Sexually transmitted infections (STIs), including gonorrhea and other pathogens causing male urethral discharge (MUD), are associated with HIV acquisition and adverse health impacts such as infertility, ectopic pregnancy, and ophthalmia neonatorum.1 In addition, HIV-1–infected men with MUD are at increased risk for transmitting HIV-1 because genital tract shedding of HIV-1 is elevated in the presence of genital tract inflammation.2,3Neisseria gonorrhoeae is the major cause of MUD in several southern African countries.4–7 However, some of these published studies also report detection of other MUD-associated sexually transmitted pathogens at a prevalence exceeding 5%, including Chlamydia trachomatis, Trichomonas vaginalis, and Mycoplasma genitalium.4 Periodic etiological surveillance of MUD, as well as other common STI syndromes, is a critical component of the syndromic management approach as it validates existing treatment algorithms. However, with the exception of South Africa, such etiological surveys are infrequently performed on a regular basis in sub-Saharan African countries and laboratory-based STI surveillance requires regional strengthening.8
Until relatively recently, single-dose oral fluoroquinolones, for example, ciprofloxacin, were used to treat gonococcal infections. However, quinolone-resistant N. gonorrhoeae (QRNG) strains emerged in the mid-1990s and fluoroquinolones were gradually abandoned as first-line treatment of gonorrhea, initially in the western Pacific and southeast Asian regions then subsequently in the United States and Europe, and most recently in east and southern Africa.9–13 Within Zimbabwe, either a single-dose oral fluoroquinolone (norfloxacin) or single-dose intramuscular kanamycin has been used as first-line therapy for presumptive gonorrhea for several years. However, there have been no recent in-country gonococcal antimicrobial susceptibility surveys despite the recent emergence and rapid dissemination of QRNG in Zimbabwe’s neighbor, South Africa.10,13
In this cross-sectional survey, undertaken in 12 clinics within the city of Harare, we determined the prevalence of STI pathogens among men with urethral discharge and, in addition, the susceptibility of cultured N. gonorrhoeae isolates to a number of antimicrobial agents.
MATERIALS AND METHODS
The survey took place within the 12 clinics in the City of Harare between November 2010 and May 2011. These clinics span the whole city and provide primary health care services, including sexual and reproductive health services, to Harare’s residents.
Study Eligibility Criteria
The target population was men 18 years or older who had been sexually active within the last 3 months and who voluntarily accessed the clinics for STI management. Men were only enrolled if they were found to have a visible urethral discharge when examined by the clinic nurse; those men complaining of dysuria alone were not recruited. All survey participants provided informed written consent as part of the enrollment procedure. No data were collected on the number of eligible patients attending the clinics.
Data collection Procedures
After enrollment, the attending nurse recorded basic demographic, clinical, and behavioral data on a questionnaire. Specifically, data were recorded concerning age, ethnicity, use of antimicrobial agents within the last 3 months, referral from another clinic with persistent urethral discharge (i.e., discharge that was unresponsive to prior treatment elsewhere), geographic location of recent sex partners for the last 3 months, history of previous urethral discharge in the last 12 months, examination findings related to the presence or absence of urethral discharge, and epididymo-orchitis, as well as antimicrobial agents prescribed at the survey visit.
Two endourethral swabs were used to collect urethral discharge specimens. The first swab was initially used to prepare a urethral smear and then placed in Amies transport medium before transport to the Biomedical Research and Training Institute laboratory for gonococcal culture. The second swab was placed in a sterile plastic tube for storage at −70°C at the Biomedical Research and Training Institute laboratory pending subsequent molecular-based etiological testing at South Africa’s National Institute for Communicable Diseases (NICD) after recruitment was completed.
Clinic-Based Management of Participants
All participants were treated for urethral discharge in accordance with current Zimbabwean STI management guidelines, specifically either with single-dose oral norfloxacin (800 mg) or with single-dose intramuscular kanamycin (2 g), together with a 1 week’s course of oral doxycycline (100 mg 12 hourly). All participants were offered access to HIV counseling and testing. Participants were requested to abstain from sexual intercourse while on medication and until their current sexual partners were treated. All participants were given partner notification slips to assist with epidemiological treatment for all sexual partners.
The first swab was used to inoculate a New York City selective agar plate for N. gonorrhoeae culture. Inoculated plates were incubated for 24 to 48 hours in a 37°C incubator in a humidified atmosphere within candle jars. Presumptive gonococcal colonies were identified by Gram staining, oxidase production, and monoclonal antibody testing (Phadebact Monoclonal GC Test; Bactus AB, Uppsala, Sweden). β-Lactamase production was determined using nitrocefin-impregnated strips.
For antimicrobial susceptibility testing, agar plates composed of GC agar base (Oxoid, Hampshire, UK) and 1% BBL IsoVitalex Enrichment (Becton Dickinson, Le Pont de Claix, France) were inoculated with a suspension of gonococci equivalent to a MacFarlane standard of 1.0. Minimum inhibitory concentrations (MICs) were determined using Etests (bioMérieux, Marcy l’Etoile, France) for ciprofloxacin (as a prototype fluoroquinolone), kanamycin, ceftriaxone, and cefixime. Minimum inhibitory concentration breakpoints were in accordance with the Clinical and Laboratory Standards Institute for ciprofloxacin, cefixime, and ceftriaxone.14 Using World Health Organization (WHO) criteria, a kanamycin MIC of 32 mg/L or less was interpreted as susceptible, an MIC of 64 mg/L was reported as intermediate susceptibility and isolates with a kanamycin MIC of at least 128 mg/L were reported as resistant.15 World Health Organization N. gonorrhoeae type strains F and K were used as internal controls to quality assure susceptibility assays.16 Based on published Etest data, WHO strain F is susceptible to ciprofloxacin (MIC = 0.004 mg/L), kanamycin (MIC = 16 mg/L), cefixime (MIC < 0.016 mg/L), and ceftriaxone (MIC < 0.002 mg/L), whereas WHO strain K is susceptible to kanamycin (MIC 16 mg/L), exhibits reduced susceptibility to both cefixime (MIC = 0.5 mg/L) and ceftriaxone (MIC = 0.064 mg/L), and is resistant to ciprofloxacin (MIC > 32 mg/L).16
Gonococcal isolates were stored at −70°C in Microbank microbial storage vials (Pro-lab Diagnostics, Ontario, Canada) and sent to the NICD at the end of the survey for external quality assurance of the Etest-based antimicrobial susceptibility testing. In addition, the NICD reference laboratory also undertook MIC determinations for azithromycin, spectinomycin, gentamicin, penicillin, and tetracycline for all viable isolates using agar dilution methodology. Plates of GC agar base (Oxoid) and 1% BBL IsoVitalex Enrichment (Becton Dickinson) were prepared with varying concentrations of these 5 antimicrobial agents according to standard methods.17 The MIC breakpoints used for azithromycin were as follows: susceptible, MIC ≤ 0.25 mg/L; intermediate, MIC 0.5 mg/L; and resistant, MIC ≥ 1.18 For spectinomycin, susceptibility was defined as MIC ≤ 32 mg/L, intermediate susceptibility as an MIC equal to 64 mg/L, and resistance as MIC ≥ 128 mg/L.14 Gentamicin susceptibility was reported as high susceptibility (MIC ≤ 4 mg/L), moderate susceptibility (MIC 8–16 mg/L), or low susceptibility (MIC ≥ 32 mg/L).19 For penicillin, the MIC breakpoints used were as follows: susceptible, MIC ≤ 0.06 mg/L; intermediate, MIC 0.12 to 1 mg/L; and resistant, MIC ≥ 2 mg/L.14 Likewise, for tetracycline, isolates were also classified as susceptible (MIC ≤ 0.25 mg/L), intermediate (0.5 mg/L to 1 mg/L), or resistant (MIC ≥ 2 mg/L).14
Once recruitment was complete, the second swab specimens, placed in plastic tubes and initially stored at −70°C in Zimbabwe, were sent to the NICD on dry ice for etiological testing using a validated in-house multiplex polymerase chain reaction (M-PCR) assay. The multiplex PCR assay was used to detect the presence of N. gonorrhoeae, C. trachomatis, T. vaginalis, and M. genitalium, as described previously.4
Data Entry and Statistical Analysis
Data were entered and cleaned using a password-protected Microsoft Office Excel 2003 database. Participants’ demographic and clinical characteristics were summarized with descriptive statistics. The Pearson χ2 test was used to determine associations between the detection of gonorrhea and demographic/clinical characteristics with the level of significance set at P = 0.05. Where cell frequencies were less than 5, the Fisher exact test was used. All analyses were done in STATA version 12 (Stata Corp, College Station, TX).
Ethics approval for the surveillance study was granted by the Medical Research Council of Zimbabwe (Approval No. MRCZ/A/1587). The Directorate of Health Services for the City of Harare also granted approval to conduct the survey in the city’s clinics.
Baseline Characteristics of Study Participants
The key baseline characteristics of the study participants are shown in Table 1. Among the 130 men surveyed, most were in the 25- to 34-year age group (59 men; 45.4%), 21.5% (28 men) had previously used an antimicrobial agent in the last 3 months, and 25.4% (33 men) had sexual partners residing outside Harare. Eighteen men (13.9%) reported a history of a urethral discharge in the last 12 months, and 7 men (5.4%) presented during the survey with additional epididymo-orchitis. Seven men (5.4%) had come from another facility with persistent symptoms. Four of these men had laboratory-confirmed gonorrhoea, whereas no pathogens were detected in the urethral specimens of the other 3 men. N. gonorrhoeae was isolated from 2 of the 4 persistent gonorrhea cases (1 susceptible strain, 1 QRNG). Three of the 4 men with persistent gonorrhea received kanamycin (including the confirmed QRNG case), whereas the fourth man was treated with norfloxacin.
Etiology of MUD
An etiological diagnosis was established in approximately 90% of participants. The most common pathogen detected was N. gonorrhoeae (106 patients; 82.8%), followed by C. trachomatis (15 patients; 11.7%), M. genitalium (6 patients; 4.7%), and T. vaginalis (2 patients; 1.6%). Most men (102; 79.7%) had only 1 urethral pathogen detected. However, 12 men (9.4%) had 2 pathogens detected and 1 man (0.8%) had 3 pathogens detected. Gonorrhea was associated with younger age (P < 0.001) and a history of urethral discharge in the past 12 months (P = 0.04).
Antimicrobial Susceptibility of Gonococcal Strains
Gonococci were cultured from urethral swabs of 66 (62.3%) of those 106 men for whom N. gonorrhoeae had been detected in urethral specimens by M-PCR. Most gonococcal isolates (38/62; 61.3%) produced β-lactamase; results were unavailable for 4 isolates. Four (6.1%) of these 66 gonococcal isolates were resistant to ciprofloxacin, whereas all isolates were susceptible to kanamycin, cefixime, and ceftriaxone. Table 2 shows the MIC data for these 66 isolates by susceptibility category, including MIC50 and MIC90 values, for each of the 4 antibiotics tested.
After completion of the survey, the NICD laboratory in South Africa received 51 (77.3%) of these 66 gonococcal isolates in a viable state. Azithromycin, spectinomycin, gentamicin, penicillin, and tetracycline MICs were subsequently determined for this subset of isolates by agar dilution. Most (46; 90.2%) of the 51 isolates were susceptible to azithromycin, whereas there were 4 (7.8%) isolates with intermediate susceptibility and 1 resistant isolate (MIC =1 mg/L). Most isolates (48; 94.1%) were also susceptible to spectinomycin, whereas 3 (5.9%) isolates exhibited intermediate susceptibility to this antibiotic. There were no spectinomycin-resistant isolates detected. For gentamicin, 18 (35.3%) isolates were highly susceptible, 30 (58.8%) had moderate susceptibility, and 3 (5.9%) isolates had low susceptibility. Most isolates were resistant to penicillin (33; 64.7%) and tetracycline (46; 90.2%), and of note, there were no penicillin- or tetracycline-susceptible isolates.
This study reports the relative prevalence of STI pathogens responsible for urethral discharge in men attending primary health care clinics in Harare, Zimbabwe. N. gonorrhoeae was the predominant STI pathogen detected, followed by C. trachomatis, M. genitalium, and T. vaginalis. This study also reports antimicrobial susceptibility data for cultured N. gonorrhoeae isolates. Although most men presenting with urethral discharge were initiated on fluoroquinolone-based antimicrobial therapy, approximately 1 in 16 gonococci tested was resistant to this antimicrobial class. The reported data support continued use of kanamycin or a switch to either cefixime or ceftriaxone as alternative therapy.
Gonorrhea is one of the most common STIs in developing countries, and the rising prevalence of gonococcal antimicrobial resistance is a major threat to control efforts. During this study, norfloxacin formed part of the empiric treatment of STIs in Zimbabwe, yet the prevalence of microbiologically predicted fluoroquinolone-resistant gonorrhea was above the WHO’s 5% resistance threshold for changing first-line therapy.20 Given the susceptibility of all N. gonorrhoeae strains tested to kanamycin, cefixime, and ceftriaxone, any of these antimicrobial agents would be a suitable alternative to norfloxacin. Given that antimicrobial susceptibility data were available for less than two-thirds of the M-PCR–diagnosed gonorrhea infections, a further survey within Harare is urgently recommended to confirm our findings in respect of QRNG prevalence. In addition, before recommending a change in national gonorrhea treatment, we would recommend that additional surveys are conducted in other regions of the country.
A report from Durban a decade ago highlighted the rapid emergence of QRNG in the KwaZulu—Natal Province in South Africa.13 This finding triggered a national survey that demonstrated that all but one site surveyed had a QRNG prevalence above the WHO’s 5% threshold.21 Between 2004 and 2007, the prevalence of QRNG rose further in 2 South African cities, specifically from 11% to 32% in Johannesburg, and from 7% to 27% in Cape Town.10 A high prevalence of QRNG has also been reported in other surveys conducted elsewhere in the southern Africa region. For example, an antimicrobial susceptibility survey performed in Namibia (2007–2008) reported a 4.5% QRNG prevalence in Windhoek, but a 10-fold higher prevalence (48%) in the northern city of Oshakati.7 There were subsequent reports of unacceptably high QRNG prevalence in eastern Africa, for example, among young men attending an STI clinic in Kisumu, Kenya (16%), and among women with high-risk sexual behavior in Kampala, Uganda (83%).11,12
The data presented in this article suggest that kanamycin can still be used with some degree of confidence to treat men with gonorrhea in Harare. Recent data on kanamycin susceptibility are few because kanamycin is a relatively old antimicrobial agent and it is used in only a very few countries.6,9 However, given that some resource-poor countries do opt to use this antimicrobial agent for presumptive gonorrhea treatment and that we are facing the threat of untreatable gonorrhea, we would argue that it remains important to test for kanamycin susceptibility in N. gonorrhoeae in more country-based surveys, where resources permit. In respect of containment of antimicrobial resistance, the use of an injectable antimicrobial agent may result in limited opportunities for abuse relative to oral agents. In contrast, in a survey that sampled men and women with genital discharges attending an STI clinic in Mozambique in 2005, when the first-line treatment of presumptive gonorrhea was single-dose intramuscular kanamycin (2 g), almost half of the 55 N. gonorrhoeae isolates tested were nonsusceptible to kanamycin (intermediate susceptibility, 40%; resistance, 7%).6 There were some differences between this latter study and our own which may have contributed to these contrasting findings. First, Apalata et al. reported that kanamycin was in use as the sole first-line therapy for presumptive gonorrhea at the time of their study, whereas kanamycin has only been used intermittently as an alternative first-line regimen to oral norfloxacin in Zimbabwe. This may have reduced the pressure on gonococci to develop kanamycin resistance within Harare. Second, there were several methodological differences between the 2 studies with respect to MIC determination, choice of N. gonorrhoeae control strains, and use of an independent reference laboratory to verify MIC testing results.
In comparison to N. gonorrhoeae, the relative prevalence of C. trachomatis, M. genitalium, and T. vaginalis as a likely cause of MUD was relatively low in the population surveyed. These data are in agreement with published data from other African countries.4,22,23 For example, in a large South African study investigating the etiology of urethral discharge among men in Cape Town (n = 290) and Johannesburg (n = 217), Mhlongo et al.4 reported that N. gonorrhoeae was detected in 85% and 71% of MUD cases, respectively. Among men in Cape Town, C. trachomatis, M. genitalium, and T. vaginalis were detected at a lower prevalence of 13%, 4%, and 3%, respectively; likewise, in Johannesburg, the relative prevalence of C. trachomatis was 24%, whereas M. genitalium and T. vaginalis infections were both detected at a lower prevalence of approximately 13%. These observations are in stark contrast to North America and Europe, where nongonococcal urethritis is more frequently diagnosed than gonorrhea among MUD cases and where C. trachomatis may account for up to 45% of nongonococcal urethritis cases.24–27
Our study has a number of limitations. First, participant recruitment from the clinics was substantially lower than had been predicted from a presurvey review of the clinics’ monthly STI statistics. This raises concerns as to the representativeness of our sample. Second, only those men with clinically evident urethral discharge, confirmed by nurses on examination, were recruited. This would most likely overestimate the prevalence of N. gonorrhoeae versus C. trachomatis or M. genitalium as a cause of urethritis. We chose not to recruit men with symptoms of dysuria alone on the grounds that this can be a nonspecific symptom and which may have several non–STI-related causes in the African setting, including urinary tract infection, schistosomiasis, and chronic nonbacterial prostatitis. Furthermore, restricting enrollment to only those men with visible urethral discharge was chosen as the most cost-effective strategy to produce a high yield of N. gonorrhoeae strains for antimicrobial susceptibility testing. Third, this survey took place in Harare only, thus limiting data interpretation for national STI treatment guideline development. Finally, despite presurvey training of nursing staff on how to fill in the short survey questionnaire, there were some missing data on participants’ demographic and clinical characteristics which limited our data analyses. This observation highlights the challenge of conducting surveys in primary health care settings using existing nursing staff who are not familiar with research methodology.
In conclusion, this study emphasizes the importance of gonorrhea as the major etiological agent for MUD and highlights the emergence of QRNG within Harare. The antimicrobial susceptibility of the gonococcal isolates tested in our study suggests that either kanamycin or ceftriaxone should be recommended to treat gonorrhea in Harare. This recommendation comes with the provision that antibiotic choice should be supported by ongoing surveillance activities because resistance to extended spectrum cephalosporins has recently emerged in South Africa.17 In resource-limited settings, where alternative treatment regimens are few, it is now more important than ever that recommended STI syndromic management treatment guidelines are based on local susceptibility data and that these are closely adhered to. In addition, potential gonorrhea treatment failures should be identified and managed effectively to prevent further spread of antimicrobial-resistant N. gonorrhoeae strains.
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