Apalata, Teke MBCHB*; Zimba, Tomas F. MBCHB*†; Sturm, Willem A. MD, PHD*; Moodley, Prashini MBCHB, PHD*
From the *Department of Medical Microbiology, Nelson R Mandela School of Medicine, University of KwaZulu-Natal, KwaZulu-Natal, South Africa; and †Central Hospital de Maputo, Maputo, Mozambique
This research project has been supported by a grant from the National Research Foundation of South Africa (Grant: 61767) (to P.M.).
Correspondence: Prashini Moodley, MBChB, PhD, Department of Medical Microbiology, Nelson R Mandela School of Medicine, University of KwaZulu-Natal, Durban, P/Bag 7, Congella, 4013, South Africa. E-mail: email@example.com.
Received for publication June 12, 2008, and accepted December 8, 2008.
The effective management of infection with Neisseria gonorrhoeae is a challenge in the face of increasing antimicrobial resistance.1–9 Penicillin and tetracycline are no longer recommended for treatment of gonorrhoea. Studies from Africa report that penicillinase-producing N. gonorrhoeae (PPNG) accounts for 15% to 82% of isolates whereas the prevalence of plasmid mediated resistance to tetracycline-resistant N. gonorrhoeae (TRNG) varies from 20% to 65%.2,8–10
Whilst aminoglycosides such as kanamycin and gentamicin continue to be used in Africa6,10–12 for treatment of gonorrhoea, there is a paucity of susceptibility data. An Ethiopian study reported kanamycin resistance in 17% of 68 tested isolates.11 In a study from Malawi, 9.5% of 63 isolates were resistant to gentamicin.2 An increase in the MICs of N. gonorrhoeae to spectinomycin, an aminocyclitol, has also been described.2,8,10
Bogaerts et al. (1993) reported on decreased susceptibility to norfloxacin in 21% and 39% of PPNG and non-PPNG isolates respectively in Rwanda.13 Moodley et al. reported on an increase in prevalence of ciprofloxacin-resistant N. gonorrhoeae in South Africa from 22% in 200314 to 42% in 2005.15 Resistance to third generation cephalosporins has not been documented in Africa.2,8,9
In Mozambique, the susceptibility profile of N. gonorrhoeae is not well documented. In 1997, 21% of tested isolates were TRNG and 64% were PPNG.2 In 2002, Mbofana et al. recommended the syndromic approach for the management of STDs.16 Genital discharge syndrome is treated with kanamycin 2 gram as a single intramuscular injection for potential infection with N. gonorrhoeae, doxycycline 100 mg twice daily for 7 days for potential infection with Chlamydia trachomatis; metronidazole as a single 2 g dose is added in nonpregnant women for potential infection with Trichomonas vaginalis and to a lesser extent bacterial vaginosis. In pregnant women, doxycycline is replaced by erythromycin 500 mg 4× daily for 5 days. Spectinomycin is used as second line treatment for patients who fail to respond to kanamycin.12 There are anecdotal reports of clinical failures following syndromic management. The aim of this study was to therefore assess the effectiveness of kanamycin for the treatment of gonorrhoea as part of the syndromic management package for genital discharge disease amongst patients attending a STD clinic in Maputo, Mozambique.
Consecutive patients presenting to the Centro de Saúde do Porto in Maputo between March 15, 2005 and April 30, 2005 with male urethritis syndrome or vaginal discharge syndrome were recruited. Written informed consent was obtained and the study was approved by the Ethics Committee of the Ministry of Health in Maputo, Mozambique (Ref: /CNBS/2005).
Probetec (Becton Dickinson) swabs were used to collect genital specimens. New York City plates [GC agar base supplemented with yeast autolysate, lincomycin, colistin, Amphotericin B, trimethoprim, (Oxoid Ltd., Basingstoke, UK), and lysed horse blood] were immediately inoculated after collection. Inoculated plates were placed in a candle extinction jar (at room temperature) and transported to the microbiology laboratory within 6 hours and incubated for 48 hours at 37°C in a CO2 incubator. Plates were only examined after 48 hours. Gram-negative, oxidase-positive organisms were subcultured onto New York City plates and incubated overnight. Overnight cultures of presumptive N. gonorrhoeae isolates were stored in Tryptic soy broth (Difco laboratories, Detroit, MI) containing 20% glycerol and transported to Durban, South Africa at −70°C. Identification of N. gonorrhoeae isolates was confirmed using carbohydrate utilisation tests in the Department of Medical Microbiology, Nelson R Mandela School of Medicine, University of KwaZulu-Natal, South Africa.
Nucleic acid amplification (Becton Dickinson Probetec Assays) tests were performed on all culture negative specimens. β-lactamase tests were performed using the chromogenic cephalosporin test (Nitrocefin, Oxoid). MICs to spectinomycin, ciprofloxacin, ceftriaxone, cefixime, tetracycline, and penicillin were determined using the CLSI methodology, formally known as NCCLS.17 MICs for kanamycin were performed using the agar dilution method and interpreted using the World Health Organization (WHO) recommendations.18 Staphylococcus aureus ATCC 29,213 and N. gonorrhoeae ATCC 49,226 were used as controls.
Susceptibility of N. gonorrhoeae was defined as follows:
Penicillin susceptible; MIC ≤0.06 mg/L.
PEN-R: chromosomally mediated penicillin resistant; MIC ≥2 mg/L in the absence of β-lactamase production.
PPNG; positive for β-lactamase production.
Tetracycline susceptible; MIC ≤0.25 mg/L.
Chromosomally mediated tetracycline resistant; MIC 2 to 8 mg/L.
High-level TRNG; MIC ≥16 mg/L.
Kanamycin susceptible: MIC ≤32 mg/L.
Kanamycin intermediate: MIC = 64 mg/L.
Kanamycin resistant: MIC ≥128 mg/L.
Spectinomycin susceptible: MIC ≤32 mg/L.
Spectinomycin resistant: MIC ≥128 mg/L.
Ciprofloxacin susceptible: MIC ≤0.06 mg/L.
Ciprofloxacin resistant: MIC ≥1 mg/L.
Ceftriaxone/cefixime susceptible: MIC ≤0.25 mg/L.
Ceftriaxone/cefixime decreased susceptibility: MIC >0.25 mg/L.
Two hundred seventy patients were recruited; 116 with male urethritis syndrome and 154 with female discharge syndrome. N. gonorrhoeae was cultured from 40 (34.5%) men and 15 (9.7%) women. BD Probetec assays yielded a further 2 positive in men and 4 more in women. The MICs of N. gonorrhoeae isolates to the various antimicrobials tested are shown in Table 1. Twenty-two (40%) were intermediate and 4 (7%) were resistant to kanamycin. Forty-two (77%) were TRNG, whilst 34 (65%) were PPNG; 51% were both TRNG and PPNG. All were susceptible to ciprofloxacin, ceftriaxone, and cefixime. Ninety-five percent displayed MIC = 64 mg/L for spectinomycin.
Twenty of the 55 (36%) culture positive patients returned within 7 days complaining of persisting symptoms. The kanamycin MICs of their isolates were: MIC ≥128 mg/L: 4/4 (100%) patients; MIC = 64 mg/L: 12/22(55%) patients; and MIC ≤32 mg/L: 4/29 (14%) patients.
The 4 patients with kanamycin MIC ≥128 mg/L, displayed MIC equal to 64 mg/L to spectinomycin. These patients did not respond to second line treatment with spectinomycin and were subsequently successfully treated with ceftriaxone.
This study illustrates that antibiotic-resistant N. gonorrhoeae is a major problem in Maputo, Mozambique. Kanamycin is the empirical choice for the treatment of gonorrhoea. Breakpoints for resistance and susceptibility to kanamycin have been based on theoretical predictions of MICs at which gonococcal infections may fail to respond to such treatment, as well as an extrapolation of breakpoints for aerobic Gram-negative bacteria.17–19 Studies have used different criteria to define kanamycin resistance and susceptibility.19–22 In this study, we used WHO criteria18 and linked this to clinical outcome. Thirty-six percent of patients failed first line therapy with kanamycin. Whilst reinfection cannot be ruled out as a cause for treatment failures, our data demonstrates that the treatment failures correspond with increasing kanamycin MICs.
The recommendation from WHO is to use drugs that are 95% effective as empirical treatment for the management of STIs.2–4 This implies that in Maputo, ineffective treatment is prescribed to patients presenting with genital discharge syndrome.
Spectinomycin is recommended as second line for patients who fail to respond to treatment with kanamycin.12 The 4 patients with kanamycin-resistant isolates failed to respond to spectinomycin. In all 4 spectinomycin, MIC was 64 mg/L. These patients were successfully treated with ceftriaxone.
Sixty-five percent of the isolates were PPNG and 33% PEN-R. In other African states, the prevalence of PPNGs ranged from 15% to 82%;2,9,10 and the prevalence of PEN-R ranged from 7% to 14%.2,9,10 The prevalence of TRNG in Mozambique increased significantly from 21% in 19972 to 77% in 2005. Although tetracyclines are prescribed as part of the syndromic management for genital discharge for potential infection with C. trachomatis, the high prevalence of TRNGs in this area makes it unlikely that this drug contributes to treatment of N. gonorrhoeae infections.
Although all isolates in our study were susceptible to ciprofloxacin, 1 isolate was found to have an increased MIC of 0.25 mg/L. The presence of 1 isolate with MIC of 0.25 mg/L is a cause for concern and may represent de novo resistance development or the introduction of strains with decreased susceptibility. The number of African countries, including neighbouring South Africa, reporting the presence of QRNG has increased.2,7,8,15,23 The situation in Mozambique about quinolone resistance has to be carefully monitored with regular surveillance to institute timely interventions when necessary.
All isolates were highly susceptible to both ceftriaxone and cefixime. There is no documented resistance to these agents from African studies.2,8,9
This is the first comprehensive study into the antimicrobial susceptibility profile of N. gonorrhoeae in Mozambique; the data presented may be useful in guiding the syndromic management protocols for genital discharge in Mozambique. Although, the study was restricted to 1 STD clinic with a limited number of N. gonorrhoeae isolates, the resistance to kanamycin as well as increased proportion of isolates with intermediate susceptibility to kanamycin remains a cause for concern. It is unlikely that these observations are restricted to this clinic. Based on this study, ciprofloxacin, ceftriaxone as well as cefixime may be suitable alternatives to kanamycin.
The limitation of this study is the small number of isolates tested over a relatively short period of time. In addition, these isolates were collected 3 years ago. These factors limit the value of any recommendation regarding the revision of the syndromic management protocols. This highlights the need for ongoing area specific surveillance to inform syndromic management protocols.
However, kanamycin is still part of syndromic management for genital discharge in Mozambique and this has major implications not only for the STI control programme, but for the HIV control programme as well.
1. World Health Organization programme for sexually transmitted diseases, Global Programme on AIDS. Recommendations for the management of sexually transmitted diseases. Geneva: WHO, 1994. WHO/GPA/TEM/94.
2. Tapsall J. Antimicrobial resistance in Neisseria gonorrhoeae.
Geneva: WHO, 2001. WHO/CDS/CSR/DRS/2001.3.
3. World Health Organization. Sexually transmitted diseases treatment strategies. Geneva: WHO, 1989. WHO/VDT/89.
5. Gerbase AC, Rowley JT, Heymann DHL, et al. Global prevalence and incidence estimates of selected curable STDs. Sex Transm Infect 1998; 74(suppl 1):S12–S16.
6. Johnson SR, Morse SA. Antibiotic resistance in Neisseria gonorrhoeae
: Genetics and mechanisms of resistance. STD laboratory program, Centres for Diseases Control, Atlanta, GA 30333. Sex Transm Dis 1988; 15:217–224.
7. Moodley P, Martin IMC, Pillay K, et al. Molecular epidemiology of recently emergent ciprofloxacin-resistant Neisseria gonorrhoeae
in South Africa. Sex Transm Dis 2006; 33:357–360.
8. Moodley P, Pillay C, Goga R, et al. Evolution in the trends of antimicrobial resistance in Neisseria gonorrhoeae
isolated in Durban over a 5 year period: Impact of the introduction of the syndromic management. J Antimicrob Chemother 2001; 48:853–859.
9. Van Dyck E, Crabbé F, Nzila N, et al. Increasing resistance of Neisseria gonorrhoeae
in West and Central Africa: Consequences on therapy of gonococcal infection. Sex Transm Dis 1997; 24:32–37.
10. Osoba AO, Johnston NA, Ogunganjo BO, et al. Plasmid profile of Neisseria gonorrhoeae
in Nigeria and efficacy of spectinomycin in treating gonorrhea. Genitourin Med 1987; 63:1–5.
11. Meless H, Abegaze B. Drug susceptibility of Neisseria gonorrhoeae
isolates from patients attending clinics for STDs in Addis Ababa. East Afr Med J 1997; 74:447–449.
12. Rui Bastos. Programma Nacional de Controle das DTS/SIDA. Maputo, Mozambique: PNCDTS/ SIDA Ministry of Health, 2002.
13. Bogaerts J, Tello WM, Akingeneye J, et al. Effectiveness of norfloxacin and ofloxacin for treatment of gonorrhoea and decrease of in vitro susceptibility to quinolones overtime in Rwanda. Genitourin Med 1993; 69:196–200.
14. Moodley P, Moodley D, Sturm AW. Ciprofloxacin-resistant Neisseria gonorrhoeae
in South Africa. Int J Antimicrob Agents 2004; 24:192–193.
15. Moodley P, Sturm AW. Ciprofloxacin-resistant gonorrhoea in South Africa. Lancet 2005; 366:1159.
16. Mbofana FS, Brito FJ, Saifodine A, et al. Syndromic management of sexually transmitted diseases at primary care level, Mozambique. Sex Transm Infect 2002; 78:E2.
17. National Committee for Clinical Laboratory Standards. Performance standard for antimicrobial susceptibility testing. Ninth Informational Supplement M2-A6. Villanova, PA: NCCLS, 1999.
18. Van Dyck E, Meheus AZ, Piot P. Laboratory Diagnosis of Sex Transm Dis. Geneva, Switzerland: World Health Organization, 1999.
19. Knapp JS, Wongba C, Limpakarnjanarat K, et al. Antimicrobial susceptibilities of strains of Neisseria gonorrhoeae
in Bangkok, Thailand: 1994–1995. Sex Transm Dis 1997; 24:142–148
20. Vorobieva V, Firsova N, Ababkova T, et al. Antibiotic susceptibility of Neisseria gonorrhoeae
in Arkhangelsk, Russia. Sex Transm Infect 2007; 83:133–135.
21. Lesmana M, Lebron CI, Taslim D, et al. In vitro antibiotic susceptibility of Neisseria gonorrhoeae
in Jakarka, Indonesia. J Antimicrob Chemother 2001; 45:359–362.
22. Marta C de Castillo, Olga A de Saab, Norma P de Fernandez, et al. Agar dilution method for susceptibility testing of Neisseria gonorrhoeae.
Mem Inst Oswaldo Cruz 1996; 91:789–793.
23. Mbwana J, Mhalu F, Mwakagile D, et al. Susceptibility pattern of Neisseria gonorrhoeae
to antimicrobial agents in Dares Salaam. East Afr Med J 1999; 76:330–334.