI. The antibiotics tested were penicillin G (10 units), nalidixic acid (30 μg), used as a marker for ciprofloxacin resistance, ceftriaxone (30 μg), spectinomycin (100 μg), and tetracycline (30 μg) (Biorad, Marne la Coquette, France). After 24 hours at 37°C, the zone of inhibition was measured. N. gonorrhoeae ATCC 49,226 was used as a control. β-Lactamase was assayed by the paper acidometric method.
Agar Plate Dilution.
All the isolates that could be recovered after freezing were tested for susceptibility to penicillin G, ciprofloxacin, ceftriaxone, spectinomycin, and tetracycline by the agar plate dilution method for determination of MICs.9 MICs of the strains collected at one site were determined by the Pasteur Institute of the same site. Two-fold serial dilutions of antibiotics were added to molten GC agar base (Oxoid) supplemented with isovitalex (1%) at a temperature of 45°C. The resulting plates were seeded with 104 cfu/spot of bacteria by means of a multipoint inoculator and incubated at 37°C in CO2 for 24 hours. Control strains (ATCC 49,226, F-28, P681E, CDC 10,328, CDC 10,329, SPJ-15, and SPL-4) kindly provided by Dr Joan S. Knapp, Sexually Transmitted Infections Branch, Centers for Disease Control and Prevention, Atlanta, were included in each run.10 The ranges of concentration of antibiotics were as follows: penicillin G, 0.008 to 128 μg/mL; tetracycline, 0.015 to 256 μg/mL; spectinomycin, 0.03 to 512 μg/mL; ciprofloxacin, 0.001 to 16 μg/mL; and ceftriaxone, 0.001 to 16 μg/mL.
Antimicrobial susceptibility was scored using breakpoint criteria defined by the CLSI guidelines for disk diffusion and MICs,11 except for nalidixic acid (disk diffusion) for which the criteria were those defined by the Antibiogram Committee of the French Microbiology Society (ACFMS).12 Resistance was thus defined as follows: penicillin MIC ≥2.0 μg/mL, tetracycline MIC ≥2.0 μg/mL, spectinomycin MIC ≥128.0 μg/mL, and ciprofloxacin MIC ≥1.0 μg/mL. Intermediate resistance to ciprofloxacin was defined as an MIC of 0.125 to 0.5 μg/mL.10,11,13 For ceftriaxone, isolates with MICs ≤0.25 μg/mL were considered to be susceptible and those with MICs ≥0.5 μg/mL were classified as having decreased susceptibility.10,11,13 Strains testing positive for β-lactamase activity were referred to as penicillinase-producing N. gonorrhoeae (PPNG).
Geometric MICs were determined using the base2 logarithms. Data were analyzed, using software EPI Info (EPI Info: a word processing database and statistics program for public health; Version 6, Atlanta, GA: Center for Disease Control and Prevention; 1995).
A descriptive analysis was followed by bivariate analysis using a χ2 test or one-way ANOVA (or Kruskall-Wallis) for comparison of the various subgroups with P <0.05 as the minimal level of significance. A multivariate analysis with logistic regression was used to the factors with P <0.20 by bivariate analysis.
Only 30 strains could be recovered in Bangui; they were isolated from 2 women and 28 men, mean age 29.0; 79 isolates were obtained in Yaoundé (39 from women, 40 from men, mean age 28.4); 126 in Antananarivo (31 women, 95 men, mean age 26.6); 126 in Ho Chi Minh Ville (47 women, 74 men, mean age 28.9); and 56 in Nha Trang (12 women, 44 men, mean age 31.4).
Of these isolates, 24 in Bangui (80%), 29 in Antananarivo (23%), 69 in Yaoundé (87%), 45 in Ho Chi Minh Ville (37%), and 26 in Nha Trang (46%) were PPNG (Table 1).
Resistance rates to nalidixic acid were much higher in Vietnam (mean 99%) than in Central Africa or Madagascar (from 0% to 4%). Rates of resistance to ceftriaxone or spectinomycin were low among isolates from all sites.
MICs could be determined only for isolates from Bangui, Antananarivo, Ho Chi Minh Ville, and Nha Trang and not those from Yaoundé, because all these strains were lost due to problems of electricity supply; not all isolates from Antananarivo and Vietnam were available for testing (Table 2).
The distribution of MIC50, MIC90, and MIC mean of the 5 antibiotics for each site is summarized in Table 2. Ceftriaxone and spectinomycin seemed to be the most effective of the 5 antibiotics tested, and ciprofloxacin remains effective in Africa but not in Vietnam. Between African centers, only resistance rates to penicillin were significantly different (P >0.001) with disc diffusion (Table 1) and MICs (Table 2). Only 1 strain was found to have a decreased susceptibility to ceftriaxone in Ho Chi Minh Ville. This strain was resistant to all other drugs except for spectinomycin.
Concordance between MICs and disc was 94.5% for ciprofloxacin, 88.9% for spectinomycin, 83.1% for penicillin, and 77.8% for ceftriaxone and tetracycline. κ Values were 0.88 for ciprofloxacine (95% CI 0.881–0.884), 0.66 for penicillin (95% CI 0.659–0.669), 0.23 for tetracycline (95% CI 0.215–0.257), 0.09 for ceftriaxone (95% CI 0.111–0.068), and 0.06 for spectinomycin (95% CI 0.121–0.001).
Antimicrobial susceptibilities of all strains, interpreted according to CLSI standards, are shown in Table 2. The strains were most susceptible to ceftriaxone and spectinomycin (Table 2). Strains from Central Africa and Madagascar were also sensitive to ciprofloxacine (Table 2). Only 1 spectinomycin-resistant gonococcal strain was isolated in Madagascar, but nearly 25% of strains isolated in Ho Chi Minh Ville had a decreased susceptibility to spectinomycin. Spectinomycin resistance was associated with a previous treatment with this antibiotic (33.3% vs. 9.9%, P <0.001).
Resistance rates did not differ significantly between age groups or between patients having received or not previous antibiotic treatment. Univariate analysis indicated a significant difference in the rate of resistance to ciprofloxacine between women (78.6% of nonsusceptible strains) and men (59.8% of nonsusceptible strains). After logistic regression, only differences between countries remained.
Although informative, this study suffers various limitations. First, the recruitment of patients was difficult in most of the countries included, resulting in only a small number of strains being collected. This may be because many patients do not attend health centers for treatment of gonorrhea either because of low income or because they prefer buying treatments from street merchants or Health Community agents. Consequently, the patients providing isolates for this study may include a large proportion of those attending health centers due to treatment failure and therefore this could introduce a bias in the study. However, there was no significant difference in the rates of resistance between strains isolated from patients having received or not a previous treatment except for spectinomycin, so it is likely that the rates of resistance obtained in this study reflect those in the general population. Another problem encountered in 2 countries, Cameroon and Madagascar, was the loss of strains due to electrical problems with the freezers. MICs could not be assessed for the Cameroonian strains and for only 68 strains from Antananarivo and 121 in Ho Chi Minh Ville. However, we think that the results concerning ciprofloxacin can be interpreted in Cameroon because (1) the concordance between the rates of resistance to nalidixic acid by disk diffusion and that to ciprofloxacin by agar dilution in other countries was good, and (2) disc tests with nalidixic acid are widely used to predict fluoroquinolone susceptibility for N. gonorrhoeae,14 and indeed are recommended by the Antibiogram Committee of the French Society of Microbiology and the British Society for Antimicrobial Chemotherapy.12,15 Interpretation for other antibiotics is more hazardous, except for PPNG.
The antibiotic resistance profile of bacterial pathogens changes constantly over time. Since the emergence of PPNG in 1976 and chromosomally mediated penicillin resistance in 1983, resistant gonococci have spread worldwide and their prevalence has increased rapidly.16–21 In South East Asia, high proportions of isolates express penicillin resistance: values of 91% in Korea, 89% in the Philippines, 80% in China, 58% in Singapore, 54% in Hong Kong, and 48% in Vietnam have been reported for 2000.22 Data concerning N. gonorrhoeae in Africa are less numerous but very high proportions of penicillin-resistant strains have been reported in South Africa (48%),23 Cameroon (60%),8 Nigeria (95%),24 Ethiopia (85%),25 Rwanda (70%),26 and Madagascar (81%).6 In our study, the proportion of PPNG was different in Asia and Africa; the highest rates of PPNG were found in the Central African Republic and Cameroon. Plasmid-mediated resistance to tetracycline was not determined but usually these strains (TRNG) can be ascertained when MICs to tetracycline are above 10 μg/mL.27 In our study, 134 strains had MICs to tetracycline above 10 μg/mL and could be considered as TRNG. Among those 66 (49%) were also PPNG.
Quinolones have been recommended by the Centers for Disease Control and Prevention (CDC) for treatment of gonorrhea from 1989 to 2007.28,29 With the widespread use of these agents, resistance has sharply increased in the United States,29 and many regions of South East Asia and resistant strains have recently spread. In Hong Kong, quinolone-resistant N. gonorrhoeae (QRNG) increased from 0.5% in 1992 to 10.4% in 199430 and from 60% in 1998 to 80% in 2000.22 Similarly, in Japan, an increasing proportion of QRNG among isolates has been reported.31 Our findings for Vietnam were similar; in 1997, only 6.6% of isolates were less susceptible or resistant,22 whereas the corresponding value for the isolates we report here was 99%.
Spectinomycin and ceftriaxone are highly effective as single-dose treatment for gonorrhea. Decreased susceptibility to ceftriaxone remains rare but begins to be described.32,33 In our strain collection, the susceptibilities to both of these antibiotics were still close to 100% in most sites. However, 1 spectinomycin-resistant strain was isolated in Antananarivo, and 25% of strains were less susceptible to spectinomycin in Ho Chi Minh Ville. More strains were found to be resistant to spectinomycin using the disk diffusion method in Antananarivo but their MICs could not be determined. Our findings indicate that spectinomycin and ceftriaxone should be the first line treatment for gonorrhea in Vietnam and that ciprofloxacin should be used to treat gonorrhea in Central Africa and Madagascar. However, the association between decreased susceptibility to spectinomycin with a previous treatment with this antibiotic indicates that susceptibility of N. gonorrhoeae to this drug must be regularly surveyed in Vietnam where spectinomycin is used.
In summary, this analysis clearly demonstrates that penicillin-resistant gonococcal strains are frequently found in all countries included in the study and that ciprofloxacin-resistant strains are relatively frequent in Vietnam, but not in Central Africa, Cameroon, or Madagascar. This study also evidences the difficulties associated with preserving the strains isolated.
The skills acquired in our institutes during this study should be maintained and the participating institutes should join the WHO network on gonococcal infections because local susceptibility data from periodic surveillance help clinicians to use appropriate antibiotics for the treatment of gonorrhea.
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