Neisseria gonorrhoeae remains a major cause of urethritis, cervicitis, pelvic inflammatory disease, and ophthalmia neonatorum, which, if untreated, may lead to serious complications such as infertility, ectopic pregnancy, adverse outcomes of pregnancy, and, rarely, blindness. In addition, it is well known now that N. gonorrhoeae infection facilitates HIV transmission and acquisition.1,2 Gonorrhea remains an important public health concern in sub-Saharan African countries, with an estimated 21.1 million new cases per year, and effective treatment is essential for its control.3
Although a curable sexually transmitted infection (STI) since the introduction of sulphonamides in the 1930s, treatment of gonorrhoea has become problematic due to the emergence and international spread of strains exhibiting resistance to many of the commonly available antimicrobial agents.4 Fluoroquinolones were introduced as first-line therapy for gonorrhea in the 1980s. Fluoroquinolone-resistant N. gonorrhoeae (QRNG) emerged in the 1990s and escalated in those regions with a history of prolonged fluoroquinolone use.4,5 In sub-Saharan Africa, where fluoroquinolones have only been used since the mid-1990s, resistance has only recently been reported.6–8 With the emergence of QRNG, most countries chose to introduce oral cephalosporins as an alternative first-line therapy. The emergence and spread of oral cephalosporin-resistant N. gonorrhoeae strains have been reported in many regions of the World, including, most recently, Africa.9 This has now resulted in a shift to the use of parenteral ceftriaxone coupled, in the case of some countries, with the use of dual therapy, to treat gonorrhea.9–11
In Uganda, between 1993 and 2010 and before its replacement with single-dose oral cefixime, the fluoroquinolone ciprofloxacin was recommended for the presumptive treatment of gonorrhea as part of syndromic management for STIs. It was recommended as treatment of vaginal discharge and male urethral discharge syndromes, as well as for recognized N. gonorrhoeae–related complications, that is, pelvic inflammatory disease and epididymo-orchitis.12 The present study was undertaken in 2008 to 2009 to determine the antimicrobial resistance pattern of N. gonorrhoeae isolates for ciprofloxacin and 6 other antimicrobial agents among female sex workers (FSWs) recruited in Kampala, Uganda.
The study was carried out at the Good Health for Women Project (GHWP), which was established in 2008. The project enrolled a cohort of 1027 FSWs in Kampala, Uganda, who were prospectively followed up at 3-month intervals to study the epidemiology of HIV and other STIs.
Specimen Collection and Transport
Sterile cotton swabs were used to collect endocervical secretions from consecutive women attending the enrollment and 3-monthly follow-up visits until approximately 200 positive gonococcal cultures were obtained. This took 18 months to achieve, and samples were collected between April 2008 and October 2009. The swabs were immediately placed in Amies’ charcoal transport medium at the GHWP clinic and promptly delivered to the on-site GHWP laboratory.
Laboratory Culture and Identification
The swabs were inoculated on in-house prepared Modified Thayer Martin Agar plates. These plates were then placed in a sealed pouch containing a CO2 generating tablet (JEMBEC system; BD Diagnostic Systems, Sparks, MD) and transferred immediately to an aerobic portable incubator at 36°C. The incubator was transported within 12 hours after specimen collection to the Medical Research Council/Uganda Virus Research Institute’s Clinical Laboratories in Entebbe, where the plates were removed from the pouch and placed in a 5% CO2 incubator at 36°C for 48 hours. All isolates were presumptively identified as N. gonorrhoeae on the basis of a positive oxidase reaction and a typical Gram stain appearance. Identification was confirmed using API NH strips (bioMérieux SA, Marcy l’Etoile, France).
Antimicrobial Susceptibility Testing
Penicillinase-producing isolates were detected using the chromogenic cephalosporin, nitrocefin (Oxoid Ltd, Basingstoke, UK). Antimicrobial susceptibility testing was performed on GC agar (Oxoid Ltd) plus 1% BBL IsoVitalex Enrichment (Becton Dickinson, Le Pont de Claix, France) using Etest strips (bioMérieux SA), which enable minimum inhibitory concentrations (MICs) to be determined for each antimicrobial agent tested. Isolates were tested in 2 consecutive MIC runs for susceptibility to ciprofloxacin, cefixime, ceftriaxone, azithromycin, spectinomycin, penicillin, and tetracycline. The N. gonorrhoeae reference strain ATCC 49226, which was fully susceptible to all antimicrobials tested, was used with each batch of MIC testing as an internal quality control during the first MIC run. Given the full susceptibility of the ATCC 49226 reference strain and the absence of inclusion in the first run of other N. gonorrhoeae control strains with intermediate susceptibility/resistant phenotypes for the antimicrobial agents tested, repeat MIC testing took place in 2011 to 2012 once the World Health Organization’s (WHO) new panel of N. gonorrhoeae control strains was made available.13 The WHO control strains used as internal quality controls in the second MIC run were strains F (fully susceptible to all antimicrobials tested), G (resistant to ciprofloxacin and tetracycline), K (resistant to penicillin, cefixime, ciprofloxacin, and tetracycline), M (β-lactamase positive, resistant to penicillin and ciprofloxacin), O (β-lactamase positive, resistant to penicillin and spectinomycin), and P (resistant to azithromycin). The European Committee on Antimicrobial Susceptibility Testing (EUCAST) MIC breakpoints (version 1.3, 2011) were used to assign susceptibility phenotypes.14 The EUCAST-derived interpretative breakpoint MIC values, adjusted for the half steps in sequential MIC doubling dilution values that occur with Etest strips, are shown in Table 1.
For the purpose of the current publication, data were analyzed and are presented with respect to the antimicrobial susceptibility profile of the first isolate cultured from women that was still viable at the time of the second MIC run.
External Quality Assurance
External quality assurance was performed on gonococcal isolates with ceftriaxone MIC values of 0.064 mg/L and higher at the Centre for HIV and STIs, National Institute for Communicable Diseases, South Africa.
Ethics and Other Approvals
Approval to conduct this study was obtained from the Science and Ethics Committee of the Uganda Virus Research Institute and the Ugandan National Committee for Science and Technology. Written or thumb-printed informed consent was obtained from all participating women.
In total, 219 positive gonococcal cultures were obtained from the enrolled study participants over the 18-month period. The first MIC run was performed on all 219 N. gonorrhoeae isolates. Only 170 (77.6%) of the 219 gonococcal isolates were still viable for the second MIC run; this run was performed in 2011 to 2012 and was quality assured using a panel of control gonococcal strains with a range of susceptibility phenotypes. These 170 gonococci were cultured from 148 women. A total of 22 N. gonorrhoeae isolates were cultured from 20 of these women at more than one follow-up visit. To avoid unnecessary bias due to persistent infections, it was decided to analyze only the MIC data for the first isolates cultured from each of these 20 women. The distribution of antimicrobial susceptibility phenotypes, as well as the MIC50 and MIC90 values, for 148 N. gonorrhoeae isolates tested against each antimicrobial agent is shown in Table 2.
Only 23 (15.6%) isolates were fully susceptible to the first-line therapeutic agent, ciprofloxacin, whereas most isolates (123; 83.1%) were resistant to this fluoroquinolone. Two isolates (1.4%) exhibited intermediate susceptibility to ciprofloxacin. Approximately two-thirds of the isolates (101; 68.2%) were penicillinase-producing N. gonorrhoeae. The remaining 47 (31.8%) non–penicillinase-producing N. gonorrhoeae isolates exhibited intermediate susceptibility to penicillin. There were no penicillin-susceptible isolates or β-lactamase–negative isolates with chromosomally mediated resistance to penicillin. Only 4 (2.7%) N. gonorrhoeae isolates were susceptible to tetracycline, whereas most isolates (144; 97.3%) were resistant to this antimicrobial agent.
All 148 isolates were susceptible to spectinomycin and ceftriaxone. One isolate was found to be resistant to cefixime (MIC, 0.38 mg/L), although the ceftriaxone MIC for this isolate was still low (MIC, 0.008 mg/L). Most isolates (124; 83.8%) were susceptible to azithromycin; however, there were 20 (13.5%) isolates with intermediate susceptibility and 4 (2.7%) azithromycin-resistant isolates.
This is one of the few published studies to report N. gonorrhoeae antimicrobial susceptibility data from Uganda for more than a decade and since the introduction of the syndromic management approach for STIs. Importantly, we report that only 16% of gonococci cultured from FSW recruited in Kampala in 2008 to 2009 were susceptible to the nationally recommended first-line agent, ciprofloxacin.15 In addition, almost all N. gonorrhoeae isolates were resistant to tetracycline, and more than two-thirds of isolates demonstrated plasmid-mediated resistance to penicillin. In contrast, all isolates were reported as susceptible to ceftriaxone and spectinomycin, whereas only 1 isolate exhibited low-level resistance to cefixime and only 4 isolates were resistant to azithromycin based on EUCAST MIC breakpoint criteria.14 Application of the Clinical Laboratory Standards Institute (CLSI) MIC breakpoint criteria would have produced identical susceptibility determinations for isolates tested against penicillin, cefixime, ceftriaxone, and spectinomycin.16 However, use of these alternative MIC breakpoints would have resulted in a higher prevalence of isolates with intermediate susceptibility to ciprofloxacin (5.4%) and a correspondingly lower prevalence of QRNG (79.1%). For tetracycline, 1 of the 4 susceptible isolates would have been reclassified as intermediate by CLSI criteria. Azithromycin does not have CLSI breakpoints, and so a comparison with the EUCAST breakpoints is not possible for this particular antimicrobial agent.
Amito Florence et al.17 recently published antimicrobial susceptibility results for 151 gonococci collected over five years (2007–2011) from the urogenital tract of women and men attending a hospital outpatients’ clinic in Gulu District (North Uganda). The authors reported a rise in QRNG prevalence from 5% (2007) to 23% (2011) using disk diffusion methodology. A second Ugandan study, contemporary with ours and conducted in Kampala among men presenting with urethral discharge to an STI clinic and a prison hospital, reported that 44 (37%) of 119 N. gonorrhoeae isolates were resistant to ciprofloxacin.18 Although the published abstract for this second study lacks sufficient methodological details to draw any firm conclusions from a comparison with our study, we did observe a much higher prevalence of QRNG in our study population of FSW. These 2 other Ugandan studies demonstrated that gonococcal resistance to ciprofloxacin, the recommended first-line treatment of gonorrhea in Uganda at the time of our study, is not only restricted to high-risk core groups but also affecting the general population at levels in excess of the WHO’s 5% threshold for changing first-line antigonococcal therapy.19
Through use of mathematical modeling, Chan et al.20 have suggested that gonorrhea can only be controlled effectively if core groups, such as sex workers, are targeted by screening and testing for gonorrhea. Paradoxically, however, they also report that this approach is likely to result in the dissemination of antimicrobial resistant strains to intermediate- and low-risk populations, ultimately undermining gonorrhea control efforts. This finding further emphasizes the importance of nontherapeutic approaches to gonorrhea control, such as increasing condom use and reducing other risk behaviors known to be associated with gonorrhea acquisition, as well as highlights the need for an effective gonococcal vaccine.
The emergence of QRNG within Africa was first reported from Kwa-Zulu Natal province in South Africa, and subsequent antimicrobial susceptibility surveys confirmed spread into several of the country’s other 8 provinces.6,7 Fluoroquinolone-resistant gonococci have also been isolated at a prevalence in excess of 5% in neighboring countries since 2006, specifically Namibia and Zimbabwe (D. Lewis, personal communication). More geographically relevant to Uganda, a rise in the QRNG prevalence from 9.5% (2007) to 50% (2009) was reported among young men with gonorrhea in Kisumu, Kenya.8
The global approach to the emergence of QRNG was to abandon oral fluoroquinolones in favor of oral cephalosporins. Gonorrhea treatment failures associated with the use of oral cephalosporins are now commonly reported from many regions of the world.21 Several countries have now abandoned oral therapy for gonorrhea completely and instead treat N. gonorrhoeae infections with intramuscular ceftriaxone at varying doses from 250 mg to 1 g.21–24 Importantly, ceftriaxone MIC creep has been observed within Kenya and South Africa, as well as in several other regions of the world.8,25,26 Higher doses of intramuscular ceftriaxone, given as part of dual therapy with azithromycin for gonorrhea in some countries, are now advised to slow down the spread of cephalosporin-resistant gonococci.21 Most alarming has been the recent emergence of extensively drug-resistant N. gonorrhoeae isolates, characterized by resistance to both oral and parenterally administered cephalosporins.11,21
Injectable treatment of gonorrhea has the disadvantage of carrying a risk of occupational needle-stick injury, which should ideally be avoided in HIV high-risk settings. Although intramuscular spectinomycin is a suitable therapeutic alternative to intramuscular ceftriaxone, it is not widely available and it would be an expensive option for resource-poor countries. Moreover, there are concerns over the ease with which resistance has developed to spectinomycin in the past.4 A cheaper alternative, single-dose intramuscular gentamicin (240 mg), has been used in Malawi for 20 years with apparent retention of antimicrobial activity against N. gonorrhoeae.27 However, a recent meta-analysis found that the pooled clinical effectiveness of gentamicin for treatment of gonorrhea was only 91.5%, lower than the clinical effectiveness of ceftriaxone and lower than the 95% effectiveness threshold used by WHO for recommended first-line regimens.28 We currently lack data regarding clinical efficacy of gentamicin monotherapy because it is usual to administer it with either doxycycline or azithromycin for presumptive chlamydial infection. Further studies are required to determine the clinical efficacy of gentamicin monotherapy at urogenital and, importantly, extragenital sites of gonococcal infection. Finally, intramuscular kanamycin is still used to treat gonorrhea in some African countries, for example, Zimbabwe and Mozambique. Recent data from Zimbabwe suggest that kanamycin may still be effective (D. Lewis, personal communication), whereas 7% of gonococci isolated in Mozambique in 2005 were resistant.29
Azithromycin, at an oral dose of 1 to 2 g, offers potential as a component of multidrug therapy with gonorrhea and would ideally be given in combination with an injectable agent, such as ceftriaxone, spectinomycin, gentamicin, or kanamycin. Although azithromycin does have a place in treating gonorrhea in certain instances, it is not suitable as a single first-line agent because resistance may develop easily rapidly, even on treatment.4,30 Importantly, low- and high-level azithromycin-resistant strains have already been reported from several regions of the world.4
The control and management of gonorrhea remains a challenging process, especially for low-income countries. Inappropriate and uncontrolled use of antimicrobial agents, inadequate regulations on quality of drugs, and lack of monitoring of gonococcal antimicrobial susceptibility can contribute to the natural selection of antimicrobial resistant N. gonorrhoeae strains. For resource-poor countries, the undertaking of quality-assured N. gonorrhoeae antimicrobial susceptibility surveillance remains a challenge because of competing demands for the health budget and limited donor funding, lack of laboratory and epidemiological capacity to undertake quality-assured national surveys, and loss of technologist competency in respect of N. gonorrhoeae culture, identification, and antimicrobial susceptibility testing. Gonococcal antimicrobial susceptibility surveys are most cost-effectively undertaken among men with urethral discharge. Ideally, gonococcal antimicrobial susceptibility surveillance programs should also include sampling of core group members, such as sex workers and men who have sex with men, as antimicrobial-resistant N. gonorrhoeae strain(s) typically emerge and amplify within these key populations.9
Our study had a few limitations. First, the antimicrobial susceptibility data relate to FSW sampled in Kampala in 2008 to 2009 and may not necessarily be representative of Uganda’s general population or the profile of N. gonorrhoeae susceptibility today. A second limitation is that molecular typing did not form part of this study, so we were unable to distinguish reinfection from persistence for women whose first and subsequent gonococcal isolates had identical antimicrobial susceptibility profiles. This resulted in our choosing only the first isolate for further analysis. Finally, because there was a substantial time interval between the first and second MIC runs, almost a quarter of the gonococcal isolates were nonviable at the time of repeat testing. However, we believe that this loss in viability was likely random because we observed a similar distribution of antimicrobial susceptibility phenotypes for those isolates tested in the first MIC run compared with the data presented here from the second MIC run (data not shown).
Our report of the emergence and widespread dissemination of QRNG among sex workers in Kampala clearly demonstrates what can happen when a country attempts to manage STIs syndromically without establishing a parallel laboratory-based national STI surveillance program. The data presented provided important evidence to underpin the Ugandan Ministry of Health’s decision to replace ciprofloxacin with cefixime as national first-line presumptive therapy for gonorrhea in 2010.
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