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Antimicrobial Susceptibility of Neisseria gonorrhoeae Isolates in Yaoundé, Cameroon From 2009 to 2014

Tayimetha, Carolle Y. MSc*†; Unemo, Magnus PhD

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doi: 10.1097/OLQ.0000000000000915
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Gonorrhea is the second most common bacterial sexually transmitted disease (STD) worldwide, with 78 million cases among adults estimated in 2012.1 Effective antimicrobial treatment is crucial to avoid the severe complications of gonorrhea.2–6 In Cameroon, Central-Western Africa, and in most other African countries, the national STD treatment guidelines recommend syndromic treatment for STDs. In Cameroon, the recommended first-line treatment of males with urethral discharge is ciprofloxacin 500 mg single oral dose and, to treat chlamydia, additionally, doxycycline 100 mg twice daily for 7 days. Second-line treatment is ceftriaxone 250 mg single intramuscular dose plus doxycycline as described above. Recommended first-line treatment of females with vaginal discharge is the same, except that also metronidazole 2 g is given for treatment of trichomoniasis.7 However, many additional antimicrobials can be used for treatment of urethral/vaginal discharge in Cameroon. It is a major global concern that antimicrobial resistance (AMR) to all antimicrobials introduced for treatment of gonorrhea has emerged in Neisseria gonorrhoeae.4–6,8 Clinical resistance, causing sporadic treatment failures, has in the recent decade emerged also to the last remaining option for empiric first-line monotherapy, that is, the extended-spectrum cephalosporin ceftriaxone.4–6,8–11 Enhanced, quality-assured AMR surveillance in gonococci is crucial globally to identify emerging AMR, monitor AMR trends, and inform revisions of STI treatment guidelines nationally, regionally, and globally.4 According to the World Health Organization (WHO), ideally, the quality-assured gonococcal AMR surveillance should include 100 or greater representative species-confirmed gonococcal isolates (one isolate per patient and, where possible, combined with epidemiological data of the corresponding patients) per year. The AMR testing should use quality-assured and recommended methods (where possible, quantitative minimum inhibitory concentration (MIC)-based methods), in accordance with the international guidelines. The 2016 WHO reference strains12 should be used for quality controls and an external quality assessment (EQA) of the AMR testing performed every year with acceptable results.8 In Africa, quality-assured gonococcal AMR surveillance has been very rare.13,14 In Cameroon, only 2 small studies have been internationally published regarding gonococcal AMR.15,16 The first study, published in 2003,15 examined 32 isolates and the most recent study examined 79 isolates cultured in Yaoundé more than one decade ago (2004–2006).16 In Yaoundé, in 2004 to 2006, a high level of resistance to benzylpenicillin and tetracycline was observed, but only a low level of resistance to ciprofloxacin (1.3%) and no resistance to ceftriaxone or spectinomycin.16

We investigated the resistance to 7 antimicrobials in gonococcal isolates cultured from 2009 to 2014 in Yaoundé, Cameroon, and recommend updates of the national STD treatment guideline in Cameroon.

From January 2009 to September 2014, clinical gonococcal isolates (one isolate/patient) were cultured at the Centre Pasteur of Cameroon; Yaoundé, Cameroon. The isolates were cultured from urethral swabs or high vaginal/cervical swabs sampled, after informed consent, from outpatients with urethral discharge (males) or vaginal discharge (females, including pregnant ones) attending 8 hospitals and 3 health centers in Yaoundé, Cameroon. Demographic data, age, and sex of all patients were collected. The isolates were cultured on selective gonococcal culture medium and species verified using Gram-stained microscopy, oxidase test, and API NH test (bioMérieux).17 The AMR testing was performed using disc diffusion method, according to Clinical and Laboratory Standards Institute (CLSI).18 The discs (Oxoid) were: ceftriaxone (30 μg), azithromycin (15 μg), spectinomycin (100 μg), nalidixic acid (30 μg; reflecting ciprofloxacin resistance17), benzylpenicillin (10 IU), tetracycline (30 IU), and chloramphenicol (30 μg). Isolates with suspected ceftriaxone resistance were confirmed with agar dilution method, according to CLSI and WHO,17,18 on Polyvitex chocolate media at 37°C in a 5% CO2-enriched atmosphere for 18 to 24 hours. Five clinical gonococcal strains with different AMR phenotypes were used for quality control. Resistance breakpoints from the CLSI18 or WHO (nalidixic acid disc)17 were applied. The study was approved by the institutional ethics committee and research to human health from the School of Health Sciences of the Catholic University of Central Africa.

During 2009 to 2014, 193 gonococcal isolates were cultured from 129 (66.8%) males and 64 (33.2%) females. The number of isolates per year was: 2009 (n = 26), 2010 (n = 18), 2011 (n = 17), 2012 (n = 46), 2013 (n = 60), and 2014 (n = 26). The mean age of the males and females was 29.5 years and 27 years, respectively. The resistance level to benzylpenicillin was 93.3% (n = 180), tetracycline was 58.5% (n = 113), ciprofloxacin was 17.6% (n = 34), chloramphenicol was 7.3% (n = 14), azithromycin was 3.1% (n = 6), spectinomycin was 2.6% (n = 5), and ceftriaxone was 1.0% (n = 2). However, the 2 isolates with suspected ceftriaxone resistance were confirmed as susceptible using agar dilution method (MIC ≤ 0.002 μg/mL) and in repeated disc diffusion testing, indicating errors or contamination in the initial disc diffusion testing. Finally, 155 (80.3%) of the isolates were penicillinase-producing N. gonorrhoeae. There were no obvious differences in resistance levels by sex or age. The resistance to benzylpenicillin and tetracycline was stably high during 2009 to 2014. However, from 2011 to 2014, the resistance to ciprofloxacin increased significantly from 3.8% to 50.0% (P < 0.05).

In the present study, the AMR in gonococcal isolates cultured in Yaoundé during 2009 to 2014 was examined. Briefly, the levels of resistance to benzylpenicillin and tetracycline were stably high, and as in most countries, globally, these antimicrobials should not be used for empiric gonorrhea treatment. The ciprofloxacin resistance increased from 3.8% in 2009 to 50% in 2014 and ciprofloxacin should clearly not either be used for empiric gonorrhea treatment in Cameroon, which is in accordance with most other countries globally.4–6,8–10 However, no isolates were resistant to ceftriaxone, and only 3.1% of the isolates were resistant to azithromycin. The results of the present surveillance will be used to inform revisions of the national STD treatment guideline in Cameroon.7 It is strongly recommended to replace ciprofloxacin 500 mg with ceftriaxone 250 to 500 mg in the empiric first-line treatment of males with urethral discharge and females with vaginal discharge. This recommendation would also be more in agreement with international gonorrhea treatment guidelines, which mainly recommend ceftriaxone 250 to 500 mg plus azithromycin 1 to 2 g.19–23 Regular, quality-assured gonococcal AMR surveillance in Cameroon is crucial to monitor the resistance to ciprofloxacin, azithromycin and spectinomycin, and particularly to obtain additional susceptibility surveillance data for ceftriaxone, for which in vitro resistance and sporadic treatment failures have been described internationally.4–6,8–11,24–30 No verified gonorrhea treatment failure with ceftriaxone has yet been reported in Africa. However, quality-assured gonococcal AMR surveillance is lacking in most African countries, and clinical resistance to the oral extended-spectrum cephalosporin cefixime has been reported from South Africa.31 The WHO Global Gonococcal Antimicrobial Surveillance Programme (WHO Global GASP), a global network of regional and subregional reference laboratories,4 was revitalized in 2009, to monitor AMR and inform revisions of treatment guidelines and public health strategies and policies worldwide.8 To ensure quality-assured and comparable data among countries, regional focal points provide technical support and training, conduct EQAs, and distribute WHO gonococcal reference strains for quality assurance.12 The latest WHO GASP publication included gonococcal AMR results from 77 countries, 2009 to 2014.8 In Cameroon and, in general, in African countries, it is crucial to consider the GASP as part of the routine clinical practice to strengthen the STD prevention and control interventions. The main limitations of the present study included the low number and likely limited representativeness of isolates each year; lack of epidemiological data for the corresponding gonorrhea patients, and international reference strains and an EQA to quality assure the AMR testing; and use of qualitative disc diffusion method for AMR testing, with suboptimal correlation to the antimicrobial MICs, instead of quantitative MIC determination using Etest or agar dilution method. Furthermore, some very rare ciprofloxacin-resistant gonococcal strains can be missed by using only nalidixic acid discs to monitor the ciprofloxacin resistance.32 If disc diffusion method is the only method possible to use, ideally also a ciprofloxacin disc should be included in the testing.17 Work is in progress to address all these issues in the future gonococcal AMR surveillance in Cameroon, making the surveillance more in agreement with the recommendations from WHO and the WHO Global GASP.8,17

In conclusion, our results from Cameroon, Western Africa will be used to inform revisions of the national STD treatment guideline,7 and it is strongly recommended to replace ciprofloxacin 500 mg single oral dose with ceftriaxone 250 to 500 mg intramuscularly in the recommended empiric first-line treatment of urethral discharge in males and vaginal discharge in females. The continued use of ciprofloxacin as first-line can result in a high number of treatment failures and severe complications, for example, pelvic inflammatory disease, ectopic pregnancy, and infertility, as well as enhanced HIV transmission. It is essential to further strengthen the surveillance of gonococcal AMR, ideally including also surveillance of gonorrhea treatment failures, in Cameroon and most countries in the WHO African Region, which requires national and international support, including technical support, and political and financial commitment.13,14

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