DIAGN MICROBIOLINFECTDIS 1992;15:627-632

627

Cefmetazole and Trospectomycin in vitro Susceptibility Testing Interpretive Criteria and Quality Control Guidelines for Neisseria gonorrhoeae Ronald N. Jones, Meridith E. Erwin, John A. Washington, Franklin P. Koontz, Patrick R. Murray, and E. Hugh Gerlach

Cefmetazole and trospectomycin were tested in a multilaboratory trial to establish Neisseria gonorrhoeae susceptibility testing criteria and quality control (QC) guidelines. Cefmetazole was active against the penicillinase-producing isolates and has an MIC9o of 16 i~g/ml, the breakpoint MIC previously used for nonfastidious species. However, a single-dose gonorrhea regimen (1 g i.m.) would require a lower 2-4 i~g/m (28-32 ram) pending more clinical experience with higher and~or prolonged cefmetazole dosing regimens. Trospectomycin was ac-

tive (MIC9o, 8 I.~g/ml) against all spectinomycin-susceptible gonococci. A susceptible breakpoint MIC of /100 Neisseria gonorrhoeae Strains a Proposed Zone Diameter Criteria (mm) for Antimicrobial Agents (Disk Content) Cefmetazole (30 ~g) Cefotetan (30 ~g)C Cefoxitin (30 ~g)d Spectinomycin (100 ~g)e Trospectomycin (30 ~g)

Regression Formula (r)

Susceptible

Intermediate

y = y = y = ND/ y =

>/33 />26 />28 />18 317

28-32 20-25 24-27 15-17 14-16

17.3 - 0.22x (0.95) 15.4 - 0.22x (0.85) 16.4 - 0.22x (0.93) 18.2 - 0.25x (0.95)

aFrom Jones et al. (1989, 1990, and 1991). bMICcorrelate in micrograms per milliliter is found in parentheses. CFromJones et al. (1991). aFrom Jones et al. (1990). eFrom Jones et al. (1989). fNot done.

(~2) b (~2) (~2) (~32) (~16)

(4) (4) (4) (64) (32)

Resistant ~27 ~19 ~23 ~14 ~13

()8) (38) ()8) (3128) ()64)

Cefmetazole and Trospectomycin versus GC

>32

F:

i

J

4 I I

I I I

I

I

211 ]

I I

--

32

--

-~, 1 2 ~

16

--

'1

8

--

6 4 ~ 3

2

1221

4-

P~ 2 1

If

3 111

629

1

O.)

20m 0.5 0.25

-

0.12

-

16 mm, both values considered to be predictive of cefmetazole susceptibility when using Q 8-hr regimens (Jones et al., 1986; NCCLS, 1991). Clinical trials have demonstrated that cefmetazole multiple-dose regimens eradicated all (100%) N. gonorrhoeae strains (Griffith et al., 1989), and that the single-dose 1-g i.m. cefmetazole regimen produced 91%-95% clinical cure rate (Thomason et al., 1990). The N. gonorrhoeae isolates in this study with cefmetazole MICs of >2 &g/ml were usually penicillinresistant by nonenzymatic mechanisms of resistance. Also, the experience of clinical case responses having correlative cefmetazole MICs appears limited. We propose that the cefmetazole susceptibility breakpoint MIC be set at -33 mm for susceptible. Furthermore, until more information can be obtained, the MICs of >2-4 ~g/ml should be considered intermediate indicating (a) the uncertain response to single-dose regimens and (b) that multiple-dose schedules may be required to achieve the highest cure rates (Griffith et al., 1989) such as those used for pelvic inflammatory disease. The use of these proposed criteria produced no falsesusceptible or -resistant errors and a 93% absolute agreement between tests (Figure 1).

Interpretive Criteria for Trospectomycin The trospectomycin MIC zone diameter scattergram and proposed interpretive criteria are shown in Figure 2 and Table 1, respectively. Trospectomycin is an aminocyclitol antimicrobial agent derived by modifying spectinomycin in an effort to reduce toxicity and improve its spectrum and potency (Zurenko et al., 1988). Like spectinomycin, trospectomycin generally remains active against ~-lactamaseproducing gonococci (Zurenko et al., 1988; Barry et al., 1989). However, spectinomycin-resistant N. gonorrhoeae were also resistant to trospectomycin, with these strains usually having MICs of ->32 ~g/ml (Zurenko et al., 1988; Barry et al., 1989). Against spectinomycin-suspectible gonococci, trospectomycin was generally four- to 16-fold more active (Zurenko et

630

R.N. Jones et al.

Trospectomycin

(30 ug)

128

64

--

32

--

8

--

4

--

2

--

1

--

E 0

1

1 1

134

11

1

331

1

1 1

1 1 55

1

I 0.5

52

2

1

1 4

i

J

~

I 10

,

I

I

I

i

I

15

20

25

30

35

I = > 40

Zone Diameter (mm)

FIGURE 2 Trospectomycin MIC-zone diameter scattergram for 100 Neisseria gonorrhoeae strains. Solid lines indicate the proposed interpretive criteria for all organisms (Barry et al., 1989) including isolates of Neisseria gonorrhoeae. The diagonal line represents the regression line calculated from this investigation. al., 1988). Thus, a lower trospectomycin susceptibility breakpoint was proposed for rapid-growing aerobic species (Zurenko et al., 1988; Barry et al., 1989) and for the N. gonorrhoeae susceptibility tests. Clinical trial results using a single 250-mg i.m. dose of trospectomycin (Colletta et al., 1990) produced a 95% gonococcal eradication rate, although the pharyngeal cure rate was only 67%. Using a trospectomycin-susceptible breakpoint of ~16 ~g/ml (->17 ram), identical to that recommended for all other species (Zurenko et al., 1988; Barry et al., 1989), no false-susceptible or -resistant errors were observed (Figure 2). The absolute agreement between the disk diffusion and MIC trospectomycin tests was 99% after applying a 3-mm intermediate range of 14-16 mm (Barry et al., 1989). However, the regression equation from our study (y = 18.2 - 0.25x) differs markedly from that published by Barry et al. (1989), as for example, y = 18.1 - 2.8x. Because Colletta et al. (1990) reported that all trospectomycin MICs were at ~8 ~g/ml, these proposed breakpoints seem appropriate and potentially predictive for trospectomycin clinical use.

Disk Diffusion Test QC The inhibitory zone diameters from the six-laboratory trial of 30-~g cefmetazole and 30-~g trospectomycin disks (three lots each, 900 total tests) are listed

in Tables 2 and 3. The cefmetazole 30-~g disk mean and median zones of inhibition were very similar, and the recommended QC zone diameter range was 3136 mm. This was calculated from the medians method and modified to >--90% confidence (described below). The trospectomycin disk QC range was 28-35 mm also by application of that range incorporating ->90% of all qualified test results. This contrasts to the use of the all-test median (mm) + one-half the average zone range of all participants (Jones et al., 1989 and 1990). In this case, only 82.0% and 85.6% of the trospectomycin and cefmetazole results, respectively, would have been within the median-calculated QC limits (Tables 2 and 3). There were no significant differences among the tabulated GC agar base lots, disk lots, or the remaining participant laboratory zones using the common lot of GC agar medium.

Agar Dilution Test QC and Drug Stability A clear modal MIC was established for each study drug (data not shown). The proposed MIC QC range for cefmetazole was 0.5-2 g.g/ml using the modal MIC - 1 log2 dilution step. The trospectomycin MIC QC range for N. gonorrhoeae ATCC 49226 was 1-4 ~g/ml. During this phase of the trial, one laboratory (R.N.J.) performed replicate tests on stored agar dilution plates (2°-5°C for 21 days) containing cefmetazole or trospectomycin. No significant decline

Cefmetazole and Trospectomycin versus GC

TABLE 2

631

G C A g a r Base D i s k D i f f u s i o n Q u a l i t y - C o n t r o l R e s u l t s f r o m F i v e Q u a l i f y i n g L a b o r a t o r i e s U s i n g 30-p,g C e f m e t a z o l e D i s k s a n d Neisseria gonorrhoeae A T C C 49226 Zone Diameter (ram)

Lots Laboratories Unique lots A B C D E Total C o m m o n Lot Total All Lots Total

Proposed QC Ranges by Method

No. Tested

Median

Range

Medians

90% Confidence

60 60 60 60 60 300

34 35 34 35 32 34

32-36 32-36 28-42 34-37 31-33 2842

32-36 a

31-36 b

75

35

32-38

375

34

28-42

~Calculated from all laboratory median (34 ram) + one-half the average range (3 mm) = 34 -+ 2 mm or 32-36 mm that includes only 85.6% of results. b90% confidence range excludes lowest 5% and largest 5% of study zone diameters, for example, 28-, 30-, 37-, 38-, 39-, 40-, and 42-mm reports. A total of 94.4% of results were within this preferred range of the two quality-control proposals.

TABLE 3

G C A g a r Base D i s k D i f f u s i o n Q u a l i t y - C o n t r o l R e s u l t s f r o m F i v e Qualified Laboratories U s i n g 30-~g T r o s p e c t o m y c i n Disks and S t r a i n Neisseria gonorrhoeae A T C C 49226 Zone Diameter (mm)

Lots Laboratories Unique lots A B C D E Total C o m m o n Lot Total All Lots Total

Proposed QC Ranges by Method

No. Tested

Median

Range

Medians

90% Confidence

60 60 60 60 60 30fF

31 34 31 33 29 32

29-33 32-36 30-33 32-35 27-31 26-36

30-34 a ------

28-35 b ------

90

32

28-35

390

32

26-36

aCalculated from all laboratory median (32 mm) _+ one-half the average range (4 ram) = 32 -+ 2 mm or 30-34 mm that includes only 82.0% of zone results. b90% confidence range excludes smallest 5% and largest 5% of study zone diameters, for example, 26-, 27-, and 36-mm results. A total of 98.3% of reported zones were within this preferred range of the two quality-control proposals. cOne laboratory was omitted from analysis because of variations using the common lot of GC agar base. The total number of zones produced in this study was 450.

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of either drug by replicate (five tests) MIC QC strain analysis was observed in 3 weeks of storage. Cefmetazole and trospectomycin both have proven in vitro (Nakao et al., 1976; Peeters et al., 1984; Jones et al., 1986; Z u r e n k o et al., 1988; Barry et al., 1989) and clinical (Griffith et al., 1989; Colletta et al., 1990; Thomason et al., 1990) activity against N. gonorrhoeae and some other sexually transmitted pathogens (Zurenko et al., 1988). The in vitro susceptibility tests for these two antimicrobial agents were established for the recently standardized NCCLS (1990a and b) methods for the gonococcus. The suggested interpretive criteria for cefmetazole were consistent with those of comparable structural entities (cefoxitin, cefotetan) and those of other "second-generation"

R.N. Jones et al.

cephalosporins such as cefuroxime used following probenicid (Jones et al., 1990 a n d 1991). Trospectomycin most resembles the previously developed antigonococcal alternative agent spectinomycin (Jones et al., 1989; NCCLS 1990a and b).

The authors thank the numerous medical technologists at each participating laboratory for their excellent support. The following persons contributed suggestions, reviews, special technical support, or organisms to this study: G. Zurenko, M. Barrett, B. Briggs, D. Johnson, and S. Putnam. The word processing was skillfully provided by L. Miller. The study was funded in part by research grants from Lederle Laboratories, G.D. Searle and Company, and The Upjohn Company.

REFERENCES Barry AL, Jones RN, Thornsberry C (1989) Antibacterial activity of trospectomycin (U-63366F) and initial evaluations of disk diffusion susceptibility tests. Antimicrob Agents Chemother 33:569-572. Centers for Disease Control (1990) Plasmid-mediated antimicrobial resistances in Neisseria gonorrhoeae: United States, 1988 and 1989. Morbid Mortal Weekly Rep 39:284293. Colletta L, Caine VA, Linnemeier P, Newmann TM, Hook EW III, Jones RB, Rompalo AM (1990) Efficacy of 250 mg trospectomycin sulfate IM versus 250 mg ceftriaxone IM for treatment of uncomplicated gonorrhea [abstr 99]. In Program and Abstracts of the Interscience Conference on Antimicrobial Agents and Chemotherapy, Atlanta, GA. Washington, DC: American Society for Microbiology. Griffith DL, Novak E, Greenwald CE, Metzler CM, Paxton LM (1989) Clinical experience with cefmetazole sodium in the United States: an overview. ] Antimicrob Chemother 23(Suppl D):21-23. Ison CA, Littleton K, Shannon KP, Easmon CSF, Phillips I (1983) Spectinomycin resistant gonococci. Br Med J 287:1827-1829. Jones RN, Barry AL, Fuchs PC, Thornsberry C (1986) Antimicrobial activity of cefmetazole (CS-1170) and recommendations for susceptibility testing by disk diffusion, dilution, and anaerobic methods. J Clin Microbiol 24:1055-1059. Jones RN, Gavan TL, Thornsberry C, Fuchs PC, Gerlach EH, Knapp JS, Murray P, Washington JAII (1989). 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 27:2758-2766. Jones RI~, Fuchs PC, Washington JAII, Gavan TL, Murray PR, Gerlach EH, Thornsberry C (1990) Interpretive criteria, quality control guidelines, and drug stability studies for susceptibility testing of cefotaxime, cefoxitin, ceftazidime, and cefuroxime against Neisseria gonorrhoeae. Diagn Microbiol Infect Dis 13:499-508. Jones RN, Gerlach EH, Koontz FP, Murray PR, Pfaller MA, Washington JA, Erwin ME, Knapp CC (1991) Neisseria

gonorrhoeae susceptibility test development for cefotetan: interpretive criteria and quality control guidelines. J Clin Microbiol 29:363-366. Ko H, Cathcart KS, Griffith DL, Peters GR, Adams WJ (1989) Pharmacokinetics of intravenously administered cefmetazole and cefoxitin and effects of probenecid on cefmetazole elimination. Antimicrob Agents Chemother 33:356-361. Nakao H, Yangisawa H, Shimizu B, Kaneko M, Nagano M, Sugawara S (1976) A new semisynthetic 7a-methoxy cephalosporin, CS-1170. J Antibiot (Tokyo) 29:554-558. National Committee for Clinical Laboratory Standards (NCCLS) (1989) Tentative guideline M23-T: development of in vitro susceptibility testing criteria and quality control parameters. Villanova, PA: NCCLS. National Committee for Clinical Laboratory Standards (NCCLS) (1990a) Approved standard M2-A4: performance standards for antimicrobic disk susceptibility tests. Villanova, PA: NCCLS. National Committee for Clinical Laboratory Standards (NCCLS) (1990b) Approved standard M7-A2: standard methods for dilution antimicrobial susceptibility tests for bacteria that grow aerobically. Villanova, PA: NCCLS. National Committee for Clinical Laboratory Standards (NCCLS) (1991) Informational supplement M100-$3. Villanova, PA: NCCLS. Peeters M, Van Dyek E, Piot P (1984) In vitro activities of the spectinomycin analog U63,366 and four quinolone derivatives against Neisseria gonorrhoeae. Antimicrob Agents Chemother 26:608-609. Thomason JL, Gelbart SM, Scaglione NJ, James JA, Leong F, Broekhuizen FF (1990) Does cefmetazole compare to cefoxitin or penicillin for treatment of gonorrhea? Results of the multicenter study [abstr 98]. In Program and Abstracts of the Interscience Conference on Antimicrobiol Agents and Chemotherapy, Atlanta, GA. Washington, DC: American Society for Microbiology, Zurenko GE, Yagi BH, Vavra JJ, Wentworth BB (1988) In vitro antibacterial activity of trospectomycin (U-63366F), a novel spectinomycin analog. Antimicrob Agents Chemother 32:216-223.