Interpretive Criteria for Susceptibilities of Haemophilus infuenzae

Transcription

1 JOURNAL OF CLINICAL MICROBIOLOGY, Nov. 994, p /94/$ Copyright C 994, Americn Society for Microbiology Vol. 32, No. Interpretive Criteri for Susceptibilities of Hemophilus infuenze to Ampicillin, Amoxicillin, nd Amoxicillin-Clvulnic Acid PTR C. FUCHS"* AND ARTHUR L. BARRY2 St. Vincent Hospitl nd Medicl Center, Portlnd, Oregon 97225, nd The Clinicl Microbiology Institute, Tultin, Oregon Received 3 My 994/Returned for modifiction 28 June 994/Accepted 2 August 994 One hundred fifty isoltes of Hemophilus influenze (including 30,B-lctmse-positive strins nd 23 IB-lctmse-negtive, mpicillin-resistnt strins) were tested for susceptibilities to mpicillin, moxicillin, nd moxicillin-clvulnic cid (A/C) by the broth microdilution method in Hemophilus test medium (HTM) nd in Mueller-Hinton medium with lysed horse blood nd by the disk diffusion method on HTM gr. Our results support the use of HTM for susceptibility testing of H. influenze but rise number of questions regrding the interpretive criteri currently in use, prticulrly with respect to the fourfold difference in MIC susceptibility brekpoints for mpicillin nd A/C nd resulting high proportion of A/C-susceptible f-lctmse-negtive, mpicillin-resistnt strins. Prior to the mid-970s, Hemophilus influenze ppered to be universlly susceptible to mpicillin nd this drug ws the tretment of choice for H. influenze infections. The first reports of mpicillin-resistnt strins of H. influenze surfced in 976 (9, 5, 28, 29). Since then, the prevlence of these,b-lctmse-producing strins of H. influenze hs progressively incresed worldwide, surpssing 25% in mny res in Americ nd urope (4, 5, 6, 27).,-Lctmse-negtive, mpicillin-resistnt (BLNAR) strins hve lso been incresingly recognized over the pst 5 yers nd constitute smll proportion of the mpicillin-resistnt H. influenze isoltes in recent surveys (5, 6, 26). The mechnisms of resistnce in these BLNAR strins pper to be primrily relted to ltered penicillin-binding proteins (9, 20, 26), but permebility brriers my ply role in some instnces (20). Although other drugs hve demonstrted good ctivity ginst H. influenze (3, 2), the proven clinicl efficcy of mpicillin ginst mpicillinsusceptible strins mkes this drug n importnt therpeutic considertion, provided the in vitro susceptibility tests cn be relibly predictive. Following the erly reports of P-lctmse-producing H. influenze, the need for nd bsence of stndrdized method for ntimicrobil susceptibility testing of H. influenze were immeditely pprent. This led to number of studies ttempting to develop such method (2, 3), culminting in the first pproved stndrdized method published by the Ntionl Committee for Clinicl Lbortory Stndrds (NCCLS) in 986 (2). This method utilized Mueller-Hinton medium supplemented with 3% lysed horse blood (LHB). Subsequently, Jorgensen et l. proposed new medium, Hemophilus test medium (HTM), consisting of Mueller-Hinton medium supplemented with specified mounts of NAD, hemtin, nd yest extrct which yielded results comprble to those obtined with LHB but with endpoints tht were esier to interpret (4). This medium ws dopted by the NCCLS in 990 for both dilution nd disk diffusion susceptibility testing (22, 23). The superior performnce of HTM for susceptibility testing of H. * Corresponding uthor. Miling ddress: Deprtment of Pthology, St. Vincent Hospitl & Medicl Center, Portlnd, OR Phone: (503) Fx: (503) influenze by both methods hs been confirmed by other studies (, 6, 27). There remin, however, few problems nd inconsistencies with the interpretive criteri for mpicillin, moxicillin, nd moxicillin-clvulnic cid (A/C) with broth dilution nd disk diffusion methods. The present study ws intended to confirm, evlute, nd propose possible solutions to some of these persistent difficulties. We tested 50 strins of H. influenze for their susceptibilities to mpicillin, moxicillin, nd A/C. These included 97 3-lctmse-negtive, mpicillin-susceptible (BLNAS) strins; 30 3-lctmse-positive (BLP) strins; nd 23 BLNAR strins. The ntibiotics (mpicillin, moxicillin, nd clvulnic cid) were procured from their Americn mnufcturers s stndrdized powders. Susceptibility studies were performed in duplicte, once ech by broth microdilution in HTM nd broth microdilution in LHB. Disk diffusion tests were lso performed on HTM with 0-,ug mpicillin disks nd 20-,ug/0-,ug A/C disks procured from Becton-Dickinson Microbiology Systems, Cockeysville, Md. The procedures for ech test were those outlined by the NCCLS (22, 23). Drug concentrtions tested in the microdilution trys were twofold dilutions rnging from 64 to 0.06,ug/ml. A/C ws tested s two prts of moxicillin plus one prt of clvulnic cid (2: rtios). Two qulity control strins (H. influenze ATCC nd ATCC 49766) were tested on ech test dy. Figure describes the distribution of MICs of the three test drugs ginst the three groups of H. influenze tested on two different broth medi (HTM nd LHB). In either medium, 3-lctmse-negtive strins were inhibited by <.0 jig of mpicillin per ml, confirming the reports of others (4, 27). However, for BLP strins, the MICs of mpicillin nd moxicillin tended to be higher on LHB thn on HTM. But in either broth medium,,-lctmse producers were clerly resistnt to mpicillin by current criteri (MIC 2 2.0,ug/ml). By definition, BLNAS strins were inhibited by s.0 jig of mpicillin per ml, but they required 52.0 jig of moxicillin per ml for inhibition (Fig. ). Jones et l. () lso reported tht mpicillin ws twice s ctive s moxicillin ginst H. influenze, nd such difference hs been shown for other orgnisms (7). However, in the current study, the distribution of MICs of moxicillin nd of mpicillin did not differ gretly when BLNAR nd BLP strins of H. influenze were tested (Fig. ).

2 VOL. 32, 994 I c IS )* Microditution MIC-in HTM broth Mficrodliutlon MIC In LHB broth CULNAS H.ep LmR U 6. vff Z lz9. J, Ampicililn MIC (Wmi Ampicillin MIC (pg/mq I --I " NOTS 2847 json I2. ji. - O.^ *.Am4o i.l M. C (g/m) " 3 " Amo)dclilin MIIC OWmQ I Amoxdclilin MIC (pg/mi) Go. -8-2s... S 536 $s So 6.6" u " 6" u 6 5" Amoxdclilln/Clvulnic Acid MIC p/mi Amoxicillln/Clvulnic Acid MIC (pg/mi FIG.. Ampicillin, moxicillin, nd A/C MIC distribution mong 50 isoltes of H. influenze tested in LHB broth versus HTM broth. Arrows designte MIC brekpoints for the susceptibility ctegories; solid lines indicte the current NCCLS brekpoints for mpicillin nd for A/C (there re no NCCLS criteri for moxicillin), nd dotted lines designte proposed MIC criteri for moxicillin nd A/C. Although mpicillin MICs re hlf those of moxicillin ginst BLNAS strins, comprble orl doses of the two drugs result in twofold-higher levels of moxicillin in blood. For exmple, 500-mg orl doses of mpicillin nd moxicillin yield men pek concentrtions in serum of 3.2 nd 7.6,ug/ml, respectively (8). Consequently, MIC brekpoints for moxicillin should be incresed by doubling concentrtion (.2.0,ug/ml for susceptibility nd -4.0,ug/ml for resistnce). In tht cse, MICs of mpicillin re predictive of moxicillin susceptibility nd mpicillin is the preferred clss representtive for routine susceptibility tests. The brekpoint for H. influenze mpicillin susceptibility is '.0,ug/ml (22, 24), which is substntilly less thn chievble levels in blood with stndrd doses nd lso less thn the.8.0-pug/ml brekpoint for other orgnisms such s members of the fmily nterobctenicee (22, 24). This.0-,ug/ml brekpoint ws selected becuse it effectively seprted BLP strins (most with mpicillin MICs of.8.0 jig/ml) from P-lctmse-

3 2848 NOTS J. CLIN. MICROBIOL. > ( 4f) e * ' I Zone Dimeter in (mm) Opg Ampicillin Disks FIG. 2. Scttergrm of mpicillin MICs (in HTM broth) nd disk diffusion zone dimeters resulting from tests with 50 isoltes of H. influenze. Verticl nd horizontl lines indicte zone dimeters nd MIC susceptibility brekpoints, respectively (20). BLNAR strins re indicted by circles. All other isoltes for which MICs were.4.0,ug/ml were f-lctmse-producing strins. negtive strins, for which prior to the ppernce of BLNAR strins MICs were -.0,g/ml. Also, prior to the ppernce of BLNAR strins, this brekpoint ws effective in predicting clinicl response. The clinicl relevnce of current interpretive criteri for BLNAR strins is uncler. The mpicillin MICs for these strins rnge from 2.0 to 6,ug/ml in HTM broth nd.0 to 8.0 pug/ml in LHB. These strins re considered resistnt becuse the mpicillin MICs re bove the.0-,ug/ml brekpoint set to distinguish,b-lctmse producers from nonproducers, nd yet they re nonproducers. Little dt re vilble on the clinicl response to mpicillin therpy of BLNAR H. influenze infections, prticulrly those for which mpicillin MICs re 2.0, 4.0, or 8.0,g/ml. At lest one instnce of filure of mpicillin in tretment of BLNAR H. influenze meningitis nd endocrditis hs been reported; the mpicillin MIC ws 6.25,ug/ml (7). In view of the pucity of such dt, the position tken by the NCCLS to consider these strins resistnt to mpicillin is clerly the sfe, conservtive one, but t the sme time, it virtully precludes the cquisition of the dt needed to chnge it. A significnt nomly occurs with A/C susceptibility testing of BLNAR strins (Fig. ). Of the 23 BLNAR strins tested, the MICs of moxicillin nd A/C were essentilly the sme (MICs were identicl for 20 strins, nd for 3 the A/C MIC ws log2 concentrtion lower). By the current NCCLS criteri (MIC c.0,g/ml for susceptibility), ll 23 isoltes were resistnt to mpicillin (nd moxicillin) yet (48%) were susceptible to A/C t the current susceptibility brekpoint of c4.0,g/2.0,g/ml (22, 24). Since clvulnic cid hs no inhibitory ctivity ginst H. influenze t these concentrtions, nd since BLNAR strins do not produce,-lctmse, there is no reson to believe tht A/C will be more effective thn moxicillin in treting BLNAR H. influenze infections. Yet current NCCLS criteri would imply tht nerly hlf of these BLNAR strins would respond to the fr more costly A/C nd not to moxicillin lone. Likewise, becuse the susceptibility brekpoint for mpicillin-sulbctm (A/S) is higher thn tht *for mpicillin, proportion of BLNAR strins would be inppropritely considered susceptible to A/S. Ironiclly, the susceptibility brekpoint for A/S ( prenterl drug) is -2.0/.0,ug/ml (23, 24), one-hlf tht for A/C (n orl drug). The proposed brekpoint for moxicillin is <2.0,g/ml for susceptibility, nd for A/C, brekpoint of s2.0,ug/.0,g/ml would seem more pproprite. With tht brekpoint, the number of BLNAR strins susceptible to A/C would be reduced to three (3%). The disk diffusion susceptibility test results for mpicillin nd A/C re summrized in Fig. 2 nd 3 on scttergrms correlting their inhibitory zone dimeters with their respective MICs. For mpicillin, there were no mjor or very mjor errors when current NCCLS criteri were pplied (24, 25). But there were 6 (%) minor errors, nd three-fourths of these were due to BLNAR strins. The former NCCLS criteri with n intermedite zone rnge of 22 to 24 mm would hve yielded minor error rte of 6%. This supports the previous chllenges to these former criteri (, 0, 8) nd grees with originl recommendtions of Jorgensen et l. (4), which were dopted by the NCCLS in 992 (24). Still, our % minor error rte is substntilly greter thn tht reported by Jones et l. (). This is most likely becuse they hd few, if ny, BLNAR strins (none for which mpicillin MICs were 2.0 or 4.0,ug/ml) mong the 250 recent clinicl isoltes they tested. Within the United Sttes, BLNAR strins currently ccount for <% of ll H. influenze isoltes encountered in clinicl mteril (5, 6, 26) ḂLNAR strins lso confounded disk diffusion susceptibility testing of A/C (Fig. 3). By current criteri, there were 2 strins (ll BLNAR) resistnt to A/C (MIC 8.0,g/4.0 pg/ml), nd 5 of these (42%) hd flse susceptibility zone dimeters rnging from 2 to 23 mm (susceptibility brekpoint 2 20 mm)

4 VOL. 32, 994 NOTS (6- *S 6/8.0-.5u o > _'~ 2.0/.0-.Od.5-0.5i025- o (2) (D)6 8 (9l / /0.06- s0.06& Zone Dimeter in (mm) 20/0 Po Amoxicillin/Clvulnic Acid Disks FIG. 3. Scttergrm of A/C MICs (in HTM broth) nd disk diffusion zone dimeters resulting from tests with 50 isoltes of H. influenze. Verticl nd horizontl lines indicte NCCLS zone dimeters nd MIC susceptibility brekpoints, respectively (20). BLNAR strins re indicted by circles. (22, 24). If the A/C brekpoint were reduced to.2.0,ug/.0,ug/ml for susceptibility, eight dditionl flse-susceptible disk test results would be recorded for BLNAR strins. For the totl popultion studied, the very mjor error rte ws 3.3%, which most would consider uncceptble. However, this popultion ws selected one in which 5% of the strins tested were BLNAR strins. At this time, clinicl isoltes of H. influenze should contin very few BLNAR strins (5, 6, 26). Consequently, the very mjor error rte for A/C disk tests should be well below %. This does not, however, del with the question of whether ny BLNAR strins should be ccepted s A/C susceptible. For 5 (65%) of the 23 BLNAR strins, A/C inhibitory zone dimeters were considered to indicte susceptibility (.20 mm) by current criteri (24). Rising the brekpoint for A/C susceptibility to.24 mm would correctly ctegorize ll BLNAR strins but would result in n uncceptbly high flse-resistnce rte of 3% of ll strins tested. Clerly, the 20-,ug/0-j,g A/C disk does not stisfctorily seprte BLNAR from BLNAS strins (Fig. 3), nd these dt support the bsence of A/C from the NCCLS H. influenze tbles for disk susceptibility testing (25). If A/C MICs re determined by dilution tests, susceptibility brekpoint of <2.0,ug/.0 jig/ml will gretly reduce the frequency of BLNAR A/C-susceptible strins. At this time, the prudent pproch for H. influenze would pper to be to test for P-lctmse production nd mpicillin disk susceptibility nd consider BLNAS nd BLP strins susceptible to A/C nd BLNAR strins resistnt. RFRNCS. Brry, A. L., J. H. Jorgensen, nd D. J. Hrdy. 99. Reproducibility of disk susceptibility tests with Hemophilus influenze. J. Antimicrob. Chemother. 27: Bergeron, M. G., P. Simmrd, nd P. Provencher Influence of growth medium nd supplement on growth of Hemophilus influenze nd on ntibcteril ctivity of severl ntibiotics. J. Clin. Microbiol. 25: Doern, G. V., nd K. C. Chpin Susceptibility of Hemophilus influenze to moxicillin/clvulnic cid, erythromycin, cefclor, nd trimethoprim/sulfmethoxzole. Dign. Microbiol. Infect. Dis. 4: Doern, G. V., J. H. Jorgensen, C. Thornsberry, D. A. Preston, nd the Hemophilus influenze Surveillnce Group Prevlence of ntimicrobil resistnce mong clinicl isoltes of Hemophilus influenze: collbortive study. Dign. Microbiol. Infect. Dis. 4: Doern, G. V., J. H. Jorgensen, C. Thornsberry, D. A. Preston, T. Tubert, J. S. Redding, nd Ls A. Mher Ntionl collbortive study of the prevlence of ntimicrobil resistnce mong clinicl isoltes of Hemophilus influenze. Antimicrob. Agents Chemother. 32: Doern, G. V., J. H. Jorgensen, C. Thornsberry, nd H. Snpper Disk diffusion susceptibility testing of Hemophilus influenze using Hemophilus Test Medium. ur. J. Clin. Microbiol. Infect. Dis. 9: Fuchs, P. C., A. L. Brry, M. A. Pfller, D. J. Hrdy, J. C. McLughlin, nd. H. Gerlch In vitro ctivity of mpicillin, moxicillin, mpicillin-sulbctm, nd moxicillin-clvulnic cid ginst consecutive clinicl isoltes of nterobctericee. Dign. Microbiol. Infect. Dis. 7: Gordon, R. C., C. Regmey, nd W. M. M. Kirby Comprtive clinicl phrmcology of moxicillin nd mpicillin dministered orlly. Antimicrob. Agents Chemother. : Gunn, B. A., J. B. Woodll, J. F. Jones, nd C. Thornsberry Ampicillin-resistnt Hemophilus influenze. Lncet ii: Helln, J. S., D. Chesnev, nd G. Gudgno Investigtion of mpicillin-intermedite strins of Hemophilus influenze by using the disk diffusion procedure nd current Ntionl Committee for Clinicl Lbortory Stndrds guidelines. J. Clin. Microbiol. 30: Jones, R. N., J. Hindler, G. V. Doern, C. Thornsberry, nd. H. Gerlch The re-evlution of the NCCLS mpicillin susceptibility tests with Hemophilus Test Medium: new interpretive criteri for the disk diffusion method. Antimicrob. Newsl. 8: Jorgensen, J. H., G. V. Doern, C. Thornsberry, D. A. Preston, J. S. Redding, L A. Mher, nd T. Tubert Susceptibility of multiply resistnt Hemophilus influenze to newer ntimicrobil gents. Dign. Microbiol. Infect. Dis. 9: Jorgensen, J. H., nd P. M. Jones Simplified medium for

5 2850 NOTS mpicillin susceptibility testing of Hemophilus influenze. Antimicrob. Agents Chemother. 7: Jorgensen, J. H., J. S. Redding, L. A. Mher, nd A. W. Howell Improved medium for ntimicrobil susceptibility testing of Hemophilus influenze. J. Clin. Microbiol. 25: Khn, W., S. Ross, W. Rodriguez, G. Controni, nd A. R. Sz Hemophilus influenze type b resistnt to mpicillin. JAMA 229: Mchk, K., I. Brveny, H. Dbernt, K. Dornbusch,. VnDyck, F. H. Kyser, B. VnKlingeren, H. Mittermyer,. Pere, nd M. Powell Distribution nd resistnce ptterns of Hemophilus influenze: uropen comprtive study. ur. J. Clin. Microbiol. Infect. Dis. 7: Mrkowitz, S. M Isoltion of n mpicillin-resistnt, non-,b-lctmse-producing strin of Hemophilus influenze. Antimicrob. Agents Chemother. 7: Mendelmn, P. M., D. 0. Chffin, C. Clusen, T. L. Stull, C. Needhm, J. D. Willims, nd A. L. Smith Filure to detect mpicillin-resistnt, non-,b-lctmse-producing Hemophilus influenze by stndrd disk susceptibility testing. Antimicrob. Agents Chemother. 30: Mendelmn, P. M., D. 0. Chffin, nd G. Klitzoglou Penicillin-binding proteins nd mpicillin resistnce in Hemophilus influenze. J. Antimicrob. Chemother. 25: Mendelmn, P. M., D. 0. Chffin, T. L. Stull, C.. Rubens, K. D. Mck, nd A. L. Smith Chrcteriztion of non-p-lctmse-medited mpicillin resistnce in Hemophilus infiuenze. Antimicrob. Agents Chemother. 26: Ntionl Committee for Clinicl Lbortory Stndrds Methods for dilution ntimicrobil susceptibility tests for bcteri tht grow erobiclly. M7-A, st ed. Ntionl Committee for J. CLIN. MICROBIOL. Clinicl Lbortory Stndrds, Villnov, P. 22. Ntionl Committee for Clinicl Lbortory Stndrds Methods for dilution ntimicrobil susceptibility tests for bcteri tht grow erobiclly. M7-A2, 2nd ed. Ntionl Committee for Clinicl Lbortory Stndrds, Villnov, P. 23. Ntionl Committee for Clinicl Lbortory Stndrds Performnce stndrds for ntimicrobil disk susceptibility tests. M2-A4. Ntionl Committee for Clinicl Lbortory Stndrds, Villnov, P. 24. Ntionl Committee for Clinicl Lbortory Stndrds Performnce stndrds for ntimicrobil susceptibility testing. M00-S4, 4th informtionl supplement. Ntionl Committee for Clinicl Lbortory Stndrds, Villnov, P. 25. Ntionl Committee for Clinicl Lbortory Stndrds Performnce stndrds for ntimicrobil disk susceptibility tests. M2-A5. Ntionl Committee for Clinicl Lbortory Stndrds, Villnov, P. 26. Reid, A. J., I. N. Simpson, P. B. Hrper, nd S. G. B. Amyes Ampicillin resistnce in Hemophilus influenze: identifiction of resistnce mechnisms. J. Antimicrob. Chemother. 20: Scriver, S. R, D.. Low, A.. Simor, B. Toye, A. McGeer, R. Jeger, nd Cndin Hemophilus Study Group Broth microdilution testing of Hemophilus influenze with hemophilus test medium versus lysed horse blood broth. J. Clin. Microbiol. 30: Thoms, W. J., J. W. McReynolds, C. R Mock, nd D. W. Biley Ampicillin-resistnt Hemophilus influenze meningitis. Lncet i: Tomeh, M., S.. Strr, J.. McGown, P. M. Terry, nd A. J. Nhmis Ampicillin-resistnt Hemophilus influenze type b infection. JAMA 229: