Article Text
PDF
Abstract
Objectives Gonorrhoea and antimicrobial resistance (AMR) in Neisseria gonorrhoeae are major public health concerns worldwide. Enhanced AMR surveillance for gonococci is essential globally. In Zimbabwe, very limited gonococcal AMR data were reported. Our aims were to (i) implement quality-assured gonococcal AMR surveillance in Zimbabwe and (ii) investigate gonococcal AMR at five health centres in 2015–2016.
Methods Gonococcal isolates from 104 men with urethral discharge were tested for susceptibility to kanamycin, ceftriaxone, cefixime, ciprofloxacin and azithromycin using Etest.
Results All isolates (102 possible to test) were susceptible to ceftriaxone and cefixime. The level of resistance (intermediate resistance) to kanamycin and ciprofloxacin was 2.0% (2.0%) and 18.6% (27.5%), respectively. The two kanamycin-resistant isolates (R≥128 mg/L) had a kanamycin minimum inhibitory concentration (MIC) of >256 mg/L. The ciprofloxacin resistance ranged from 9.5% to 30.8% in the five sentinel sites. Only 10 (9.6%) of the isolates were tested for susceptibility to azithromycin and 1 (10.0%) was resistant (MIC=4 mg/L).
Conclusions The emergence of multidrug-resistant gonorrhoea internationally is a major public health concern and gonococcal AMR surveillance is crucial globally. In Zimbabwe, gonococcal AMR surveillance has now been implemented and quality assured according to WHO standards. The results of this first surveillance will be used to directly inform revisions of the national treatment guidelines. It is imperative to further strengthen the surveillance of gonococcal AMR, and ideally also treatment failures, in Zimbabwe and most countries in the WHO African region, which requires continuous national and international support, including technical support, and political and financial commitment.
- NEISSERIA GONORRHOEA
- GONORRHOEA
- ANTIMICROBIAL RESISTANCE
- TREATMENT
- AFRICA
Statistics from Altmetric.com
Introduction
Gonorrhoea, aetiological agent Neisseria gonorrhoeae, is a public health concern globally. In 2012, the WHO estimated 78 million new global cases of gonorrhoea and 11.4 million of these cases were in the WHO African region.1 In southern Africa, including Zimbabwe, the most frequent aetiology of urethral discharge in men is N gonorrhoeae, followed by Chlamydia trachomatis, Mycoplasma genitalium and Trichomonas vaginalis infections.2–4 Furthermore, in 2015 urethral discharge in men accounted for nearly a quarter of all reported STI cases in Zimbabwe.3 The Zimbabwe National STI Treatment Guidelines of 2012,5 which will be revised based on the results of the present study, recommend syndromic treatment for all men presenting with urethral discharge, which includes treatment for both gonorrhoea and C trachomatis infection. Accordingly, all men with urethral discharge are recommended to be treated with ceftriaxone 250 mg single dose or kanamycin 2 g single dose intramuscularly and, to cover C trachomatis infection, additionally doxycycline 100 mg twice daily for 7 days. Alternative therapeutic options for gonorrhoea are cefixime 400 mg single dose orally or spectinomycin 2 g single dose intramuscularly and, for C trachomatis infection, azithromycin 1 g single dose orally, erythromycin 500 mg four times daily for 7 days orally or ofloxacin 300 mg twice daily for 7 days.5
It is a great concern that N gonorrhoeae has shown an extraordinary capacity to develop resistance to all antimicrobials introduced since the mid-1930s, using mainly all known resistance mechanisms. Currently, in many countries the resistance levels are high to sulfonamides, penicillins, early generation macrolides, tetracyclines and fluoroquinolones.6 ,7 In vitro (including high-level) resistance and rare treatment failures have now emerged to the last remaining option for first-line empirical monotherapy in most countries globally, that is, the extended-spectrum cephalosporin (ESC) ceftriaxone.6–9 Accordingly, strengthened, regular and quality-assured surveillance of antimicrobial resistance in N gonorrhoeae is imperative worldwide to monitor the resistance trends, identify emerging resistance and inform development of, or a regular update of, the STI management guidelines locally, nationally and globally. This is in strict accordance with the Global Action Plan to Control the Spread and Impact of Antimicrobial Resistance in Neisseria gonorrhoeae published by the WHO in 2012.10 ,11 Most worryingly, in Africa, with the exception of South Africa, quality-assured N gonorrhoeae antimicrobial resistance surveillance has been exceedingly rare,12 and, as previously emphasised,13 significantly more support to strengthen the N gonorrhoeae antimicrobial resistance surveillance in Africa is crucial. In Zimbabwe, during the last decades only one study including examination of antimicrobial susceptibility in N gonorrhoeae has been conducted.4 This was an important aetiological study with the primary aim to describe the relative prevalence of N gonorrhoeae, C trachomatis, M genitalium and T vaginalis in a sample of 130 Zimbabwean men with urethral discharge, but a secondary aim was to investigate the antimicrobial susceptibility in N gonorrhoeae. This study was carried out in 2010–2011 and demonstrated a low level of resistance to ciprofloxacin (6.1%), but no resistance to kanamycin or ESCs. However, only 66 N gonorrhoeae isolates were examined and the study was limited to clinics in the capital city, Harare.4
The aims of the present study were to (i) design and implement quality-assured sentinel surveillance of antimicrobial susceptibility in N gonorrhoeae in Zimbabwe and (ii) investigate the antimicrobial susceptibility of N gonorrhoeae isolates obtained from five health centres in Zimbabwe (two in the capital city Harare, two in Bulawayo and one in Beit Bridge) in 2015–2016.
Material and methods
Patients and patient management
The sentinel surveillance sites were Mbare and Budiriro Polyclinics in Harare, Nkulumane and Khami Road Polyclinics in Bulawayo and Dulibadzimu Clinic in Beit Bridge. Men attending these clinics from April 2015 to July 2016 were enrolled in the study if they were ≥18 years of age, they provided written informed consent to take part in the study and they had a visible urethral discharge on examination. Urethral discharge was collected for Gram-stained microscopy and culture. The smears prepared on site and urethral swabs placed in Amies transport medium were transported to the central laboratories in Bulawayo and Harare for microscopy and culture.
Subjects with urethral discharge were managed according to the standard treatment protocol for male urethral discharge in Zimbabwe.5 Enrolled participants were given kanamycin 2 g single dose intramuscularly or ceftriaxone 250 mg single dose intramuscularly, and, additionally, they were given doxycycline 100 mg orally twice daily for 7 days (to cover C trachomatis infections). Subjects with persistent or recurrent urethral discharge were given, in addition, a single oral dose of metronidazole 2 g.
Microscopy, culture and antimicrobial susceptibility testing
On arrival at the laboratory, the urethral smears were examined using Gram-stained microscopy. The urethral swab was inoculated onto Modified New York City selective agar plates, which subsequently were incubated at 37°C in a humid 5% CO2-enriched atmosphere (in candle jars) for 24 hours. Culture plates with no suspected gonococcal growth after 24 hours were reincubated for additional 48 hours. Species confirmation of suspected N gonorrhoeae colonies was performed using Gram-stained microscopy, oxidase test and the Phadebact Monoclonal GC Test (Bactus AB, Uppsala, Sweden).
Antimicrobial susceptibility testing was performed using Etests (bioMérieux, Marcy l'Etoile, France), according to the manufacturer's instructions, on GC media (Oxoid, Hampshire, UK) supplemented with 1% BBL IsoVitalex Enrichment (Becton Dickinson, Le Pont de Claix, France). The minimum inhibitory concentration (MIC; mg/L) of ceftriaxone, cefixime, kanamycin, ciprofloxacin and azithromycin was determined and, with the exception of kanamycin, categorised into susceptibility, intermediate resistance, reduced susceptibility (only for ESCs) and resistance according to the current WHO14 and Clinical Laboratory and Standards Institute (CLSI) criteria.15 For kanamycin, CLSI has not stated any susceptibility or resistance breakpoints and, accordingly, breakpoints earlier stated by the WHO were applied, that is, susceptibility MIC≤32 mg/L and resistance MIC≥128 mg/L.16 Only a small number (n=10) of isolates were examined for azithromycin susceptibility because of problems acquiring azithromycin Etest strips. For quality control, the 2008 WHO N gonorrhoeae reference strains were used.17 ,18
penA sequencing to confirm ESC resistance/susceptibility
Genomic DNA was isolated using the Wizard Genomic DNA Purification kit (Promega), according to the manufacturer's instructions. The penA gene was sequenced as previously described.19
Results
Demographic data of patients, treatment and laboratory diagnosis
A total of 425 men with urethral discharge were enrolled in the study. Of these men, 70 (16.5%) were administered kanamycin 2 g single dose intramuscularly, 318 (74.8%) ceftriaxone 250 mg single dose intramuscularly and 388 (91.3%) were additionally given doxycycline 100 mg twice daily for 7 days. Twenty-seven (6.4%) subjects additionally received metronidazole 2 g single dose orally. Treatment information was not recorded for 29 (6.8%) of the subjects. Table 1 summarises the distribution of subjects by clinic of enrolment, age of subjects and results of diagnostics.
Distribution of enrolled men with urethral discharge by clinic, city, age and diagnostics
Briefly, each sentinel site enrolled from 58 to 125 subjects. The mean age, median age and age range of the subjects was 28.9±7.2 years, 28 years and 18–63 years, respectively. Gram-negative intracellular diplococci were identified in the urethral smears from 348 (81.9%) subjects; however, a N gonorrhoeae isolate was only obtained from 104 (24.5%) subjects (table 1). Also, 21 (20.2%) of these isolates belonged to serogroup WI (PorB1a), 81 (77.9%) to serogroup WII/III (PorB1b) and 2 (1.9%) were not tested.
Antimicrobial susceptibility
Antimicrobial susceptibility testing was performed on 102 (98.1%) of the N gonorrhoeae isolates. The results of this testing are summarised in table 2.
Antimicrobial susceptibility of Neisseria gonorrhoeae isolates (n=102) from five sentinel sites (cities) in Zimbabwe, 2015–2016
Briefly, all isolates were susceptible to ceftriaxone (MIC range: <0.002 to 0.064 mg/L) and cefixime (MIC range: <0.016 to 0.064 mg/L) using WHO/CLSI criteria14 ,15 as well as the criteria stated by the European Committee on Antimicrobial Susceptibility Testing (http://www.eucast.org). The level of resistance to kanamycin was 2% and an additional 2% of the isolates showed intermediate resistance. The kanamycin-resistant isolates were from Nkulumane (n=1) and Dulibadzimu (n=1) and both had a kanamycin MIC of >256 mg/L. The two patients with these kanamycin-resistant strains were treated with ceftriaxone 250 mg single dose plus doxycycline 100 mg twice daily for 7 days and kanamycin 2 g single dose plus doxycycline 100 mg twice daily for 7 days, respectively. Unfortunately, no information regarding treatment outcome for these patients was available. Resistance to ciprofloxacin was 18.6% and 27.5% of the isolates had an intermediate resistance. Ciprofloxacin resistance was found in isolates from all sentinel sites and ranged from 9.5% in Mbare to 30.8% in Nkulumane (table 2). Only 10 (9.6%) of the N gonorrhoeae isolates (from Budiriro (n=6) and Khami (n=4)) were tested for susceptibility to azithromycin and 9 (90%) of these were susceptible (MIC≤0.25 mg/L). The only azithromycin-resistant (MIC≥1 mg/L) isolate was from Budiriro and had an azithromycin MIC of 4 mg/L.
Discussion
Antimicrobial resistance in N gonorrhoeae is a global public health concern and enhanced antimicrobial resistance surveillance is imperative globally. In Africa, except South Africa, quality-assured N gonorrhoeae antimicrobial resistance surveillance has been exceedingly rare.12 ,13 In Zimbabwe, during the last decades only one study has been performed, which examined antimicrobial resistance in 66 N gonorrhoeae isolates from Harare in 2010–2011.4 In the present study, sentinel surveillance of antimicrobial susceptibility in N gonorrhoeae isolates in Zimbabwe was designed, implemented and quality assured using WHO standards10 ,11 ,14 and gonococcal WHO reference strains.17 ,18 The antimicrobial susceptibility of N gonorrhoeae isolates obtained from five health centres in Zimbabwe from 2015 to 2016 was examined. The sentinel sites were established in the two major cities in the country (two sites in Harare and two in Bulawayo) and in a location close to the border with South Africa (one site in Beit Bridge). The present surveillance showed that 18.6% of the gonococcal isolates were resistant to ciprofloxacin, ranging from 9.5% to 30.8% resistance in the sentinel sites, and 2% were resistant to kanamycin. No isolates showed reduced susceptibility to ceftriaxone or cefixime. In the previous study from 2010 to 2011, only 6.1% of the gonococcal isolates were resistant to ciprofloxacin and all isolates were susceptible to kanamycin, ceftriaxone and cefixime.4 Accordingly, the resistance to ciprofloxacin appears to have rapidly increased across Zimbabwe and, evidently, resistance and intermediate resistance to kanamycin, first-line treatment for urethral discharge syndrome in men,5 exist in Zimbabwe. Obviously, ciprofloxacin should not be used for empiric treatment of gonorrhoea in Zimbabwe or in most other countries globally where resistance is prevalent.6–10 The low resistance (2%) and intermediate resistance (2%) to kanamycin found in 2015–2016 is also a concern. The results of the present surveillance will be used to inform the revision of the Zimbabwe National STI Treatment Guidelines from 2012,5 and most likely it should be considered to recommend only ceftriaxone, ideally in a 500 mg intramuscular dose, as first-line treatment, and instead recommend kanamycin 2 g intramuscularly as the second-line treatment. This recommendation would also be more evidence-based and in accordance with international gonorrhoea treatment guidelines, which mainly recommend ceftriaxone 250–500 mg intramuscularly plus azithromycin 1–2 g orally.20–24 Kanamycin has been used in Zimbabwe for the treatment of gonorrhoea for some decades now.25 However, kanamycin has rarely been used for gonorrhoea treatment in other countries, no randomised controlled clinical trials have been performed, appropriate pharmacokinetic/pharmacodynamic data for the treatment of urogenital and extragenital gonorrhoea are lacking and quality-assured data regarding clinical efficacy are exceedingly limited, and mainly lacking for women and in extragenital gonococcal infections. Furthermore, despite that the in vitro resistance to kanamycin is rarely examined internationally, resistance and intermediate resistance to kanamycin have been described from many regions worldwide, for example, in Russia,26 Armenia,27 Indonesia,28 Benin,29Mozambique,30 which is one of the neighbouring countries to Zimbabwe, and also previously in Zimbabwe in the 1990s.31 Nevertheless, the in vitro resistance to kanamycin in Zimbabwe appears to have remained relatively low despite its use for the treatment of gonorrhoea for several decades and additional infections such as multidrug-resistant tuberculosis. No gonococcal isolates with resistance or reduced susceptibility to ceftriaxone or cefixime have yet been found in Zimbabwe. This might reflect that no gonococcal strain with ESC resistance has been imported and/or managed to establish any domestic spread. Furthermore, in Zimbabwe cefixime or other less potent oral ESCs have never been widely used for the treatment of gonorrhoea, which has resulted in a lower selection pressure for emergence and spread of ESC resistance. Nevertheless, cefixime has been used for the treatment of gonorrhoea in many countries worldwide including the neighbouring country South Africa, where also cefixime resistance resulting in treatment failures has been verified.32 Accordingly, the susceptibility to ESCs needs to be carefully monitored in Zimbabwe.
Regular, at a minimum every 18–24 months, quality-assured surveillance of antimicrobial resistance in N gonorrhoeae to monitor the resistance trends, identify emerging resistance and inform regular updates of the STI management guidelines in Zimbabwe is crucial. This is imperative to follow the spread of ciprofloxacin resistance, monitor azithromycin resistance and particularly to obtain more data regarding kanamycin and ceftriaxone susceptibility. Ideally, this in vitro surveillance of resistance should be supported by monitoring also of treatment failures using recommended therapies, which could be implemented at least at some of the sentinel sites. The WHO Global Gonococcal Antimicrobial Surveillance Programme (WHO GASP), supported by the WHO global action plan, was revitalised in 2009.10 ,11 The WHO GASP has since developed and expanded significantly. However, it is a major concern that quality-assured GASP programmes remain sporadic or completely lacking in large parts of Africa,12 ,13 which suffers from a high burden of gonorrhoea, creating prerequisites for rapid emergence and spread of antimicrobial resistance in N gonorrhoeae. The present study, which includes Zimbabwe in the WHO GASP, might illustrate the way forward and help to catalyse enhanced national and regional GASP networks essential for the whole WHO African region. In Zimbabwe and, in general, in the WHO African region, it is important to consider the GASP work as part of the routine clinical practice to assist and strengthen the STI prevention and control intervention, and not only as rarely performed research projects requiring ethical approval and separate research funding. In the WHO African region, it is evident that skills for sample collection, sample transportation and culturing of N gonorrhoeae have been lost among many healthcare providers and laboratory staff. This was evident also in the present surveillance by the low yield of gonococcal isolates where only 104 (24.5%) N gonorrhoeae isolates were cultured from urethral discharge samples from 425 men, while Gram-negative intracellular diplococci were identified in the urethral smears from 348 (81.9%) of these men. Another reason for this low yield in culture might be that some of the subjects had taken antimicrobials before culturing and, accordingly, no viable N gonorrhoeae remained. However, the main reasons appeared to be inappropriate storage of samples before and during transportation and far too long transportation times. Accordingly, one of the lessons learnt, at least at some sentinel sites, is the need of additional training reinforced by continual collection and transportation of samples, gonococcal culture and antimicrobial resistance testing, ideally at a minimum of a weekly basis. Such an undertaking should include both the public and private sector clinics and laboratories. In Zimbabwe, initiatives to improve the GASP such as additional training of healthcare providers and laboratory staff, increasing the number of gonococcal isolates examined, representativeness (consecutive patients, collection across the country), linkage to a higher level of epidemiological data and including testing for susceptibility to doxycycline/tetracycline and spectinomycin, as well as molecular resistance testing, that is, to be able to predict the antimicrobial resistance in samples with non-viable gonococci (which would significantly increase the number of samples tested) and confirm resistance in the viable ones, are considered for the future surveillance. Finally, continuous national and international support, including political and financial commitment, is essential to establish new GASPs and/or strengthen the few existing GASPs, for example, in Zimbabwe and other countries in the WHO African region.
Key messages
Antimicrobial resistance in Neisseria gonorrhoeae is a major public health concern globally, which significantly compromises the treatment of gonorrhoea internationally.
Increased surveillance of antimicrobial resistance in N gonorrhoeae internationally is imperative to identify emerging resistance, monitor resistance trends and inform treatment guidelines.
With the exception of South Africa, exceedingly limited antimicrobial surveillance data in N gonorrhoeae exist from Africa.
In the present study, quality-assured antimicrobial resistance surveillance according to WHO standards was implemented in Zimbabwe, southern Africa.
Acknowledgments
The authors are grateful to the Ministry of Health and Child Care, AIDS and TB Unit for coordinating the surveillance and to the Mbare, Budiriro, Khami, Nkulumane and Dulibadzimu clinics for implementing the surveillance. They are also grateful to the Medical Directors of both Harare and Bulawayo cities and the Provincial Medical Director of Matabelel and South Province and the District Medical Officer for Beit Bridge District for supporting the surveillance.
References
Footnotes
Handling editor Jackie A Cassell
Contributors ASL, assisted by LG, designed the surveillance protocol, performed the initial data analysis and wrote the first draft of manuscript together with MU. AM provided overall coordination during the surveillance. FN, TW and CCM provided technical support during the surveillance. AT, MG-M and FDS performed all the laboratory work. MU provided laboratory training, quality assurance and confirmatory testing. All authors contributed to the finalisation of the manuscript.
Funding The Department of Reproductive Health and Research, WHO, Geneva.
Competing interests None declared.
Patient consent Obtained.
Ethics approval The protocol and consent forms were reviewed and approved by the Medical Research Council of Zimbabwe (MRCZ; ref: MRCZ/A/1883).
Provenance and peer review Not commissioned; externally peer reviewed.