ANTIMICROBIAL SUSCEPTIBILITY TESTING Kemwth J. Ryan, M.D.,* and Jolm C. Sherris, M.D.-?

Abstract Considerable advances ltave been m a d e in antimicrobial susceptibility testing. This review emphasizes tire continued efforts toward standardization of methods for dilution and diffusion testing, particularl)' in tire area of variation in m e d i u m performance, methods for detection of ampicillin resistant Haemophihts influenzae, and attempts to develop rapid a u t o m a t e d systems for susceptibility testing. Susceptibility testing of anaerobes continues to be controversial from tim standpoint of both tim selection of methods and the application of susceptibility results to the clinical situation. It is expected that these and other problems in susceptibility testing will be solved by continued application of the investigative approaches that have brought us wltere we are today.

O f the considerations involved in the selection of an appropriate antimicrobic d r u g s to treat an infection, the a m o u n t required to inhibit or kill the organism in vitro and the concentrations attained in body fluids d u r i n g treatment m'e subject to direct measnrentent in the clinical microbiology laboratory. Recent developments in the use of the first group of tests for bacteria other than mycobacteria are the subject of this review. Recent books mad reviews lmve covered all aspects of this subject, allowing us to concentrate on selected developments since their publication) -4 For details of methndology the reader is referred to tire literature cited and the susceptibility testing chapters of the ASM Mamtal ~!["Clinical Microbiol%%'-'

Antimicrobial assays are reviewed elsewhere in this issue. Other considerations in antimicrobic d r u g selection, such as details of clinical pharmacology (toxicity, protein binding, absorption, distribution), clinical experience and efficacy, and consideration of the tmlque features presented b)' particular infections or host alterations, are beyond the scope of this review. T h e recent developments selected for discussion are: f u r t h e r progress in standardization o f methodolog)', new problems of antinficrobial resistafice, and progress in the developntent of rapid and autonmted susceptibility tests.

+The term antitnicrobic is used throughout this discussion to describe both antibiotic and chemotherapeutic drugs.

T h e resuhs of both dilutioi1 and diffusion susceptibility tests may be inlluenced markedly by the reagents and condi-

STANDARDIZATION

OF M E T H O D S

*Assistant l'rol'essor of l'atlaologyand Microbiology,University of Arizona Coli;zgeof Medicine, Chief, Clinical MicrobiologyLaboratories, Arizona MedicalCenter, Tucson, Arizona. "lProfes~or. and Chairman, Department of Mic!'obiology, University of Washington School of Medicine, Seattle, Washington.

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IIUMAN I'ATIIOLOGY-VOLUME 7, NUMBER 3 May 1976 ,ions of .testing. Irmculum size, medium composition and pH, incubation time and temperattrre, atmosl)here, and antimicrobic drug stability ntay have an effect on any of these tests. In addition, diffusion tests are influenced by the growth rate of the organism, dilt'nsibility of tile antimicrobic drug, and the type, depth, and concentration of agar used. For these reasons considerable effort has beera directed toward achieving nmthodologic standardization and tim establishment of reference procedures. T h e report of the International Collaborative Study (ICS) includes recommendations for" reference dilution procedures, '~ and a diffnsion procedure has beera accepted by the Food and Drug Administration and adopted as a standard by the National Committee on 9Clinical Laboratory Standards Subcomntittee on Antimicrobial Susceptibility Tests. c-8 These methods Imve now been shown to give good interlaboratory and intralaboratory reproducibility when performed as described arid under control with recommended standard strains. However, special problems remain, and some limitations must be recognized.

Medium Performance and Variability

278

There is, unfortunately, some variation in performance of the Mueller-Hinton ntediunt produced by different manufacturers, particularly with regard to results with the tetracyclines, aminoglycosides, and polymyxins?-~7 The Mueller-Hinton media produced by earlier ntanufacturers (BBL and Difco) show generally comparable results, except with Pseudomonas and gentamicin and with tim tetrac)'clines,T M ' 5 but those of manul;acturers who have more ~;ecently begun to produce tim medium may differ sufficiently to produce some 1-esults with the standard strains that are outside the FDA l'ecommended limits. ~7 Certain Pseudomonas aeruginosa organism-antimicrobic drug contbinations can present nnique problems in which small differen~:es in medinm constitution carl cause variations that could not be predicted fl'om experience or quality control with other species. One of the best defined is the inlluence of calcium and ntagnesium medium content on susceptibility to the aminogtycosides and polyifiyxins. A

nmrked reduction in tile susceptibility of

I'. aeruginosa to gentamicin ira the p,'eseuce of increased conceutrations of calcium and magnesium was first reported I)y Garrod and Waterworth? who found significant variations in inhibition zones and nainimal inhibitory concentrations on different media with 1'. aeruginosa but not with E. coll. The variations were found to depend on the general COmlmsition of the mediunl and of the agar used to solidify it, and greater" resistance could be produced by the addition of xnagncsium st, Ifate to the same medium. Others have found the calcium concentration to be important as well and have extended these findings to the polymyxins.~~ Tbis plmnomenon is peculiar to P. aeruginosa and is not the result of antibiotic inactivation.9-12 This is an important consideration in tile susceptibility testing of !'. aeruginosa, because different media have beeu shown to vary significantly in their magnesium and calcium conteut, as have different batches of the same medium whetlter produced by the same or different manufacturers.9, x0,~4 Differences of as much as 16-fold in tim results of aminoglycoside dilution tests and of 8 to 9 ram. in agar difflrsion zone sizes with gentamicin discs have been correlated with variations in the calciun~ and magnesium content of the Mueller-Ilinton medium. Dilution tests with currently available Mueller-Hinton broth have beera found to give lower minimal inhibitory concentrations than in agar dilution tests unless they were prel)ared to contain equivalent amounts of calcium and lnagnesium ions? 4 These differences are great enough to alter the interpretation of results, potentiall), leading to errors in antibiotic selection by the clinician, since tim margin between tile gentamicin mininml inhibitory concentration for ninny strains of P. aeruginosa and the achievable serum concentrations is not large. For example, a strain showing increased resistance to gentamicin could be reported as sensitive on the basis o f t broth dihHion test because broths often have a low calcium and magnesium content in r'elationghil~ m agar media or physiological concentrations. Better reproducibility in testing tim susceptibility of P. aeruginosa t o amino-

ANTIMICROBIAL SUSCEPTIBILITY TESTING--RvAx, SIIERRIS glycosides with the Mueller-Hinton mediuln can be" achieved with g r e a t e r u n i f o r m i t y o f tim calcium a n d m a g n e s i u l n content o f comlnercially available media than presently exists. Relier et al) a have r e c o m m e n d e d concentrations o f 25 to 35 rag. o f m a g n e s i u m and 50 to I 0 0 rag. o f calcium p e r liter, levels that a p p r o x i m a t e the physiological range. T h e y showed that s u p p l e m e n t a t i o n o f M u e l l e r - l l i n t o n b r o t h a n d agar, when necessary, could bring results into c o n f o r m i t y with those o f a r e f e r e n c e batch a n d r e c o m m e n d e d that m a n u f a c t u r e r s m a k e such s u p p l e m e n t a tion. H o w e v e r , the ultimate m e a s u r e o f a d e q u a c y m u s t be p e r f o r m a n c e . T h e r e is n e e d for the estab)ishment o f l ) e r f o r m a n c e s t a n d a r d s for M u e l l e r - H i n t o n xnedia. It seems p r o b a b l e that quite m i n o r xnodifications" o r additions could eliminate the m o r e serious problems. PSI"UIIOMONAS AERUGINOSA CONTROL

STRAINS. Reller et al) 4 evaluated a n u m ber o f potential P. aeruginosa control strains f o r sensitivity to the c a l c i u m - m a g nesium effect a n d stability on r e p e a t e d subculture. All t h r e e strains tested were sensitive a n d stable, but the strain designated Boston 41.501 was r e c o m m e n d e d for quality control on the basis o f ease a n d clarity o f e n d p o i n t reading. T h i s strain has been e n t e r e d in the Anterican T y p e Culture Collection as A T C C 27853 a n d is r c c o m n t e n d e d for quality control o f aminoglycoside a n d p o l y m y x i n susceptibility tests. It should be used in tire s a m e

m a n n e r as is r e c o m m e n d e d for Escherichia colt A T C C 25922 a u d Staphylococcus aureus A T C C 259233 since tim latter two strains c a n n o t be used to detect the mediunl variations already described for Pseudomonas. T a b l e 1 shows the resnhs o b t a i n e d when this strain was used for r o u t i n e quality control o f diffusion testing at the University o f A r i z o n a and University o f W a s h i n g t o n hospitals and in the studies o f Reller et al.: 4 as well as the r e c o l n m e n d a tions o f the National C o m m i t t e e for Clinical L a b o r a t o r y Standards. s T h e s e figures m a y serve as a guide until better m e d i u m s t a n d a r d i z a t i o n is achieved. Carbenicillin Testing Recent recognition o f difficulties with the use o f the 50 meg. carbenicillin disc originally r e c o m m e n d e d by FDA has led to reinvestigation o f its use in diffusion testing. Carbenicillin presents u n i q u e l a b o r a t o r y testing p r o b l e m s owing to its wide r a n g e o f activity and relatively high attainable s e r u m levels, so that infections by l'seudomonas strains with nfinimal inhibitory c o n c e n t r a t i o n values in the 100 to 200 meg. p e r ml. r a n g e may be considered treatable with high closes o f carbenicillin. Susceptibility m e t h o d s m u s t t h e r e f o r e enable o n e to discriminate these strains f r o m m o r e resistant isolates as well as those that are highly susceptible. T i m 50 meg. carbenicillin disc did not p e r f o r m this fimction, because it sometimes p r o -

ZOXEDIAMETERSOF PSEUDOMONASAERUGINOSAATCC 27853 (BosTON 41501) TESTED ON M UEI.LER-HtXTOX AGAR (M H A)

T A B L E I.

Antibiotic~Disc

R e l l e r et al. I

University of

University of

Arizona"-

lVashington a

NCCLS 4 v

Gentanficin (10 mcg.)

18.2 • '2.0s

17.2 -----'2.'2

19.8 • 2.5

16-21

Tobramycin (10 meg.)

20.7 +__1.4

-

-

19-25

Carbenicillin (i 00 meg.)

21.4 --+-1.7

19.9 ___2.6

'2'2.'2 +--2.4

20-24

I'olymyxin B (300 units)

15.'~ + 1.5

-

15.6 -r 2.0

-

-

12.9 ___1.6

-

-

Colistin (10 meg.)

I. Resuhs based on .t8 observations with six batches o f MIIA (Difco) (i-t). '2. Results fi-om 75 consecutive routine qt,ality control tests with a single batch o f M 11A (Difco). 3. Results o f 1()0 consecutive quality control tests encompassing four batches o f MItA (l)ifco). -1. National Committee for Clinical I.aboralory Standards r e c o n m m n d e d test limits, s 5 . . M e a n zone'size (toni.) -r 2 SI).

279

HUMAN I'ATHOI.O(;Y--VOI.UME 7, NUMBER 3 May 1976 duced little or no zone of inlfibition with strains that were potentially susceptible to high closes of carbenicillin. The perfl~rmance of a 100 mcg. disc has now been evaluated and found to produce both larger inhibition zones and much better discrimination between sensitive, intermediate,* and resistant strains) s With ibis disc, sensitive sir~fins of Pseudomonas aeruginosa produce zones of 17 ram. in diameter or gn'eater whereas resistant strains show zones 13 ram. or less in diameter. Owing to differences in bacterial populat!on distributions and otlter factors, a separate set of interpretive criteria is required when E. eoli and Proteus are tested, with zone diameters 23 ram. or greater considered sensitive and 17 mm. or less, resistant. The FDA has approved a 9100 Incg. disc (50 mcg. was previously the only one available), which should now be used by all clinical laboratories that test susceptibility to carbenicillin by the diffllsion method using the recomnlended interpretive standards, s It should be noted that these interpretive standards apply only to gram negative organisms, since solne gram positive organisms Stlch as penicillinase producing staphylococci may be inhibited into "sensitive" ranges with the 100 mcg. carbenicillin disc.

Susceptibility Testing of Anaerobes The increased interest in isolation of anaerobic bacteria and their correlation with a variety of infectious syndromes have been accompanied by an increased demand for the kind of therapeutic guidance that susceptibility testing provides for most aerobic and facuhative organisms. Ahhougll considerable effort is being directed toward this goal, its achievement is complicated by some problems that are unique to tim anaerobes and others that are common to standardization of susceptibility testing generally) 9 Methods employing the same principles used in dilution and diffusion testing of aerobic and facultative bacteria have been applied to anaerobes, but substantial modifications have been necessary to satisfy their growth requirements. There is no general agreement about specific

280

*Minimal inhibitoryconcentration between'125 a,,a 23o incg. per ml.

details of dilution procedures as yet, and investigators luwe used marl)' different media, inocula, and endpoint criteria. Methods used have included macrobroth dilution, microbroth dilution, and agar cfilution techniques.2"2t'zz Agar diffusion procedures have also been described, but these are complicated by the generally slower and more varied growth rate of the anaerobes as compared to rapidly growing aerobic and facuhative organisms.2~ Results with all methods are influenced directly by the carbon dioxide content in anaerobic jars or chambers in the case of antimicrobic drugs such as the macrolides and tetracyclines. A decision about the effectiveness of susceptibility tests of anaerobes is thus currently difficult for most laboratories since no uniform practical approach can be recommended. Testing should be restricted to isolates that are clearly related to an anaerobic infection (e.g., surgical specimens, deep aspirates) and to methods that have been shown to be reproducible. To date, the ntost reproducible methods have been dilution procedures, such as the ones recommended in the ASM Mamtal of Clinkal MicrobioIo~9',z ahhough diffusion tests have worked reasonably well with the more rapidly growing species. In a comparative evaluation of four diffusion methods, Barry and Fay25 found the methods of Sutter and coworkers 23 to yield the best growth and the best minimal inlfibitory concentration-zone size correlation, although in a large number of tests the zones were found to be in the intermediate category when their interpretive criteria were applied. This method should not be ditficuh to introduce in most laboratories, since it utilizes the same approach as the FDA recommended diffusion procedure using different media and interpretive standards3, TOther modifications of the Bauer-Kirby and agar overlay procedures for anaerobes did not give acceptable results and were not recommended for clinical u s e . 2 5 - 2 7 None of the diffusion procedures described to date is entirely satisfactory for testing nutritionally fastidious strains, and it is reasonable until more experience is gained to limit their application to strains such as Bacteroidesfi'agilis and some

ANTIMICROBIAL SUSCEPTIBILITY TESTING- RVAN, SHERRIS clostridia that show adequate growth after 16 to 18 hours' incubation. If the clinical laboratory is not able to utilize one of the procedures that have been shown to give reproducible resnlts, it is better that the clinician base his treatment on statistical data developed by others rather than attempt to perform an unstandardized test. In one of our laboratories (KJR) the data of Martin et al.2z have been abstracted and circulated to tile medical staff, providing both a rational basis for initial therapy and data for use when information about susceptibilities of individual strains is not available. Such reports should be updated periodically but can serve until data relating to local strains are available. Another consideration for the clinician is" the impact that susceptibility testing of anaerobes can be expected to have on patient care, given reproducible standardized methods. Owing to the slow growth of many anaerobic strains, resuhs are often delayed, perlmps to the point at which the clinical situation lnay differ enough to make their application irrelevant or even lmzardous. For exanaple, in a staphylococcal infection, isolation and preliminary identification can be expected in 18 to 24 hours, with standardized susceptibility results within 48 hours. With an anaerobic isolate requiring 48 hours for good growth, tiffs time may be extended to four to six days during which the clinical status of the patient may have changed dranmtically. Some clinicians also feel that the application of susceptibility tests to anaerobic infections is not as clearcut as with other organisms, because infcctions are frequently mixed and the effect of therapy on the interaction of these strains Icannot be tested directly in tile laboratory,zs Thus, more experience with the clinical application of susceptilfility tests must be gained in addition to agreement about practical reproducible methodology before laboratory results can be reliably interpreted. N E W P R O B L E M S OF B A C T E R I A L RESISTANCE

The use of antimicrobial agents to treat infections has been complicated from the 6utset by the developmefit of

clinical and in vitro resistance in groups of organisms previously thought to be uniformly susceptible. In s()me instances resistance has appeared only after tlle drug has been in use for many )'ears, as with the sudden emergence of sulfonamide resistance in meningococci in the early 1960s, the more recent increase in the United States in Shigella resistant to ampicillin,29'3~ and the isolation of strains of Salmonella typhi resistant to ampicillin and chlol,'amphenicol in Mexico? ~ Some organism-antimicrobic drug combinations have presented particular problems in metlmdology, such as methicillin resistance in staphylococci, which is more readily detected at incubation temperatures of 35 ~ C. or below than at 37 ~ C.3" or the special precautions and interl)retative standards needed when meningococci are tested against the sulfonamides by the diffusion naethor ~3 More recently investigation of resistance to the macrolide group of agents has revealed some conaplexities, including a pattern of "zonar' resistance in wifich strains of group A beta hemolytic streptococci were resistant to high and low but not to intermediate concentrations of lincomycin and clinclam)'cin34"35 and the induction of resistance to the lincomycins by erythromycin in erythromycin resistant strains of Staphylococcus attreus. 36"3T The clinical significance of this phenomenon in the absence of erythromycin therapy is nnknown, but tile property may be detected by juxtaposing erythromycin and lincomycin or clindalnycin discs when a distortion of the lincomycin zone is seen on the side of the erythromycin disc. In such cases ahernatives to the lincomycin drugs in theral)y would be al)prol)riate. The emergence of aml)icillin resistant strains of Haemophilus ilzJluenzae i n , t h e past two years is of particular interest because of its clinical importance and because it presents unique problems in susceptibility testing. Although occasionall}, suspected on clinical gronnds and carefldly searched for in the laboratory, in vitro resistance of H. bfluetlzae to ampicillin was essentially unknown until cases reported from Maryland and the District of Columbia in November and December of 1973 were confirmed by the Center for Disease Control.38 Four of these patients

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IIUMAN I'ATIIOLOGY--VOI,UME 7, NUMBER 3 May 1976 and others reported subsequently presented with meningitis or sepsis that did not respond to ampicillin therapy. The strains of H. influenzae isolated lind ampicillin minimal inhibitory concentrations 10 to 100 times those of previously studied isolates39 Although still rm'e, these strains have continued to appear u n d e r the same circumstances in various parts of the country, leading inany pediatricians to conclude that initial therapeutic considerations in serious Haemophilus infections must assume the organism to be ampicillin resistant until proven sensitive. a~ In most cases this means the substitution or addition of cldoramphenicol, the duration of theral) )' depending upon the results of susceptibility tests. Because of the special growth requirements of H. inJluenzae, modifications in methods used for less fastidious organisms are necessary. Both dilution and diffusion methods for mnpicillin testing described prior to the emergence of ampicillin resistant strains have proven usefifl in detecting resistance, and additional methods have been described with the entergence of resistant strains.39,41,42 Resistant strains described to date have minimal inhibitor)' concentrations ranging from 8 meg. per ml. to more than 32 meg. per ml. with sensitive strains inhibited by 0.5 meg. per ml. tar less. In addition, resistant strains have been shown to possess a beta lactamase enzyme, which inactivates penicillin and ampicillin?3"4a Although there are reports of dilticulty with the disc diffusion method,45-4r all the resistant strains tested by Thornsberry and Kirven s9 showed inhibition zones of I0 ram. or less, whereas all sensitive strains lind zones larger than 25 nlm. with J0 meg. ampicillin discs. Our experience is in agreelnent with these findings, with the additional observation tlmt resistant strains lmve consistently shown no zone of inhibition with a 10 unit penicillin disc. As with all susceptibility tests, it is important to realize that the methods used by these investigators for difft|sion testing of Haemophihts must be tollowed exactly if ampicillin resistant strains are to be detected. These steps are: (I) preparation of the inoculum by adjusting a suspension of the organisms (e.g., from a

282

chocolate agar plate) to match a MacFarland 0.5 turbidity standard, (2) swabbing of tile inocuhun over tile surface of plates containing chocolatized MuellerHinton agar with 1 per cent Isovitalex (BBL), (3) incubation for 24 hours in a candle jar, and (4) reading of the inhibition zones by rellected light. Inocuhun size has been shown to affect endpoint reading with susceptible strains and has a marked effect on minimal inhibitory concentrations and zone sizes with resistant strains because of enzymatic inactivation of the ampicillin? 9"4~ An additional test that has proven of value is the detection of the beta lactamase enzyme produce d by resistant strains. Thornsberry and Kirven44 have shown that when tested by a capillary tube method, ampidllin resistant strains demonstrate beta iactamase activity within five to 15 minutes whereas sensitive strains do not. Tiffs test is simple and rapid, requiring a nfinimum of materials and reagents (capillary tubes, penicillin vial, phenol red), all of which are available in every hospital laboratory. Since the test is set up by scraping colonies fi-om the primary isolation plate directly into the capillary tube, results are available 18 to 24 hours before dilution or diffusion susceptibility tests. It is our practice to report presumptive ampicillin susceptibility or resistance on the basis of this test, confirming with the standardized disc diffusion procedure already outlined. Thus, ampicillin resistant strains of Haemophilus influenzae studied to date have demonstrated a number of clmracteristics that enable them to be recognized in the clinical laboratory: ampicillin zone (10 meg. disc) less than 13 Into. by diffusion testing, no zone of inhibition with a penicillin disc (I0 unit), l)l'oduction of betalactamase fl'om isolated colonies within five to 15 minutes, mad minimal inhibitory concentrations greater titan 8 meg. per ml. by dilution testing. Since the mq)ortant theral)eutic decisions are made in the first few hotlrs and days, clinical microbiology laboratories in hospitals likely to encounter serious Haemophilus infections (meningitis, epiglotitis, septicemia) should be prepared to perform a combination of these tests.

ANTIMICROBIAL SUSCEIrI'II$11.1TY TESTING-RYAx, SHERRIS T H E D E V E L O P M E N T OF R A P I D AND AUTOMATED TESTS

As in all areas of clinical microbiolog)', there Ires recently been increasing interest in developing rapid, mechanized, and automated procedures for susceptibility testing that can give results the same day the test is set up rather than after the overnight incubation that currently recommended procedures require?'n Rapid modifications have been described for regular agar diffusion tests using early reading of standard plates and plates sprayed with tetrazolium dyes to enhance delineation between areas of growth and inhibition and for broth dilution methods with the addition of tetrazolium.4s-5~ Rapid automated procedures lmve been developed tlmt depend upon the release of radioactively labeled substances, laser light scattering, electrical impedance measurements, light scattering measurements, and direct bacterial counting? 2-5s Rapid automated methods generally place the organism in the presence of a single concentration or series of concentrations of an antimicrobic agent to determine whether inhibition has taken place within four hours or less. These new methods have been evahmted by comparison with the resuhs of a traditional overnight dilution method, and the conditious of the rapid procedure have been nmnipulated to obtain the best possible correlation. With two of the procedures most studied this has involved pragmatic lowering of the antimicrobic concentrations used in the rapid test. Exact correlation in terms of an absolute minimal inhibitory concentration is not good when the same inoculum is used because the minimal~ inhibitory concentrations tend to increase with the duration of incubation.a~,5,~ Tiffs is particularly striking with organisms producing extracellular antibiotic inactivating enzymes (such as b e t a lactamase producing stal)hylococciC~ with antibiotics that deteriorate during incubation,GI and when high mutation rates to resistance or hetcroresist,race exist. Using the International Collaborative Study broth dilution procedure, 5 Lampe et alp ~- conlirmed these findings and found that approximately 28 p e r cent of" the

tests showed fourfold or greater increases in mininlal inhibitory concentrations after 18 hours of incubation when compared with three hour readings. Some differences were as great as 100-fold, and no overnight minimal inhibitory concentrations were lower than the early readings of the same test. T h e correlation was not improved by replacing visual endpoint readings with those read by a mechanical particle counter. T h e correlation between three hour and overnight minimal inhibitory concentrations was improved considerably, however, when the inoculum for the three hour test was increased to 107 organisms per ml. while the inocuhnn for the comparison overnight test was held at 105 organisms per nd. Altllough it may be argued tlmt overnight incubation for determination of the minimal inifibitory concentration is an artificial situation in itself, comparability of early read automated tests with traditional methods is important because of the quantitative data and concepts that have been developed cumulatively over the years. Although complete correlation with all organismantibiotic combinations may not be possible, it appears that inoculum manipulation should be considered as a means to improve the correlation between rapid tests and the overnight minimal inhibitory concentrations determined under standardized conditions, which must remain the comparative yardstick. Of the rapid autonmted s)'stems currently under investigation, the Autobac I system (l'lizer Diagnostics) has undergone the most comprehensive evaluation and is currently approved by the FDA for clinical use. Tiffs system utilizes forward light scattering to detect the proliferation of bacteria in cuvettes to wlfich antimicrobic drugs Imve been added by elu~tion from paper discs. T h e inocultnn f o r the test is standardized with the aid of a light scattering photometer and dispensed into a multichambered cuvette in which the growth in a control cuvette (no antimicrobic drug) can be compared with that in cuvettes containing 12 different antimicrobic drugs. Results read after three hours' incubation are-automatically expressed as a light scattering index, which is a numerical ratio between 0 and 1 of test to control. Since the system in its cur-

283

HUMAN P A T H O L O G Y - V O L U M E 7, NUMBER 3 rent f o r m tests each agent at a single concentration, the atnottnt o f antitnicrobic drt,g in the ehttion discs has been adjt, sted to ntaxitnize correlation with currently r e c o m m e n d e d dihttion and diffusion tests. This systent was evahtated for perf o r m a n c e witlt 17 antimicrobic drugs (exchtding sulfonamides, which are not available for the systent) in a collaborative study designed to c o m p a r e the Autobac light scattering index resnhs with those obtained with the FDA r e c o m t n e n d e d diffusion and the ICS agar dilution procetlures.S-7, ss Complete interpretive a g r e e n t e n t between Autobac I and disc ditfilsion results was 90 per cent or higher for 13 drugs; with three, between 85 and 90 p e r cent; and with one, 77 per cent (nitrofurantoin). Contparison o f Autobac I attd disc diffttsion resuhs with those with the ICS agar dihttion p r o c e d u r e showed both to yield levels o f a g r e e m e n t with tire ICS dihttion test tltat were reasonably high and eqttivalent. T h a t is, Autobac I a g r e e d with a r e f e r e n c e dilution proc e d u r e as well as the FDA r e c o m m e n d e d dilfttsion procedt,re did. Tlte overall agreem e n t o f 16,000 tests c o n d u c t e d with tire Autobac I and disc diffusion methods was a p p r o x i m a t e l y 90 p e r cent. Difl'erences were observed, however, in correlations between the diffnsio,a and dihttion p r o c e d u r e s and the Autobac I and dihttion p r o c e d u r e s wlten discrepancies r a t h e r than a g r e e m e n t s were exantined. With the diffnsion test, ntost discrepancies involved the "interntediate" category (sensitive to intertnediate, resistant to intermediate) and, because they are less likely to lead the therapeutic errors, were called minor, q'he fi'equency o f major discrepancies, especially tltose in which Ihe Antobac s)'stetn indicated sensitivity and the dilution test, resistance, was considerably greater than with the disc test. Tire f r e q u e n c y o f tiffs type o f discrepancy was particttlarly high with certain atttimicrobic drttg-orga,tism comlfinations, such as gentamicin-l'seudomonas aeruginosa,

ampicillin-Emerobacter, cepbalotbin-Enterobatter, tet racycline-enterococcus, and

284

gentatnicin-enterococcus, in which m o r e than 10 per cent o f the tests were sensitive by tim Autobac m e t h o d but resistant by the r e f e r e n c e dihttion method. T h e s e findhags resulted in part bechttse o f the

May

1976

very n a r r o w intertnediate categot-)' for the Autobac I test in which each test cttvette contains a single concentration o f an atttimicrobic drug, in contrast to tim disc diffusion p r o c e d u r e , wlfich provides a continttotts concentration gradient with an i n t e r m e d i a t e category large e n o u g h to p r e v e n t small technical variations f r o m resulting in major difl'erences in interpretation. However, it also reflects the fact tltat manipulation o f the antimicrobic d r u g c o n t e n t alone catmot correct the differences between the three h o u r and overnight minimal inhibitory concentration results already discussed. T h e Autobac I systetn is a major technical advance in susceptibility testing toward the provision o f rapid a u t o m a t e d results that correlate well with r e f e r e n c e methods. As with all tnethods, t h e r e are sotne limitations. In the case o f tim At, tobac I system the tnost significant a p p e a r to be the lack o f a sulfonamide test and tim tendency to classify sonte organisms that are resistant in an o v e r n i g h t test as "sensitive." Sttlfonamides can be tested by anotlter tnethod when there is need for sttch tests, and still better correlation with traditional tests may be acltieved by m i n o r technical modifications. At present, resistance as detected by the Autobac I m e t h o d is highly reliable, and false sensitivity is restricted to a few organism-antimicrobic d r u g combinations in particular. Additional e x p e r i e n c e is n e e d e d before it can be d e t e r m i n e d w h e t h e r m o r e specialized sttsceptibility testing problems such as detection o f mctllicillin-resistant staphylococci can be solved by early read systems.

REFERENCES

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Clinical Microbiology Laboratories University of Arizona College of Medicine "I'ucsot,, Arizona 8572-t (Dr. Ryan)

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