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Abstract
Objectives The aims of this study were to assess antimicrobial resistance in Neisseria gonorrhoeae infections and update the treatment in the national guidelines for the syndromic management of sexually transmitted infections in Morocco.
Methods 171 men complaining of urethral discharge were recruited from basic health services during 2009. Urethral swab samples were collected and N gonorrhoeae identification was performed by culture. Antimicrobial susceptibility testing was performed using the Etest method and the antimicrobial agents tested were ciprofloxacin, penicillin, spectinomycin, tetracycline, ceftriaxone and cefixime.
Results A total of 72 isolates were examined. Significant resistance to tetracycline (92.8%) and ciprofloxacin (86.8%), which was used as first-line treatment in gonococcal infections, was noted. No resistance to spectinomycin, ceftriaxone or cefixime was detected in all the isolates.
Conclusions Following these results the Ministry of Health of Morocco replaced ciprofloxacin and introduced ceftriaxone 250 mg as a single dose in the treatment of gonococcal infections. Using funds from the Global Fund to Fight AIDS, Tuberculosis and Malaria (the Global Fund), a surveillance programme was set up for antimicrobial resistance testing in N gonorrhoeae.
- NEISSERIA GONORRHOEA
- SURVEILLANCE
- ANTIMICROBIAL RESISTANCE
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Introduction
Gonorrhoea, which is caused by Neisseria gonorrhoeae, is one of the most common sexually transmitted infections (STIs) in most countries.1 Gonococcal infections represent 106 million of the estimated 498 million new cases of curable STIs that occur globally every year.2
Gonorrhoea continues to be a public health problem because of high disease prevalence in resource-constrained countries and lower, but still substantial and increasing, rates in developed countries, compounded by increasing resistant gonococcal strains.3 Accurate diagnosis with effective treatment to prevent further transmission is one of the essential elements for the control of gonococcal infections.4 Treatment strategies should be designed to utilise appropriate and preferably single-dose therapy that could be conveniently administered at the time of diagnosis.5 Over the years, the gonococcus has demonstrated the ability to develop resistance to antimicrobial drugs used for therapy, and the worldwide spread of resistant strains has shown that the antimicrobial susceptibility of this bacterium needs to be monitored closely.6 The surveillance of antimicrobial resistance (AMR) is used to guide therapy in the individual patient, monitor AMR trends and track the emergence and spread of new resistant strains. Additionally, in diseases of public health importance such as gonorrhoea, AMR surveillance helps establish and maintain the efficacy of standard treatment regimens to inform public health control and help to fight against the disease.7–9
Some countries in different parts of the world established a Gonococcal Antimicrobial Surveillance Programme (GASP) a long time ago. For example, Australia has had such a program since 1981, the USA since 1986 and the European Union (EU) countries since 2004.10–14 GASPs have helped these countries to follow trends of AMR in N gonorrhoeae over time and to adapt the treatment of these infections according to the susceptibility profiles of the isolates. Unfortunately, these programmes have not been universally adopted. Consequently no regular monitoring of antimicrobial susceptibility of N gonorrhoeae is carried out in many countries, especially in developing countries. There are multiple reasons for this, including the invasive nature of specimen collection procedures, the fastidious nature of the organism which makes transportation from one place to another and culture difficult, the need for specialised culture media and trained personnel, and the cost of reagents.15 Moreover, the adoption of the syndromic management of STIs, which stipulates that treatment of gonorrhoea should be on the basis of diagnosis according to symptoms and signs, has resulted in a significant decrease in laboratory tests on N gonorrhoeae and the loss of skills to culture the pathogen. The end result has been a big decrease in the isolation of N gonorrhoeae isolates on which to determine antimicrobial susceptibility trends.
More recently, resistance to currently recommended first-line third-generation cephalosporins—cefixime and ceftriaxone—has emerged, and treatment failures with cefixime have been verified in Japan, in several European countries and in South Africa.3 ,16–19 The observation of decreasing susceptibility of N gonorrhoeae to the ‘last-line’ third-generation cephalosporins, together with the longstanding high prevalence of resistance to penicillins, sulfonamides, tetracyclines and, more recently, quinolones and macrolides (including azithromycin), indicate that gonorrhoea has the potential to become untreatable.2
In 2012, in response to this grave threat, WHO prepared and published the ‘Global Action Plan to Control the Spread and Impact of AMR in N gonorrhoeae’.2 ,3 ,20 ,21 The objective of this global action plan is to control the spread and minimise the impact of AMR in N gonorrhoeae.
Morocco has implemented the syndromic approach for the management of STIs at the national level since 1998. All patients presenting with genital complaints receive antibiotic treatment following the appropriate syndromic management algorithms and using a single dose of ciprofloxacin (500 mg) as the drug of choice for treatment of gonococcal infection. The surveillance system for STIs was developed and implemented in the same year and the current system is based on universal syndromic case reporting and prevalence studies for STIs, and on N gonorrhoeae AMR monitoring. Several prevalence studies have been conducted by the Ministry of Health but only two studies on AMR in N gonorrhoeae were performed in 2001 and 2009. All these studies have helped to establish and evaluate the syndromic approach to manage STIs in the country.
The second study on AMR in N gonorrhoeae conducted 8 years after the first was enabled using resources from a grant to Morocco provided by the Global Fund to fight AIDS, Tuberculosis and Malaria (the Global Fund). The Global Fund proposal by Morocco identified the importance of STIs and AMR as a component of HIV control and funds were thus, received and duly allocated by the Moroccan government to support this work.
This article summarises data from the study on AMR in N gonorrhoeae conducted by the Ministry of Health of Morocco in 2009 and the change in the susceptibility of gonococcal strains in Morocco between 2001 and 2009. The aim of this second study was to validate the efficacy of the therapeutic recommendations adopted for the syndromic management of gonococcal infections in Morocco.
Materials and methods
The study was conducted between July and December 2009 in five cities situated in north, central, west and south Morocco so that the sample was a good representation at the national level. A total of 171 men complaining of urethral discharge were recruited from several basic health services (BHS) in these cities. A clinical examination was performed and urethral specimens were collected by physicians in the BHS and sent to the hospital laboratories in each city. Laboratory staff were trained to perform culture and identification of N gonorrhoeae.
Diagnostic methods, culture and preservation of N gonorrhoeae isolates
Isolation and identification of N gonorrhoeae was performed by culture on Thayer–Martin medium (Oxoid, UK) with 1% Vitox (Oxoid), incubated at 37°C in a 5% CO2 atmosphere for 18–20 h.
The isolates were stored in trypticase soy broth with 20% glycerol at −80°C.
Antimicrobial susceptibility testing
All isolates from the study were sent to the STI National Reference Laboratory at the National Institute of Hygiene in Rabat and evaluated for their antimicrobial susceptibility profile.
Antimicrobial susceptibilities were determined using the Etest method (AB Biodisk) according to the manufacturer's instructions: a gonococcal suspension of approximately 104 cfu/μL (turbidity equivalent to that of a 0.5 McFarland standard) was inoculated on GC agar (Oxoid) supplemented with 1% Vitox, and Etest strips were applied to the plate surface and the minimum inhibitory concentrations (MICs) were recorded following incubation at 36°C in 5% CO2 for 18–20 h. MICs were defined as the concentration point at which the zone of inhibition intercepted the Etest strip.
The antimicrobial agents tested were ciprofloxacin, penicillin, spectinomycin, tetracycline, ceftriaxone and cefixime.
Quality control was performed using the ATCC 49226 and CDC reference strains (F28 (SpcR), CDC 10328 (cipR) and SPL-4 (CfxDS)) at first because the WHO panel was not initially available and once received, the WHO K and L strains, which exhibit decreased susceptibility to ceftriaxone and cefixime, were used in a second round of testing of all the isolates.
The breakpoints for all these drugs were those indicated by the Clinical and Laboratory Standards Institute (formerly National Committee for Clinical Laboratory Standards).22 Accordingly, the MIC breakpoints for susceptibility or resistance were ≤0.06/≥1 for ciprofloxacin, ≤32/≥128 for spectinomycin, ≤0.25 (susceptible) for cefixime and ceftriaxone, ≤0.25/≥2 for tetracycline and ≤0.06/≥1 for penicillin.22 Penicillinase production was determined by the chromogenic cephalosporin method using the nitrocefin disc (Oxoid).
All the laboratory results were aggregated on Epi info V.6. Data analysis was performed on the same software.
Results
In the 171 men recruited in the five cities involved in this study, gonococcal infection was confirmed in 63% by PCR; 82 N gonorrhoeae strains were identified by culture from which 72 isolates were studied for MICs. The majority of the men recruited were symptomatic patients with an average age of 30 years.
The proportions of N gonorrhoeae isolates that displayed resistance and intermediate susceptibility to the antimicrobial agents used in this study are shown in table 1. Resistance to ciprofloxacin was identified in 86.8% of N gonorrhoeae strains, 16.2% were resistant to penicillin and 92.6% were resistant to tetracycline. All the isolates were 100% susceptible to ceftriaxone, cefixime and spectinomycin. β-lactamase production was demonstrated in 37.8% of the N gonorrhoeae isolates.
Antimicrobial susceptibility profile of Neisseria gonorrhoeae isolates in 2009 in Morocco
MIC values of ceftriaxone and cefixime are very low, ranging between 0.008 and 0.002 mg/L for ceftriaxone and are all less than 0.016 mg/L for cefixime.
A similar AMR study was conducted in 2001 in Morocco.23 On comparing susceptibility profiles between the two studies it is apparent that in 2009 there were more isolates resistant to ciprofloxacin and tetracycline (table 2). Cefixime and spectinomycin were not tested in 2001.
Evolution of resistance in Neisseria gonorrhoeae strains isolated in 2001 and 2009
Discussion
The second study on AMR conducted in 2009 took 6 months to recruit 171 patients with urethral discharge from whom 72 gonococcal isolates were identified and tested for antimicrobial susceptibility. Even though the sample of isolates tested was small, the data show an important evolution of the antimicrobial susceptibility in N gonorrhoeae in Morocco.
The AMR study in 2009 demonstrated an increasing trend of resistance in N gonorrhoeae to tetracycline (from 59.7% in 2001 to 92.6% in 2009) and to ciprofloxacin (from 2.6% in 2001 to 86.7% in 2009).
Tetracycline is cheap and is therefore used to treat many other infections, including N gonorrhoeae and Chlamydia trachomatis infections. Tetracycline-resistant N gonorrhoeae were first reported in the USA in 1985.15 Tetracycline resistance, chromosomal and plasmid mediated, has increased and attained a high prevalence in many countries such as the USA,24 India, Thailand, Indonesia,25 ,26 Scotland, Rwanda and DRC.27 Despite this, tetracyclines remain important and effective agents in the treatment of other STIs, notably C trachomatis.15
In Morocco, before 1998, the recommended treatment for gonococcal infection was penicillin, but many cases of treatment failure were identified. Fluoroquinolones became a popular therapy during the 1980s and were widely used as effective oral therapy against penicillin-resistant N gonorrhoeae. The most widely used quinolone against uncomplicated gonorrhoea was ciprofloxacin and the efficacy of this agent was 100% with a single dose of 500 mg. On the basis of data showing high efficacy, safety and convenience as single-dose therapies, oral fluoroquinolones were recommended for gonorrhoea treatment by the Centers for Disease Control and Prevention (CDC) in the USA in 1993.15
When Morocco implemented the syndromic approach in 1998 at the national level, ciprofloxacin was chosen as the drug of choice for the treatment of gonococcal infection based on the CDC and WHO recommendations. Resistance to fluoroquinolones was first detected in the mid 1980s in Asia, and then it spread to other areas in Europe, the Western Pacific, South and South-East Asia and the Americas.28–31 The results of the 2001 AMR study demonstrated that N gonorrhoeae isolates were still susceptible to ciprofloxacin in Morocco.
From 2001 to 2009, the recommended treatment for gonococcal infections in the different algorithms in Morocco was the single, oral dose of 500 mg of ciprofloxacin. The data reported in the latter study on AMR showed that N gonorrhoeae had a high level of resistance to ciprofloxacin, far in excess of the cut-off of 5% as recommended by WHO.5 ,16 WHO recommends the substitution of a drug in treatment regimens for gonorrhoea when the rate of resistance is 5% or higher in a particular community. This cut-off point defines the requirements for an effective treatment in individuals and for the purposes of public health control of the infection.16 Consequently, based on the 2009 findings, ciprofloxacin, tetracycline and penicillin were no longer recommended for the treatment of gonorrhoea in Morocco.
The local isolates were uniformly susceptible to spectinomycin and to the third-generation cephalosporins (ceftriaxone and cefixime). Spectinomycin played a central role in the control of gonococcal infection following the emergence of penicillinase producing N gonorrhoeae. It has been shown to be effective for the treatment of gonococcal infections15 but it is expensive and not available in Morocco.
Due to the high prevalence of fluoroquinolone resistance in many parts of the world, ceftriaxone is currently the drug of choice for the treatment of gonorrhoea.32 The third-generation cephalosporins have proved highly effective for more than a decade in this regard. Ceftriaxone is recommended as the drug of choice for gonorrhoea at all anatomical sites and it is favoured in comparison to other cephalosporins for its long serum half life, and relatively infrequent and mild side effects. Based on information from other international guidelines, Morocco recommended the use of a single dose of 250 mg of ceftriaxone administered intramuscularly as first-line treatment for gonococcal infection in the syndromic approach, commencing in 2010.19
In view of recent reports of decreased susceptibility of N gonorrhoeae to third-generation cephalosporins and reports of treatment failures in Japan,17 ,33 Norway,34 Austria,35 France,20 the UK and Sweden,36 and in some countries in the Western Pacific and South-East Asian regions,37 the Ministry of Health of Morocco has launched a national surveillance programme of AMR monitoring in N gonorrhoeae. Although the MICs of the cephalosporins in the 2009 study in Morocco are very low, the speed with which the MICs have crept up in other countries and regions of the world demands that such a programme of vigilance be instituted. This Gonococcal Antimicrobial Surveillance Programme in Morocco (Mor-GASP) was one of the components of the Global Fund proposals that Morocco submitted and was approved for funding from the 10th round. The Global Fund money will support the Mor-GASP for 5 years from 2012 to 2016 and then the Ministry of Health of Morocco will subsequently assume responsibility.
Mor-GASP aims to set up a system which will allow national biennial reporting of AMR. Its objectives are the timely detection of emerging trends in gonococcal resistance across Morocco and to provide a robust reference for informing national guidelines for therapy. The programme started in March 2013 and the monitoring has been implemented in five cities. The surveillance will be carried out over a period of 5 months every 2 years. The urethral specimens will be collected from men who present with urethral discharge in BHS and in some non-governmental health clinics in the five cities. The N gonorrhoeae isolates will be examined in the hospital laboratories in each city then sent to the National Reference Laboratory at the National Institute of Hygiene in Rabat to record the MICs.
Mor-GASP aims to provide susceptibility data for a range of therapeutically relevant antimicrobial drugs in a timely manner, to include an external quality assessment scheme, and to provide training in STI laboratory diagnosis, especially in monitoring AMR in N gonorrhoeae, to the laboratory staff involved in this programme.
Acknowledgments
We are thankful to our regional directors, physicians and nursing staff for their selfless patient care, regional laboratory staff and to our fellow doctors who helped us to perform this study. Special thanks to the Global Fund for their financial support.
References
Footnotes
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Handling editor Jackie A Cassell.
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Contributors AH participated in the design of the study protocol and the completion of the experimental work. She analysed and interpreted the data. She performed the literature search and drafted the article. FN participated in the critical revision of the manuscript for important intellectual content and gave final approval of the version to be published. BB participated in the design of the experimental work regarding the study. AB participated in the conception and design of the study protocol and has assured the coordination with healthcare facilities. AK participated in the conception and design of the study protocol and assured the coordination with healthcare facilities. RC assured the coordination of the teamwork. REA participated in the design of the study, revised the initial study protocol and assured the coordination of the teamwork.
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Funding The study was funded by the Global Fund to Fight AIDS, Tuberculosis and Malaria, Grant No Mor 607G02H.
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Competing interests None.
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Provenance and peer review Commissioned; externally peer reviewed.