Development of Neisseria gonorrhoeae in vitro susceptibility test methods for cefixime including quality control guidelines

DIAGN MICROBIOLINFECTDIS 1991;14:383-388

383

Development of Neisseria gonorrhoeae in vitro Susceptibility Test Methods for Cefixime Including Quality Control Guidelines Ronald N. Jones, E. Hugh Gerlach, Frank P. Koontz, Patrick R. Murray, Michael A. Pfaller, John A. Washington, Meridith E. Erwin, and Cynthia C. Knapp

Cefixime was tested in a multilaboratory study to establish susceptibility testing interpretive criteria and quality control (QC) guidelines for Neisseria gonorrhoeae. Interpretive criteria were established by using triplicate testing of 102 representative gonococcal strains. Only a susceptible category was proposed for cefixime (931 mm and 40.25 I~g/ml) because of very rare, resistant isolates. QC ranges [N. gonorrhoeae American Type Culture Collection (ATCC) 49226] were estab-

lished using multiple GC agar lots, three disk lots, and a number of test replicates consistent with the National Committee for Clinical Laboratory Standards (NCCLS) M23-T guidelines. Cefixime was stable in GC agar medium for at least 21 days when stored at 2°-5°C. Oral third-generation cephalosporins, such as cefixime, appear to represent potentially useful clinical alternatives to parenteral drugs of the same class for single-dose therapy of gonorrhea.

INTRODUCTION

mycin, or drug combinations (CDC, 1987). Most recently, a nationwide N. gonorrhoeae susceptibility surveillance study (CDC, 1990b) documented an increasing frequency of plasmid-mediated penicillin and tetracycline resistance and the emergence of chrornosomally mediated penicillin resistance. As these resistance mechanisms become more prevalent among the gonococci, the requirement for accurate susceptibility testing also becomes very important for surveillance, antimicrobial research, epidemiology, and formulation of therapeutic guidelines for treatment failures (CDC, 1990a; Jones et al., 1991). The National Committee for Clinical Laboratory Standards (NCCLS) Gonococcal Working Group has published a standardized procedure for the disk diffusion and agar dilution methods (NCCLS, 1990a and b). Criteria have been proposed for penicillin, tetracycline, spectinomycin, ceftriaxone, cefotaxime, cefoxitin, ceftazidime, and cefuroxime (CDC, 1990a; Jones et al., 1989 and 1990). Among the orally administered cephalosporins, cefuroxime axetil has in vitro testing criteria (Jones et al., 1990) and acceptable cure rates reported from clinical trials (Fong et

The therapy of uncomplicated Neisseria gonorrhoeae infections has markedly changed to now include 13lactamase-stable cephalosporins [Centers for Disease Control (CDC), 1987, 1989, and 1990a and b]. These changes are recommended therapy and were prompted by the fact that gonococci had evolved several antimicrobial resistance mechanisms requiring increases in penicillin dosing or the use of alternative regimens such as tetracycline, spectinoFrom the Universityof Iowa Hospitals and Clinics, College of Medicine (R.N.J., M.E.E., F.P.K., M.A.P.), and the Department of Veterans AffairsMedicalCenter (R.N.J., M.A.P.), Iowa City, Iowa; St. Francis RegionalMedical Center (E.H.G.), Wichita, Kansas; Barnes Hospital (P.R.M.), St. Louis, Missouri; and the ClevelandClinicFoundation(J.A.W., C.C.K.), Cleveland, Ohio, USA. Address reprint requests to Prof. R.N. Jones, Department of Pathology, Universityof Iowa Collegeof Medicine, Iowa City, IA, 52242, USA. Received 14 August 1990; revised and accepted 1 November 1990. © 1991 ElsevierScience PublishingCo., Inc. 655 Avenue of the Americas, New York, NY 10010 0732-8893/91/$3.50

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aI., 1986; Gottlieb and Mills, 1986; Reichman et al., 1985; Schift et al., 1986). Newer oral cephalosporins (cefixime, cefetemet), more structurally similar to parenteral third-generation agents, have also been proven to be effective in early clinical studies (Kuhlwein and Nies, 1989; Megran et al., 1990; Tio et al., 1990). Thus, in vitro tests for cefixime appear to be needed. The subject of this report is to present interpretive criteria, stability determinations in agar, and quality control (QC) guidelines for cefixime, based on studies in six laboratories.

MATERIALS A N D METHODS Antimicrobial Agents Cefixime was obtained from Lederle Laboratories of Pearl River, NY. Ceftriaxone was provided by Hoffman-LaRoche (Nutley, NJ). Cefixime and cefotaxime (control) disks for regression comparisons were manufactured by Difco Laboratories (Detroit, MI). Diffusion test disk lots for the QC studies were obtained from Becton Dickinson Microbiology Systems (BDMS, Cockeysville, MD) and Difco Laboratories. Three lots for each drug were utilized: two Difco lots (783855 and 691346) and one BDMS lot (907554).

R.N. Jones et al.

o m m e n d e d by the NCCLS (1990a and b). All tests were performed on GC agar base using a defined supplement (NCCLS, 1990a and b) without cysteine (Prepared Media Laboratories, Tualatin, OR). Incubation was carried out at 35°C in 5%-7% CO2, and minimum inhibitory concentrations (MICs) were read at 20-24 hr. All strains tested in the regression-interpretive criteria studies were processed in triplicate by each susceptibility testing method. The mean zone diameter rounded to the nearest whole millimeter and modal log2 dilution step were used to calculate regression statistics. In nearly all cases, this represented a single MIC value from all three tests or two identical MIC results with the remaining endpoint at an adjacent dilution step. QC trials generated a minimum of 60 zones and 20 MICs per drug-organism pair at each participating facility using their unique GC agar lot. Each participant also used a common lot of GC agar base. Thus, the study sites produced 90 zone diameters and 30 MICs determined on that lot (BDMS A2DVHH). The other GC agar lots used successfully in this protocol were BDMS lots A3DWXC and K2GWPM, Difco lots 774555, 774960, and 783222, and Oxoid lot 292-41686. A detailed description of the study design was cited previously (Jones et al., 1989 and 1990).

Organisms

Statistical Methods

Two groups of organisms were tested, one for the quality control trials and the second for the determinations of cefixime test interpretive criteria. The three QC organisms currently recommended by the NCCLS (1990a and b) were provided to the participants for replicate testing. The organisms included N. gonorrhoeae ATCC 49226 and Staphylococcus aureus strains ATCC 25923 or ATCC 29213. The 102 strains of N. gonorrhoeae tested to determine susceptibility breakpoints were selected to possess various drug resistances as follows: penicillinase-producing N. gonorrhoeae (PPNG, 31 strains), penicillin-resistant ~-lactamase-negative isolates (CMRNG, 42 strains), and recent penicillin-susceptible clinical isolates (29 strains). In addition, several strains were resistant to tetracycline and spectinomycin (Jones et al., 1989). The organism collection strains came from worldwide locations and included strains kindly provided by the US Navy research facilities (San Diego, CA). All strains were confirmed to be gonococci by established procedures. All organisms were isolates from human genital cultures.

Cefixime and cefotaxime MICs were compared with their zone diameters around commercially prepared 5- and 30-~g disks, respectively. Where possible, suggested susceptible breakpoints conformed to those of similar agents published in NCCLS or international documents (CDC, 1990a; Jones et al., 1989 and 1990; NCCLS, 1990a and b). The control cefotaxime regression was consistent with that previously published (Jones et al., 1990).

Susceptibility Testing Methods The susceptibility tests were performed by the agar dilution and disk diffusion methods recently rec-

RESULTS Interpretive Criteria for Cefixime Figure 1 and Table 1 present the results of the cefixime interpretive criteria studies. By GC agar base dilution tests, cefixime was very active against all strains of N. gonorrhoeae with an MIC10o of 0.03 ~g/ml and an MIC50 of 0.008 ~g/ml. Direct comparative cefixime MICs with ceftriaxone (Table 2) revealed that cefixime was equal to or more potent than ceftriaxone against penicillin-resistant strains. All 5-~g cefixime disk zone diameters were ---37 mm (Figure 1). Organisms having elevated penicillin MICs were also less susceptible to cefixime (data not shown). The Figure 1 regression-line scattergram dem-

Cefixime versus Gonococci

385

.5 .25

~

.12

CEFIXIME (5 ug)

.06

tO

.03 12

.015

1 1

~

1 2 4

.008

/

~ ,/

: - ~ ~ 1

I

2

;, .004 .002 i~ 25

I

!

1

L

4

30

35

40

45

50

Cefixime

Zone Diameter

I

-)55

(ram)

FIGURE 1 Cefixime scattergram comparing 5-p,g disk zone diameters to MICs. Vertical line is p r o p o s e d susceptible zone interpretive criterion. No resistant strains have been isolated.

TABLE 1

R e g r e s s i o n S t a t i s t i c s a n d P r o p o s e d S u s c e p t i b i l i t y C r i t e r i a for C e f i x i m e , C e f o t a x i m e , a n d C e f t r i a x o n e D e r i v e d f r o m S t u d i e s o f 100 o r 102 Neisseria gonorrhoeae S t r a i n s Proposed Criteria in m m (~g/ml) for

Antimicrobial Agent (Disk Content)

Regression Formula (r)

Cefixime (5 ~g) Cefotaxime (30 ,,g)b Ceftriaxone (30 ~g)C

Y = 7.8 - 0.14x (0.54) Y = 15.4 - 0.26x (0.79) Y = 10.7 - 0.19x (0.65)

Susceptible

Intermediate or Moderately Susceptible a

Resistant

1>31 (~0.25) I>31 (~0.5) >t35 (~0.25)

NA NA NA

NA NA NA

NA, not applicable. "Moderately susceptible organisms have a documented lower clinical response rate (85%-95%) compared with >95% for susceptible strains (CDC, 1987). An intermediate or indeterminate result for an antimicrobial agent indicates either a technical problem that should be resolved by repeated testing or a lack of clinical experience in treating organisms with these zones or MICs. bData modified from Jones et al. (1990). CData modified from Jones et al. (1989).

TABLE 2

D i r e c t C o m p a r i s o n R a t i o s of C e f i x i m e - C e f t r i a x o n e A c t i v i t y a n d G e o m e t r i c M e a n M I C s T e s t i n g S e l e c t e d P e n i c i l l i n - R e s i s t a n t Neisseria gonorhoeae S t r a i n s Cefixime-Ceftriaxone MIC =

Geometric Mean MIC

Resistant Mechanism

No. of Isolates

/>4

2

1

0.5

~<0.25

Cefixime

Ceftriaxone

Penicillinase p r o d u c i n g CMRNG b All strains

20 17 37

0 1 1

6 0 6

11 5 16

3 9 12

0 2 2

0.005 a 0.016 0.010

0.005 0.030 0.017

"MIC in ~g/ml. bCMRNG, chromosomal-resistant N. gonorrhoeae;most strains were also resistant to tetracycline,

386

R.N. Jones et al.

onstrates a modest linear relationship between the MIC and zone diameter; however, this had a poor correlation coefficient (r = 0.54). As with other previously studied third-generation cephalosporins (cefotaxime, ceftriaxone, ceftazidime), no documented resistant isolates have been observed clinically. Therefore, we suggest (Table 1) only susceptible criteria (/>31 mm and 40.25 t~g/ml) to be consistent with the interpretive breakpoints of comparable cephalosporin agents (Jones et al., 1989 and 1990). No interpretive errors were identified using those test strains that had a very high incidence of [3-1actam resistance.

Agar D i l u t i o n Test Quality Control and Cefixime Stability Table 3 lists the cefixime MIC results from the six collaborating laboratories testing the N. gonorrhoeae ATCC 49226 and Staphylococcus aureus ATCC 29213 QC strains (NCCLS, 1990b). The common lot internal control revealed that one laboratory had lower MIC results compared with all other participants. The results from that location's common and unique lot were still utilized in the analyses without compromising the study conformance with NCCLS (1989) M23-T guidelines for QC. The N. gonorrhoeae ATCC 49226 cefixime MIC mode was established at 0.015 t~g/ml leading to a suggested QC range of 0.008-0.03 t~g/ml. The second NCCLS-recommended QC strain (S. aureus ATCC 29213) did not have a cefixime MIC within the clinically relevant range (-<2 I~g/ml). The Mueller-Hinton agar cefixime QC range for that staphylococcal organism was ->32 t~g/ml (NCCLS, 1990b). The cefixime stability in GC agar plates was tested by storing prepared plates at 2°-5°C for 21 days. Five replicate MIC tests of N. gonorrhoeae ATCC 49226 were made on days 0, 2, 7, 14, and 21. No significant decline in cefixime potency (mean MICs, 0.008-0.015 t~g/ml) was identified within 21 days. These results were comparable to those reported for other structurally similar cephalosporins (Jones et al., 1989 and 1990).

D i s k D i f f u s i o n Test Quality Control The zone diameters from the six-laboratory study of 5-p,g cefixime disks (three lots, 449 total replicates) are summarized in Table 4. The statistical method described in earlier gonococcal susceptibility test standardization studies (Jones et al., 1989) was also applied to the cefixime disk test. Only the N. gonorrhoeae ATCC 49226 strain was used as the staphylococcal strain was of little utility (see results above). The cefixime 5-p,g disk mean and median data were similar, and the recommended QC zone range was 37-45 mm. Using this guideline, 96.7% of all studygenerated zone diameters (common and unique lots) were within control. There were no significant differences among GC agar lots, disk lots, or participant laboratory zones.

DISCUSSION Cefixime (formerly FR 17027, FK027, and C1284,635) is a so-called oral third-generation cephalosporin with a spectrum of activity most appropriate for therapy of respiratory tract and urinary tract infections (Neu et al., 1984). However, the high-cefixime potency against N. gonorrhoeae strains (Bowie et al., 1986; Neu et al., 1984) showed its promise as an alternative to the very effective intramuscular use of ceftriaxone or cefotaxime (Korting and Abeck, 1989). Guay et al. (1986) demonstrated that a 400-rag oral dose produced a cefixime serum level at or greater than the gonococcal cefixime MIC100 for - 24 hr (Bowie et al., 1986; Neu et al., 1984). Some new cephalosporin esters, such as cefetemet and cefpodoxime, also appear applicable in this sexually transmitted disease clinical setting (Schaadt et al., 1990; Tio et al., 1990). Two clinical trials used cefixime for treatment of gonorrhea at a single dose of 400 or 800 mg (Kuhlwein and Nies, 1989; Megran et al., 1990) resulted in the cure or improvement in 126 (99.2%) of 127 patients. These results confirmed the successful use of cefixime for therapy of gonorrhea in Japan (Saito, 1985). The cefixime cure rates were comparable to parenteral doses of 250-500 mg of ceftriaxone or ce-

TABLE 3 GC Agar Dilution Test Results from a Six-Laboratory Trial (150 Total Results) Using Quality Control Strains Neisseria gonorrhoeae ATCC 49226 and Staphyalococcus aureus ATCC 29213 Cefixime MIC (l~g/rnl) Occurrences" Quality Control Strain

0.004

0.008

0.015

0.03

0.06

>0.06

N. gonorrhoeae ATCC 49226 S. aureus ATCC 29213

18 0

51b 0

76b 0

5b 0

0 0

0 150b

qncludes results reported from seven lots of GC agar base. The common lot results were also included. bRecommended mode + 1 log2 dilution quality control ranges.

387

Cefixime versus Gonococci

TABLE 4

GC Agar Disk Diffusion Test Results from the Six-Laboratory Trial Using T h r e e Lots of 5-p~g Cefixime Disks a n d the Quality Control Strain Neisseria gonorrhoeae ATCC 49226

5-p,g Cefixime Disk Zone Diameter (mm) 33 34 35 36 37a 38 39 40 41 42 43 44 45a 46 Median Mean Range of zones

Zone Diameter Occurrence by Site A

2 4 8 23 16 6 1

38 38.2 6

B

4 7 11 14 9 4 4 6 1 40 40.4 8

C

14 13 11 14 8

42 41.8 4

D

E

6 10 21 7 6 6 2 2 41 41.6 7

F

Total

1 1 1

1 1 3 4 15 46 46 64 62 45 43 25 3 2

1 1 3 8 25 22 8 5 11 2 3

41 40.5 13

41 41.2 10

1 14 11 7 6 8 9 4

2 2 2 13 12 15 10 1

40 40.4 7

41 40.8 11

Common Lot

aLimits of proposed QC range.

fotaxime (Korting and Abeck, 1989). P P N G strains were eradicated b y cefixime but nongonococcal urethritis (NGU) was unaffected (Kuhlwein and Nies, 1989; Megran et al., 1990). The rates of p o s t t h e r a p y N G U were similar for cefixime, ceftriaxone, cefotaxime, and cefetemet (Korting and Abeck, 1989; Schaadt et al., 1990). In vitro tests with cefixime against N. gonorrhoeae strains performed acceptably, and the QC limits were readily established. The cefixime activity against penicillin-resistant (PPNG or CMRNG) strains was equal or in some cases superior to parenteral thirdgeneration cephalosporins. This fact, coupled with acceptable pharmacokinetics, potentially lower cost, and ease of administration, m a y m a k e orally administered third-generation c e p h e m s the drugs of choice

for uncomplicated gonococcal infection in the 1990s. More clinical evaluations of cefixime and similar drugs are clearly required for gonococcal disease. Also the interpretive criteria for cefixime, ceftriaxone, cefotaxime, and ceftazidime s h o u l d be reevaluated as resistant isolates emerge.

The authors thank the numerous medical technologists at each participating laboratory for the excellent support. The following persons contributed suggestions~reviews or organisms to this study: Dr. R. Testa, Dr. B. Lavin, S. Putnam, and M. Barrett. The word processing was skillfully provided by L. Schneekloth. This study was funded in part by research grants from Lederle Laboratories, Hoechst-Roussel Pharmaceuticals, and G.D. Searle and Company.

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Centers for Disease Control (1990a) Disk diffusion antimicrobial susceptibility testing of Neisseria gonorrhoeae. Morbid Mortal Weekly Rep 39:167-169. Centers for Disease Control (1990b) Plasmid-mediated antimicrobial resistances in Neisseria gonorrhoeae: United States, 1988 and 1989. Morbid Mortal Weekly Rep 39:284293. Fong JW, Linton W, Simbul M, Hinton NA (1986) Comparative clinical efficacy of single oral doses of cefuroxime axetil and amoxicillin in uncomplicated gonococcal infections. Antimicrob Agents Chemother 30:321-322.

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R.N. Jones et al.

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