Gonorrhea remains a significant health problem worldwide; recent WHO figures estimate a global total of 62 million new cases annually, with 17 million in Sub-Saharan Africa.1Neisseriagonorrhoeae infection facilitates HIV transmission,2–4 and effective treatment of gonococcal urethritis reduces HIV shedding in the genital tract.5 Thus, control of gonorrhea and other STDs is critical to efforts to prevent and control HIV transmission. Effective treatment eradicates infection in the affected individual, prevents the development of complications, and also reduces the duration of infection, thus decreasing transmission and eliminating reservoirs of infection. Antimicrobial resistance can compromise effective treatment of N. gonorrhoeae, lead to an increase of cases and burden the healthcare system.
Resistance to multiple classes of antimicrobials has developed over the past 60 years, and diminishing treatment options are a global concern.6 Internationally, several antimicrobial surveillance projects exist to monitor the development and spread of resistance. Two major programs are the Gonococcal Antimicrobial Susceptibility Program (GASP) monitored by the World Health Organization, and the Gonococcal Isolate Surveillance Project (GISP) monitored by the US Centers for Disease Control and Prevention. The Western Pacific Region GASP is a multicenter long-term program of continuous surveillance in 19 countries, while more limited regional WHO surveillance efforts are ongoing in Central and South America and South East Asia.7 Additionally, Australia, Canada, Great Britain, and several other European countries engage in antimicrobial resistance surveillance of gonorrhea. Periodic assessments of gonococcal susceptibility have been performed in African countries.8–12 As antimicrobial susceptibilities of prevailing gonococcal strains can change rapidly due to the nature of the bacteria, accurate assessments of susceptibility patterns are needed.
In Malawi, STD syndromic management was adopted in 1993 and Gentamicin 240 mg intramuscularly was selected as the first line regimen to treat urethritis in the syndromic algorithm,13 although neither the CDC nor the WHO has ever recommended gentamicin as either a primary or alternative choice for the management of gonococcal urethritis. The decision was based on cost and high clinical efficacy, demonstrated by a clinical cure rate of 95%.14 Clinical cure rates from 1994 to 2004 ranged from 91.8% to 94.8%15,16 and the prevalence of gonococcal urethritis in a variety of populations in Malawi has not increased over time and in most cases has declined.14–17 There are no interpretive criteria for Minimum Inhibitory Concentration (MIC) for gentamicin and N. gonorrhoeae, however the overall pattern of susceptibility remained unchanged during the same time period. Continued N. gonorrhoeae antibiotic susceptibility monitoring is important to inform STD syndromic treatment strategies. The objectives of this project were to (1) determine and monitor antimicrobial susceptibility patterns of N. gonorrhoeae strains isolated in Malawi, and (2) to compare results obtained by disk diffusion, E-test, and agar dilution MIC for gentamicin.
METHODS
Consecutive men presenting at the Kamuzu Central Hospital STI clinic in Lilongwe, Malawi with urethral discharge had history, genital exam, and urethral swabs taken. The patients were treated syndromically for urethritis with a single dose of gentamicin 240 mg intramuscularly and doxycycline 100 mg twice daily for 7 days. Consistent with the diagnostic standard of care, Gram stains were performed on urethral swab smears in the microbiology laboratory at the UNC Project in Lilongwe, Malawi. Urethral swabs from specimens containing intracellular Gram-negative diplococci consistent with N. gonorrhoeae infection were directly inoculated onto selective Thayer Martin agar media (GC agar base, bovine hemoglobin, IsoVitaleX, vancomycin, colistin, nystatin) for recovery of gonococcal isolates. Cultures were incubated at 35°C in a 5% CO2 atmosphere and inspected for growth indicative of N. gonorrhoeae after 24, 48, and 72 hours. From plates containing growth with oxidase positive colonies with morphology consistent with N. gonorrhoeae, isolated colonies from each specimen were confirmed to be Gram-negative diplococci and subcultured onto nonselective chocolate agar media (GC agar base, bovine hemoglobin, IsoVitaleX). Gonococcal culture stocks were used on site for disc diffusion and E-test susceptibility testing, and portions of each culture were frozen at −80°C and shipped frozen to the University of North Carolina (Chapel Hill, NC) for agar dilution MIC testing.
For disc diffusion and E-test susceptibility testing, inocula were prepared with N. gonorrhoeae isolates suspended in saline to a turbidity equivalent to a 0.5 McFarland BaSO4 standard. An Oxoid Turbidometer was used to verify the turbidity of the suspension. GC agar plates were streaked over the entire surface with a cotton swab to deliver the bacterial suspension. A 10 μg gentamicin disc (BBL Sensi Disc Antimicrobial Susceptibility test discs; Becton Dickinson) and a gentamicin E-test strip (AB Biodisk North America Inc., Piscataway, NJ) containing an exponential antibiotic gradient on one side and an MIC reading scale marked on the other side were firmly placed on the plate. Discs containing ceftriaxone (30 μg), cefixime (5 μg), spectinomycin (100 μg), kanamycin (30 μg), ciprofloxacin (5 μg), azithromycin (15 μg), penicillin (10 units), and tetracycline (30 μg) were also placed. Plates were incubated for 24 hours at 35°C in a 5% CO2 atmosphere. The diameter of the growth inhibition zone surrounding the antibiotic disks was read to the nearest millimeter and interpretation of susceptibility was based on guidelines provided by the Clinical Laboratory and Standards Institute (CLSI).18 There are CLSI criteria for determining susceptible, intermediate susceptibility, and resistant categories by disc testing for penicillin, spectinomycin, and ciprofloxacin; susceptibility breakpoints for ceftriaxone and cefixime; and resistance breakpoints for azithromycin. The E-test MICs for gentamicin were determined by reading the value at the point of intersection between the edge of the zone of growth inhibition and the E-test strip. The reference strain ATCC 49,226 was used before the monitoring began and monthly during the sample collection period.
MIC determination by agar dilution followed the current Centers for Disease Control and Prevention guidelines for the National Gonococcal Isolate Surveillance Project. The agar dilution MIC was defined as the lowest concentration of the antibiotic that completely inhibited the growth of the inoculum. Breakpoints for susceptible, intermediate susceptibility, and resistant were based on CLSI guidelines.18 Resistance breakpoints for azithromycin are based on CDC prevention guidelines as currently there are no CLSI-established criteria.19 There are no CLSI or CDC criteria for kanamycin or gentamicin. Ranges presented for gentamicin are based on previous clinical cure and MIC comparisons.14N. Gonorrhoeae ATCC 49,226 was used as a control.
The κ test statistic for interrater agreement between the agar dilution MICs and E-test MICs for individual isolates was calculated using Stata v. 10 (College Station, TX).
RESULTS
Between May 15 and August 10 2007, 126 urethral swabs were obtained from men presenting with urethral discharge, and 108 contained Gram-negative diplococci. N. gonorrhoeae was cultured from 106 (84%), and MICs were performed on 100.
Zone inhibition sizes on disk diffusion ranged from 8 to 29 mm (gentamicin); 0 to 48 mm (penicillin); 10 to 47 mm (tetracycline); 0 to 50 mm (azithromycin); 0 to 61 mm (ceftriaxone); 0 to 60 mm (cefixime); 16 to 44 mm (spectinomycin); 0 to 40 mm (kanamycin); and 18 to 52 mm (erythromycin).
In agar dilution MIC determination isolates were uniformly susceptible to ceftriaxone, spectinomycin, and gentamicin. Only one isolate (1%) was observed to be not susceptible to cefixime, and 1% of isolates demonstrated intermediate resistance to ciprofloxacin and kanamycin. High levels of resistance were observed to penicillin and tetracycline as seventy-seven (77%) isolates demonstrated resistance to tetracycline and 19 (19%) isolates were resistant to penicillin (Tables 1 and 2).
;)
TABLE 1: Susceptibility Profile for Gonococcal Isolates, 2007
TABLE 2: Antimicrobial Susceptibilities From Lilongwe, Malawi, 2007 (n = 100)
Agreement was observed in the susceptibility patterns identified by E-test and agar dilution MICs for gentamicin (Table 3).
TABLE 3: Comparison of Agar Dilution Assay and E-test Results for N. gonorrhoeae Susceptibility to Gentamicin, 2007
DISCUSSION
After 14 years of consistent use in treating urethritis, gonococcal isolates in Malawi continue to be sensitive to gentamicin. Effective treatment of gonococcal urethritis is essential to prevent adverse complications in the individual patient and to interrupt transmission chains. Single-dose therapy on first presentation is recommended by the WHO7 in order to achieve high compliance rates and to reduce further disease transmission as quickly as possible. When syndromic management principles are guiding treatment, knowledge of the local resistance patterns in gonorrhea is necessary to develop standard treatment regimens.
Urethritis is a common STI syndrome in Malawi, accounting for almost a quarter of all male STI clinic visits.20 Consistent with previous studies, gonorrhea continues to be a significant cause of urethritis in Malawi accounting for 85% of urethritis.21 Gentamicin was selected as the recommended treatment for urethritis in the syndromic format in Malawi in 1993 because it demonstrated high clinical efficacy,14 was inexpensive relative to other treatment options, and administered as a single injection, is not subject to abuse. Although intramuscular injection reduces the potential for abuse, the route of administration raises concerns about the risk of used needles and the transmission of blood-born pathogens, including HIV and hepatitis. Additionally, there are no existing data on the possible cumulative side effects such as ototoxicity and nephrotoxicity related to recurrent gentamicin use for those who are repeatedly treated. However, Malawi has now used gentamicin extensively and exclusively for the treatment of urethral discharges for 14 years without any such obviously related toxicities. Aminoglycosides are currently under consideration as an alternative treatment for gonorrhea in the United States6 and have been recommended for use as primary drugs for gonorrhea in Indonesia,22 where data from over a decade of susceptibility surveys provides additional evidence for the lack of emerging resistance to these agents.23–25
Our results are consistent with regional data, which demonstrate high levels of tetracycline and penicillin resistance while most isolates are susceptible to ciprofloxacin and cephalosporins.8,10,12 In addition to gentamicin, ciprofloxacin is currently effective against gonococcal urethritis in Malawi. However, increasing resistance to ciprofloxacin used as part of syndromic regimens has been described regionally. In Durban South Africa, where ciprofloxacin is a first line agent in the syndromic management of gonorrhea, all isolates collected between 1995 and 2002 were completely susceptible to ciprofloxacin. In 2003 the prevalence of ciprofloxacin-resistant N. gonorrhoeae was 22% and had increased to 42% by 2005.26 Quinolone resistance also increased rapidly in Cape Town and Johannesburg, and South Africa has now replaced ciprofloxacin with cefixime as first line treatment for gonorrhea.27 To date there has been no documented resistance to cefixime in the African setting. If the resistant isolate we observed is confirmed, it would be a most worrying development. Investigation to confirm and describe the nature of this resistance is ongoing.
E-test MICs have compared favorably to agar dilution for gonococcal isolates15,28 and the high level of agreement we observed between the susceptibility classification based on agar dilution MICs and disk diffusion and between agar dilution MICs and E-test MICs suggest future susceptibility monitoring can be performed locally in a developing country laboratory setting. The use of a turbidometer to standardize turbidity of the culture suspension on the media likely improved the correlation between methods compared to previous susceptibility surveys.15 However, the majority of our isolates were in the susceptible range and discrepancies may become more apparent at higher ranges of MICs. In addition, while high agreement was observed between the overall susceptibility classification between methods, the correlation between the MIC as measured by E-test and agar dilution was low. Given the absence of interpretive criteria for aminoglycoside susceptibility for N. gonorrhoeae, incompletely resolved comparisons between the test methods, and the difficulty in obtaining clinical/in vitro correlates in this difficult setting, the current study provides only indicative information regarding the interchangeability of the test methods used. Although not available at the time of this study, the new set of WHO control strains which has been fully characterized phenotypically and genotypically for relevant resistant determinants will improve future antimicrobial susceptibility studies and allow comparisons of data.29 Susceptibility surveillance can and should be done more frequently and over a wider geographic area in order to tailor local treatment recommendations to local trends.
These data are from a single STI clinic in an urban area. However, this population would be expected to have a higher prevalence of antimicrobial resistance than the general Malawi population as STI rates are higher in urban areas in Malawi30 and STI clinics manage a disproportionate number of referral cases.31 Another limitation of these results is the absence of individually linked clinical cure data. However, a retrospective review of the clinic visits during the sample collection period showed only one patient returned to the clinic with continuing symptoms.
Effective treatment of gonococcal urethritis is important to reduce the burden of STIs and to reduce HIV transmission. Malawi has a high burden of both STI and HIV, with an estimated 7% STI prevalence and 14% HIV prevalence.30 Continued monitoring of treatment efficacy in Malawi and other countries in the region with a high dual burden is a critical component of a comprehensive STI control and HIV prevention strategy. More data are also urgently needed on the safety profile and proper dosing of alternative regimens for gonococcal urethritis, such as gentamicin, that are accessible to low income countries.
REFERENCES
1. World Health Organization. Global Prevalence and Incidence of Selected Curable Sexually Transmitted Infections. Geneva, Switzerland: WHO; 2001.
2. Laga M, Manoka A, Kivuvu M, et al. Non-ulcerative sexually transmitted diseases as risk factors for HIV-1 transmission in women: Results from a cohort study. AIDS 1993; 7:95–102.
3. Zhang J, Li G, Bafica A, et al.
Neisseria gonorrhoeae enhances infection of dendritic cells by HIV type 1. J Immunol 2005; 174:7995–8002.
4. Chen A, Boulton IC, Pongoski J, et al. Induction of HIV-1 long terminal repeat-mediated transcription by
Neisseria gonorrhoeae. AIDS 2003; 17:625–628.
5. Cohen MS, Hoffman IF, Royce RA, et al. Reduction of concentration of HIV-1 in semen after treatment of urethritis: Implications for prevention of sexual transmission of HIV-1. Lancet 1997; 349:1868–1875.
6. Workowski K, Berman M, Douglas J. Emerging antimicrobial resistance in
Neisseria gonorrhoeae: Urgent need to strengthen prevention strategies. Ann Intern Med 2008; 148:606–613.
7. WHO. Management of sexually transmitted diseases. Document WHO/GPA/TEM94.1 Rev. 1. Geneva, Switzerland: World Health Organization; 1997.
8. Apalata T, Zimba TF, Sturm WA, et al. Antimicrobial susceptibility profile of
Neisseria gonorrhoeae isolated from patients attending a STD facility in Maputo, Mozambique. Sex Transm Dis 2009; 36:341–343.
9. Cao V, Ratsima E, Van Tri D, et al. Antimicrobial susceptibility of
Neisseria gonorrhoeae strains isolated in 2004-2006 in Bangui, Central African Republic; Yaounde, Cameroon; Antananarivo, Madagascar; and Ho Chi Minh Ville and Nha Trang, Vietnam. Sex Transm Dis 2008; 35:941–945.
10. Mbwana J, Mhalu F, Mwakagile D, et al. Susceptibility pattern of
Neisseria gonorrhoeae to antimicrobial agents in Dar es Salaam. East Afr Med J 1999; 76:330–334.
11. Tadesse A, Mekonnen A, Kassu A, et al. Antimicrobial sensitivity of
Neisseria gonorrhoeae in Gondar, Ethiopia. East Afr Med J 2001; 78:259–261.
12. Van Dyck E, Karita E, Abdellati S, et al. Antimicrobial susceptibilities of
Neisseria gonorrhoeae in Kigali, Rwanda, and trends of resistance between 1986 and 2000. Sex Transm Dis 2001; 28:539–545.
13. Management of Sexually Transmitted Infections Using Syndromic Management Approach: Guidelines for Service Providers. Lilongwe, Malawi: Malawi Ministry of Health; 2007.
14. Lule G, Behets FM, Hoffman IF, et al. STD/HIV control in Malawi and the search for affordable and effective urethritis therapy: A first field evaluation. Genitourin Med 1994; 70:384–388.
15. Daly CC, Hoffman I, Hobbs M, et al. Development of an antimicrobial susceptibility surveillance system for
Neisseria gonorrhoeae in Malawi: Comparison of methods. J Clin Microbiol 1997; 35:2985–2988.
16. Price MA, Zimba D, Hoffman IF, et al. Addition of treatment for trichomoniasis to syndromic management of urethritis in Malawi: A randomized clinical trial. Sex Transm Dis 2003; 30:516–522.
17. Taha TE, Brown ER, Hoffman IF, et al. A phase III clinical trial of antibiotics to reduce chorioamnionitis-related perinatal HIV-1 transmission. AIDS 2006; 20:1313–1321.
18. CLSI. Performance standards for antimicrobial susceptibility testing. 15th Informational supplement. Wayne, PA: Clinical and Laboratory Standards Institute; 2005.
19. CDC B88.
Neisseria Gonorrheae Reference Strains for Antimicrobial Susceptibility Testing. Atlanta, GA: Centers for Disease Control and Prevention; 2005.
20. Brown L, Krysiak R, Kamanga G, et al. HIV testing and
Neisseria gonorrhoeae antimicrobial susceptibility among men with urethritis in Lilongwe, Malawi. In: XVII International AIDS Conference; 2008; Mexico City. Abstract no MOPE0379.
21. Hobbs M, Kazembe P, Reed AW, et al. Trichomonas vaginalis as a cause of urethritis in Malawian men. Sex Transm Dis 1999; 26:381–387.
22. Ieven M, Van Looveren M, Sudigdoadi S, et al. Antimicrobial susceptibilities of
Neisseria gonorrhoeae strains isolated in Java, Indonesia. Sex Transm Dis 2003; 30:25–29.
23. Joesoef MR, Knapp JS, Idajadi A, et al. Antimicrobial susceptibilities of
Neisseria gonorrhoeae strains isolated in Surabaya, Indonesia. Antimicrob Agents Chemother 1994; 38:2530–2533.
24. Lesmana M, Lebron CI, Taslim D, et al. In vitro antibiotic susceptibility of
Neisseria gonorrhoeae in Jakarta, Indonesia. Antimicrob Agents Chemother 2001; 45:359–362.
25. Sutrisna A, Soebjakto O, Wignall FS, et al. Increasing resistance to ciprofloxacin and other antibiotics in
Neisseria gonorrhoeae from East Java and Papua, Indonesia, in 2004—implications for treatment. Int J STD AIDS 2006; 17:810–812.
26. Moodley P, Sturm AW. Ciprofloxacin-resistant gonorrhoeae in South Africa. Lancet 2005; 366:1159.
27. Lewis DA, Scott L, Slabbert M, et al. Escalation in the relative prevalence of ciprofloxacin-resistant gonorrhoeae among men with urethral discharge in two South African cities: Association with HIV seropositivity. Sex Transm Infect 2008; 84:352–355.
28. Van Dyck E, Smet H, Piot P. Comparison of E test with agar dilution for antimicrobial susceptibility testing of
Neisseria gonorrhoeae. J Clin Microbiol 1994; 32:1586–1588.
29. Unemo M, Fasth O, Fredlund H, et al. Phenotypic and genetic characterization of the 2008 WHO
Neisseria gonorrhoeae reference strain panel intended for global quality assurance and quality control of gonococcal antimicrobial resistance surveillance for public health purposes. J Antimicrob Chemother 2009; 63:1142–1151.
30. Malawi Demographic and Health Survey 2004. Calverton, MD: National Statistical Office (Malawi) and ORC Macro; 2005.
31. Tapsall J. Antibiotic Resistance in
Neisseria gonorrhoeae. Clin Infect Dis 2005; 41(suppl 4):S263–S268.
