Current Trends Disk Diffusion Antimicrobial Susceptibility Testing of Neisseria gonorrhoeae

Antimicrobial resistance in Neisseria gonorrhoeae has developed to each of the agents that have been recommended for gonorrhea therapy (1). As a result, a well-standardized laboratory method to monitor the susceptibilities of gonococcal isolates has been recommended by the National Committee for Clinical Laboratory Standards (NCCLS). The NCCLS recently completed a multicenter study to standardize disk diffusion (and agar dilution) susceptibility tests for N. gonorrhoeae and to establish interpretive criteria and quality-control guidelines (2). The recommended test medium is GC base agar with a defined "XV-like" supplement. Control organisms are N. gonorrhoeae ATCC 49226 (CDC F-18), N. gonorrhoeae WHO V, and Staphylococcus aureus ATCC 25923.

Interpretive criteria based on expected treatment failure rates for single-agent therapy with penicillin, tetracycline, spectinomycin, or ceftriaxone have been selected (Table 1). Resistance to penicillin is defined as a zone diameter of less than or equal to 26 mm (10-U disk), corresponding to a minimum inhibitory concentration (MIC) of greater than or equal to 2 ug/mL. Strains producing beta-lactamase (penicillinase-producing N. gonorrhoeae (PPNG)) produce zone sizes of less than or equal to 19 mm. Resistance to tetracycline is defined as a zone diameter of less than or equal to 30 mm (30-ug disk), also corresponding to an MIC of greater than or equal to 2 ug/mL. Strains producing zone diameters of less than or equal to 19 mm may be presumptively identified as having high-level plasmid-mediated resistance to tetracycline (tetracycline-resistant N. gonorrhoeae (TRNG)); the corresponding MIC of these strains is greater than or equal to 16 ug tetracycline/mL. Spectinomycin-resistant isolates produce zone sizes of less than or equal to 14 mm (MIC greater than or equal to 128 ug spectinomycin/mL) with a 100-ug disk. Only a susceptible criterion for ceftriaxone has been established (30-ug disk) because of the absence of treatment failures in patients treated with ceftriaxone, 250 mg, IM.

Cure rates of less than or equal to 85% would be expected for patients infected with organisms resistant to an antimicrobial agent when treated with that agent alone. Cure rates of greater than or equal to 95% would be expected for patients infected with susceptible organisms. Cure rates lower than those for infections caused by susceptible organisms may be expected for patients infected with moderately susceptible* organisms (2). Reported by: RN Jones, MD, Univ of Iowa College of Medicine, Iowa City, Iowa. Subcommittee on Antimicrobial Susceptibility Testing, National Committee for Clinical Laboratory Standards, Villanova, Pennsylvania. Div of Sexually Transmitted Diseases Laboratory Research, Center for Infectious Diseases; Div of STD/HIV Prevention, Center for Prevention Svcs, CDC.

Editorial Note

Editorial Note: Antimicrobial resistance in N. gonorrhoeae has been due either to multiple chromosomal mutations or to R-factor plasmids. The development of resistance to tetracycline due to chromosomal mutations (1) prompted a CDC recommendation in 1985 that tetracycline not be used as single-drug therapy for gonococcal infection (3). The subsequent emergence of plasmid-mediated resistance to tetracycline (4) affirmed that recommendation. In addition, increasing prevalence of strains containing beta-lactamase plasmids prompted the virtual abandonment of penicillins as single-dose therapies for gonorrhea in 1987 (5). Isolates with chromosomal resistance to alternative drugs such as spectinomycin have also been reported (1).

NCCLS criteria for interpreting disk diffusion susceptibility test results update previous CDC recommendations (5). Criteria for resistance to the four listed anti microbial agents are only slightly different from those previously published by CDC (5). For penicillin and spectinomycin, the criteria for resistance have been modified from less than or equal to 25 mm to less than or equal to 26 mm and less than or equal to 15 mm to less than or equal to 14 mm, respectively. The criteria for distinguishing moderately susceptible from susceptible organisms have undergone the greatest changes. The criteria for interpreting MIC values (5; Table 1) were modified either because of changes in the procedure for determining MICs (penicillin) or reevaluation of treatment outcome data (tetracycline and spectinomycin).

This report does not alter the recommended methods for detecting PPNG; such strains may be identified easily by the detection of beta-lactamase. Strains of N. gonorrhoeae that have chromosomally mediated resistance to antimicrobial agents or plasmid-mediated resistance to penicillin and/or tetracycline may be detected by measuring their susceptibilities by disk diffusion tests. Disk diffusion (or agar dilution) susceptibility tests alone can only identify TRNG isolates presumptively; TRNG can be confirmed only with genetic probes that specifically detect the TetM determinant.

Determining resistance is primarily a laboratory responsibility that affects both surveillance and patient care. The standardized test method and interpretive criteria permit comparison of results obtained in different health jurisdictions. Surveillance of susceptibilities based on carefully collected information permits the detection of emerging resistance that may necessitate revision of therapy recommendations.

Based on surveillance of gonococcal susceptibilities in 1988 and 1989, greater than 20% of isolates were resistant to penicillin and/or tetracycline (CDC, unpublished data); thus, single-drug therapy with these agents would be expected to result in unacceptably low cure rates. The use of a beta-lactam and tetracycline, in combination, may be expected to improve cure rates over those obtained with each agent individually. However, such dual beta-lactam/tetracycline therapy may be inadequate to cure infections caused by strains with chromosomal resistance to multiple agents or plasmid-mediated resistance (PPNG and/or TRNG) (5,6). Thus, it may be more difficult to correlate zone sizes or MICs with clinical outcome when dual therapies are used.

State and local health departments are encouraged to determine antimicrobial susceptibilities of isolates from selected patients. Isolates should be tested from patients with disseminated gonococcal infection or neonatal disease and from persons thought to have failed initial therapy. In addition, laboratories are encouraged to systematically monitor local patterns and trends of antimicrobial susceptibilities of isolates from uncomplicated infections (e.g., a sample such as the first 20 isolates each month) (7).

Ideally, susceptibilities to penicillin, tetracycline, spectinomycin, and ceftriaxone should be determined. At a minimum, susceptibilities to the antigonococcal agents used locally should be determined. If ceftriaxone is the primary antigonococcal agent, susceptibilities to penicillin, as well as ceftriaxone, can be used as a marker for possible emerging ceftriaxone resistance. Although all gonococcal strains are susceptible to ceftriaxone, strains chromosomally resistant to penicillin have exhibited decreased relative susceptibility to ceftriaxone (1). Susceptibility testing to tetracycline may be included to detect TRNG.

The disk diffusion testing protocol and supplemental control organisms that define individual types of resistance are available to laboratories from the Technical Services Branch, Scientific Resources Program, Center for Infectious Diseases, CDC, Mailstop C21, Atlanta, GA 30333; telephone (404) 639-3354.

References

1. Whittington WL, Knapp JS. Trends in antimicrobial resistance in Neisseria gonorrhoeae in the United States. Sex Transm Dis 1988;15:202-10.

2. Jones RN, Gavan TL, Thornsberry C, et al. Standardization of disk diffusion and agar dilution susceptibility tests for Neisseria gonorrhoeae: interpretive criteria and quality control guidelines for ceftriaxone, penicillin, spectinomycin, and tetracycline. J Clin Microbiol 1989;27:2758-66.

3. CDC. 1985 STD treatment guidelines. MMWR 1985;34(no. 4S).

4. Morse SA, Johnson SR, Biddle JW, Roberts MC. High-level tetracycline resistance in Neisseria gonorrhoeae is the result of the acquisition of the streptococcal tetM determinant. Antimicrob Agent Chemother 1986;30:664-70.

5. CDC. Antibiotic-resistant strains of Neisseria gonorrhoeae: policy guidelines for detection, management, and control. MMWR 1987;36(no. 5S).

6. CDC. 1989 Sexually transmitted diseases treatment guidelines. MMWR 1989;38(no. S-8).

7. Rice RJ, Hook EW III, Holmes KK, Knapp JS. Evaluation of sampling methods for surveillance of Neisseria gonorrhoeae strain populations. In: Poolman JT, Zanen HC, Meyer TF, et al., eds. Gonococci and meningococci. Dordrecht, Netherlands: Kluwer Academic Publishers, 1988:167-73. *The term "intermediate," used previously by CDC, has been replaced by the term "moderately susceptible"; "intermediate" is now used only for spectinomycin susceptibilities of 15-17 mm (MIC of 64 ug/mL) for which clinical experience is insufficient.

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