Cumitech 25: Current Concepts and Approaches to Antimicrobial Agent Susceptibility Testing

25

CUMITCCH

Cumitech

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l

Blood

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Laboratory

Cultures

Cumltech

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Practical

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September

II

June

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DiagnoG Quality

1982

of Urinary

Control

Tract

Procedure\

Infections

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for the Clinical

1987

Microbiology

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1976

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New

Diagnoslr

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Anaerobic Development\

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Susceptibliity

Te\ting

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Detection

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DiagnoGs

of Lower

of Microbial

Respiratory

Antigen\

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Infections

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Collection

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I I

and

Laboratory

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Practical

MIcrobIology

Processing

of Bacteriological

Diagnose\

of Upper

Methods

Laboratory

for

Culture

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Respiratory and

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Laboratory

Diagnosi\

of Bacterial

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of Ocular

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of Central

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Diagnosis

of Viral

Infections

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Mycobacterioses

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Laboratory

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Immunoserology

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Infections

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Rapid

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Skin

in

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Clinical

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and

Infection\

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1982

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Tract

Viruses

March

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Disease

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Infection\

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Mycoplasmal

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Antimicrobial

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of Viruses

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Genital and

May

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Nervous

of Chlamydlal Monitoring:

of the

Fungi

October

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Infection\

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1979 December

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Cumltech

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Infections

Identification

Cumitech

Cumitech

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by Immunofluorescence

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August

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1984 1986

1987 June l

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1988

Cumitechs should be cited OS follows, e,g.: Sahm, D. F., M. A. Neuman, C. Thornsbeq, and J. E. McGowan, Jr. 1988. Cumitech 25, Current concepts and approaches to antimicrobial agent susceptibility testing. Coordinating ed., J. E. McGowan, Jr. American Society for Microbiology, Washington, D.C.

Editorial Board for ASM Cumitechs: Steven C Specter, William J Martone, John E. McGowan, Jr., Rick S Nolte, Glenn Thomas J Tinghttella, and Alice S. Welssfeld

Otarman; D Roberts,

Carl Abromson, Ellen Baron, James W. Smith, John A Smith,

The purpose of the Cumitech series ISto provide consensus recommendaBons by the authors as to approprvzte stateof-the-art operating procedures for clinical microbiology laboratories which may lack the facilities for fully evaluating routine

or new

methods.

The procedures

given are not proposed

CopyrIght

as “standard”

6 1988

methods.

American Society 1913 I St. NW WashIngton. DC 20006

for MIcrobIology

CURRENT CONCEPTS AND APPROACHES TO ANTIMICROBIAL AGENT SUSCEPTIBILITY TESTING DANIEL F. SAHM, Medical Center, MARK Inc., CLYDE

Clinical Chicago,

A. NEUMAN, and Department THORNSBERRY,

Microbiology Laboratories Illinois 60637

and Department

of Pathology,

University

of Chicago

Clinical Microbiology, Immunology and Virology Laboratories, Diagnostic Services, of Pathology and Laboratory Medicine, Naples Hospital, Naples, Florida 33940 Antimicrobics

Branch,

Centers

JOHN E. MCGOWAN, JR., Department Medicine, and Clinical Laboratory,

of Pathology Grady Memorial

JOHN E. MCGOWAN, JR., Department Medicine, and Clinical Laboratory,

of Pathology Grady Memorial

COORDINATING

Testing bacteria for susceptibility to antimicrobial agents (“susceptibility testing”) remains one of the most useful procedures in clinical microbiology. “Few other reports can dictate therapeutic direction so completely” (73). Rapid and major changes in available technology and in health care financing have led to many changes in diagnostic microbiology laboratories (6), and testing of the susceptibility of microorganisms is one of the areas where major changes have occurred, This was inevitable in view of the new pathogens encountered, the frequent introduction of new antimicrobial agents for the treatment of infections, and the rapid development of equipment to support this need. This Cumitech is intended to update the concepts and approaches presented in a previous publication in this series (65) regarding tests used to determine susceptibility, with particular reference to the ways in which a given laboratory decides how susceptibility testing is conducted and reported. The presentation is intended as a general guide to help laboratories decide what testing methods will be used and how the methods will be implemented. Thus, it attempts to supplement publications such as the American Society for Microbiology’s Manual of Clinical Microbiology (32) and the documents of the National Committee for Clinical Laboratory Standards (NCCLS) (37-45), to which readers are referred for detailed descriptions of the most recently recommended testing methods. Publications of NCCLS can be obtained directly by writing to NCCLS, 771 East Lancaster Avenue, Villanova, PA 19085, or by telephoning (215) 525-2435. A companion Cumitech (no. 6A) deal-

for Disease

Control,

Atlanta,

Georgia

and Laboratory Medicine, Emory University Hospital, Atlanta, Georgia 30335

30333 School

of

School

of

EDITOR and Laboratory Medicine, Emory University Hospital, Atlanta, Georgia 30335

ing with susceptibility testing of special problem bacteria will follow this one. SELECTING DRUGS FOR ROUTINE TESTING The need to choose certain drugs and to exclude others from routine testing is a relatively recent problem. In the past, there was a relatively small number of antimicrobial agents available for clinical use, and laboratories could test susceptibility to each of these agents as a routine practice. Today, however, the number of antimicrobial agents available to prescribers has risen dramatically. Many of the current agents may have only minor, or even no, differences in activity against microorganisms; they may be marketed because they differ from drugs of a similar spectrum in attributes such as pharmacology, toxicity, and cost. These trends have made it impossible for most laboratories to routinely test all available agents, or even all agents possibly active against a given class of organism (e.g., all antibiotics active against gram-positive cocci or all those active against gram-negative aerobic bacilli) (23, 46). The increasing use of commercial automated systems for susceptibility testing, rather than agar disk diffusion methods, has added to the need for selecting certain agents (1). When an agar disk diffusion system is used, the introduction of a new antibiotic can be handled by adding another disk to the test battery, assuming that space still remains in the dispenser and on the plate. For many commercial systems, however, testing the new antimicrobial agent may require the manufacturer to design customized testing

2

SAHM

ET AL.

modules (cards, panels, etc.) that incorporate the most recently released drugs. Such units are expensive and laboratories may have to discard or use up stocks of previously purchased modules containing older drug configurations before the new panels can be used. Such changes in drug test panels may be desirable, but today’s financial climate, which demands cost-effective and efficient laboratory operation, often renders these time-consuming and expensive changes less feasible. Because of these factors, laboratories must now carefully consider the drugs that they include in their routine test panels. The selection of drugs to be included in susceptibility testing panels requires the consideration of several factors (46,51). The first of these is the type and size of the laboratory. A small laboratory in a community hospital may have different testing needs than a university hospital or a reference laboratory (29). The difference in needs would reflect a variation in the patient population and in the services offered by the laboratory. For example, a laboratory may serve an institution with a large number of chronic care patients, in whom patterns of infection vary from those of a population requiring acute care. The second consideration is the kind of organisms frequently recovered in a given laboratory and their resistance patterns (i.e., antibiograms). For example, a high prevalence of gram-negative aerobic bacilli resistant to gentamicin and tobramycin would make the routine testing of amikacin more reasonable than in hospitals not harboring such resistant strains. The most likely susceptibilities can and should be predicted from data at the institution (23). Susceptibility patterns from another hospital or from national data cannot be used, as the patterns may vary markedly, even when the patient populations in the hospitals are similar (17). Third, formulary decisions of hospitals have a great impact. Prospective reimbursement is leading many hospitals, health maintenance organizations, and other health care institutions to be more restrictive about the drugs available in the hospital formulary (58). This makes it all the more important for the drugs in the testing panel to match the drugs available in the formulary (34). How well a laboratory can achieve this goal depends on the flexibility of the testing methods, which should allow the laboratory to introduce inexpensive and relatively trouble-free changes of drug batteries. Selection Process A good place to begin the task of selection is to review the medical literature. A number of groups have published lists of drugs to consider for routine testing. The most influential of these perhaps is NCCLS, which lists the agents that should be considered for routine testing bv ei-

CUMITECH

25

ther the agar disk diffusion method (37) or dilution tests (38). In these listings, drugs are grouped into categories of primary, secondary, and urinary tract infections and the categories are further divided into drugs that may be tested against members of the family Ertterobacteriaceae, Pseudomonas spp., enterococci, nonenterococcal streptococci, and staphylococci. A list for Haemophilus spp. is soon to be added. These listings are updated periodically (42) and may include drugs that have not been released for routine use in the United States, but are expected to be approved soon. To choose the best test panel for a given institution, microbiologists should also obtain information from outside the laboratory (35). Guidelineslike those of NCCLS are intended as a general guide for all types of laboratories throughout the country. While clinical microbiologists are qualified to evaluate the technical features and validity of laboratory procedures, the ideal application of theseprocedures should be established*by microbiologists, clinicians, and pharmacistsworking together (29). A specialist in infectious diseases,if present at the institution, or other staff physicians with an interest in infectious diseasesshould be part of the decision-makingprocess(34,58). Depending on the size of the hospital and patient populations, clinicians from other services (pediatrician, obstetrician, ophthalmologist, surgeon, etc.) may contribute valuable insights about which agents are needed and which can be omitted without compromising the quality of patient care. In the samefashion clinical microbiologists need to be included in making decisionsabout the antimicrobial drugsin the hospital formulary (34, 35). This is especially important when a laboratory is using automatedor other commercial test methods.In this casesomedrugs being proposedfor inclusion in the formulary may not be available for testing by the susceptibility testing equipment and materials of the laboratory. This problemneedsto be known assoonas the proposalfor inclusion of the drug is made. Certain antimicrobial agents are unsatisfactory for the therapy of certain infections because they fail to achieve effective concentrations at the body site. Testing of these drugs, then, should be performed only when the organism tested has beenisolated from sites where effective concentrationscan be achieved. For example, recommendedsusceptibility panelsfor isolates from urine (Table 1) include drugs not tested when infection is suspectedat any other site’,becausethe drugs achieve therapeutic levels only in urine. Likewise, certain cephalospor-inswhich do not achieve therapeutic levels in cerebrosninalfluid (e.g.. cenhalothin. cefaman-

CUMITECH

TABLE

25

SUSCEPTIBILITY

TESTING

3

1. Examples of suggested groupings of antimicrobial agents that should be considered for routine testing and reporting by clinical microbiology laboratories” Organism

Drug group

Enterobacteriaceae

P. aeruginosa

Staphylococci

Primary drugs

Amikacin, ampicillin, cefazolin, cephalothin, chloramphenicol, gentamicin, tetracycline, tobramycin

Amikacin, [azlocillin or Cephalothin, clindamycin, piperacillin] , [carbenicillin or erythromycin, [oxacillin or mezlocillin or ticarcillin] , methicillin] , penicillin G , gentamicin, netilmicin, tetracycline, vancomycin tobramycin

Secondary drugs

Amoxicillin-clavulanic acid, ampicillin-sulbactam, [aztreonam or cefotaxime or ceftazidime or ceftizoxime or ceftriaxone or moxalactam], [carbenicillin or ticarcillin] , [cefamandole or cefonicid or cefuroxime], cefoperazone, cefotetan, cefoxitin, ciprofloxacin, imipenem, kanamycin, [mezlocillin or piperacillin], netilmicin, ticarcillin-clavulanic acid, trimethoprimsulfamethoxazole

Aztreonam, [cefoperazone or ceftazidime] , [cefotaxime or cefiriaxone or moxalactam], imipenem, ticarcillinclavulanic acid

[Amikacin or gentamicin or kanamycin or netilmicin or tobramycin], amoxicillinclavulanic acid, ampicillinsulbactam, chloramphenicol, ciprofloxacin, imipenem, rifampin, ticarcillinclavulanic acid, trimethoprimsulfamethoxazole

Urine

[Cinoxacin or nalidixic acid], ciprofloxacin, nitrofurantoin, norfloxacin, sulfisoxazole, trimethoprim, trimethoprimsulfamethoxazole

Ceftizoxime, ciprofloxacin, norfloxacin

Nitrofurantoin, ciprofloxacin, norfloxacin , sulfisoxazole, trimethoprim, trimethoprimsulfamethoxazole

“Adapted from NCCLS standard supplement MlOO-S2 (42). See this reference for footnotes and cautions; listing here is only to exemplify recommendations for primary and secondary groups of drugs, testing of different drugs at different sites, and use of spectrum groups (shown in boldface within brackets).

dole, and cefoperazone) should not be included in panelsfor the testing of isolatesfrom cerebrospinal fluid becausethese drugs do not achieve therapeutic levels at this site. SpectrumGrouping Concept To permit a wider choice of antimicrobial agentsfor prescribers, yet maintain an efficient testing program, NCCLS hasincluded in recent guidelines recommendations that establish “spectrum” drug groupings (37, 38, 42). These designationsare intended to “prevent duplication of susceptibility test information, wasted laboratory costs, and physician confusion” (R. N. Jones, Antimicrobic Newsl. 3:81-86, 1986). Ordinarily, the testing of one representative drug of each “spectrum group” should be sufficient (23, 25). Within a given spectrum grouping a laboratory should test the specific drug most often employed at the institution. To illustrate, assumethat a hospital formulary includesmezlocillin and ticarcillin but not carbenicillin and that ticarcillin is used much more frequently than mezlocillin. NCCLS guidelines suggestthat thesethree drugs can be considered

a spectrumgroup for the testing of P. aeruginosa (42). In this instance, the testing of ticarcillin alonewould permit efficient and satisfactory laboratory service for predicting susceptibility to carbenicillin, mezlocillin, and ticarcillin but not azlocillin or piperacillin. Similarly, cefoperazone and ceftazidime constitute a spectrumgroup for P. aeruginosa, and amikacin, gentamicin, kanamycin, netilmicin, and tobramycin constitute a spectrum group for staphylococci. These and other spectrum groups are given in boldface in Table 1. The same principle of spectrum group has been included in recent versions of NCCLS standardsfor dilution testing (38), as well as for agar disk diffusion testing (37). In each of these, however, certain drugs are listed with warnings that they cannot satisfactorily serve asspectrum group drugs. For example, the standardon disk diffusion testing states that “cefazolin should not be usedto predict the susceptibility of other first-generation cephalosporins(cephalothin remainspreferred) becauseof a significantnumber of false-susceptibleerrors, principally among the E. coli isolates” (42).

4

SAHM ET AL. TABLE

CUMITECH

2. Example?

of sequential testing and reporting of antimicrobial gram-negative bacteria

25

susceptibility results for

Example

1. Enterobacteriaceae

Ampicillin, ticarcillin,’ cefazolin, cefuroxime,b cefotetan,b gentamicin,b trimethoprim-sulfamethoxazole, tetracycline, chloramphenicol

Ticarcillin-clavulanic acid, piperacillin, ceftriaxone, imipenem, amikacin

2. P. aeruginosa

Mezlocillin ,b gentamicin, tobramy tin’

Piperacillin, ceftriaxone, ceftazidime , imipenem, amikacin

“The examples shown are taken from protocols in use by at least one of the authors at the time this Cumitech was written. They are intended as an illustration only and are not being recommended for adoption by others; the best system for each hospital depends on a number of factors (see discussion in the text). They vary from drug panels recommended by NCCLS (42). ‘Resistance to this drug is a signal that leads to the testing and reporting of the entire battery of drugs contained in the secondary panel.

of drugs to be tested. Of course, such an approach is easierto implementwhen a laboratory employs methodsthat permit same-daydetermination of results (seesection below on selecting methodsfor routine testing), so that for resistant organismsresultsof both primary and secondary test panels can be available for the patient’s physicianby the next morning after the organism is recovered. In laboratories where the great majority of organismsare susceptible to routinely tested drugs, the type of “process control” (68) afforded by sequentialtesting can provide efficient yet satisfactory handling of most organisms. However, in a hospital where the prevalence of resistant organisms is so great that frequent testing with secondary drugs is needed, it may be more cost-effective to simultaneously test primary and secondary agents and selectively report results as needed(see section below on selective reporting of susceptibility results). By this approach, when resistant isolates are enSequential Testing countered, susceptibility results for other possiSomehospitalstoday use a sequentialsystem bly active drugs will not be delayed 16 to 24 h. of testing antimicrobial resistance (52). This Clinical laboratoriesfrequently do not receive concept is included in some of the NCCLS all pertinent information neededto make approstandards (37, 38, 42); one group of drugs is priate decisionsabout which drugs should be recommendedfor the routine testing of all orga- tested. Instancesmay arise (e.g., polymicrobial nisms of a given type (gram-positive cocci, infections, drug allergy, and altered excretion gram-negative aerobic bacilli, etc.), and a sec- mechanisms)where data on a drug included in ond list of antimicrobial agentsis employed for the secondary panel may be more important further testing when the initial battery reveals than usual. Therefore, if sequential testing is unusual resistance of the organism or at the undertaken, the flow of information between a request of the patient’s physician. Examples of laboratory and the medical staff must be such sequentialtesting protocols are given in Table 2. that, when needed, exceptions to the protocol In these examples, resistanceto certain drugs may be instituted promptly. from the routine (primary) test battery may be SELECTING ORGANISMS FOR used to automatically signal the testing of seROUTINE TESTING lected drugs from a backup (secondary) battery of drugs; no further request by the patient’s Laboratories must select specific organisms physician would be neededfor the secondgroup for susceptibility testing on a routine basisand Although NCCLS does not advocate the extrapolation of results, a categorization of findings (i.e., susceptible, resistant, etc.) for each drug in a spectrumgroup is likely to be the same. This does not mean, however, that the MIC or zone size is going to be identical for each drug in a spectrum group. The spectrum group concept must be distinguishedfrom the idea of a “class disk.” In the latter, one drug is chosenfor the testing of all drugsthat are so closein spectrumthat each will respondin the samefashion to testing. Thus, for example, cephalothin is used as the class drug for the testing of all other “first-generation cephalosporins”(42),and resultsfor cephalothin are used to predict susceptibility to all drugs of the class. This concept varies from that of the spectrumgroup, as drugsin the spectrumgroup do not necessarily have interchangeable test results, as would drugs for which a classdisk is used.

CUMITECH

25

SUSCEPTIBILITY

TESTING

5

designate the situations in which other orga- standards(they will be returned to the standards nisms will be tested. These criteria should be if such information becomesavailable) (42). based on the clinical importance of the organism For a numberof drug-organismcombinations, and on the predictability of the drug susceptibilmultiple problems may exist with testing; the ity profile of the organism. Although staphylotest may not be standardizedwell, or a correlacocci, Enterobacteriaceae, and Pseudomonas tion with a favorable clinical responsehas not aeruginosa usually are tested routinely, the pro- been established(4, 8, 30). However, clinical liferation of other organismscausinginfections situations arise in which a “best estimate” of in today’s patients and the lossof predictability likelihood of responsecan be useful to a pain susceptibility of many “old” pathogens(e.g., tient’s care (66). In thesesettingsrecent NCCLS Haemophilus influenzae and Neisseria gonorguidelines (42) and recommendationsfrom the rhoeae) have complicated decisionsconcerning Centers for DiseaseControl (66) suggest test which microorganismsto test. methods for a number of organisms in this category. Results of such testing should be interpreted with discretion (22), and reporting OrganismsThat Should Not should reflect that these tests may not predict Be TestedRoutinely the likely clinical responseas well as the stanCertain organismshave characteristicswhich dard tests that are routinely recommended. precludethe necessityfor susceptibility testing. Organismsfor Routine Testing This limits the number of organismsthat are in Certain Situations tested for susceptibility. Some organismshave such a constant responseto certain antimicroCertain organismsmay be tested only when bial agents that susceptibility can be reliably they are isolated from certain body sites (51). predicted from their identity. Thus, because For example, pneumococcihave predictable regroup A streptococci and Neisseria meningitidis sponsesto penicillin G in laboratory testing. In have almost always been susceptibleto penicil- casesof meningitis, however, a relative resislin G (51), testing susceptibility of these orga- tance to penicillin G may predict a clinical nisms against penicillin G is necessary only failure. Therefore, routinely testing pneumowhen strains inconsistent with this predictive cocci isolatedfrom cerebrospinalfluid or certain response are encountered or when a patient other usually sterile body fluids is recomcannot tolerate penicillin. mended, while pneumococcal isolates from Certain organismsshouldnot be tested against other body sites(sputum, etc.) shouldbe tested someagents becauseavailable testing methods only in specialcircumstances. are unreliable and do not accurately reflect the Routine susceptibility testing of some orgalikelihood of favorable patient response (23). nismsmight be performed by certain laboratoFor example, Salmonella typhi is almost always ries but not by others. For example, the testing shown to be susceptibleto cefamandolein lab- of anaerobesis a time-intensive procedure that oratory testing, but a clinical responseto cefa- often does not correlate well with a clinical mandole in patients with typhoid can be ex- response(20; Jones, Antimicrobic Newsl. 3:81pected in fewer than 50% of patients whose 86, 1986).Anaerobe susceptibility testing might isolates are susceptible by laboratory testing be routinely performed by a referenceor referral (13). Similarly, the NCCLS standardsfor disk laboratory, but somehospital clinical laboratodiffusion testing list a number of drugs (cepha- ries might choose not to perform these tests losporins, clindamycin, and aminoglycosides) routinely (20, 51). A second example is fungal that should not be tested against enterococci susceptibility testing, which requires intensive “because the reporting of their results can be time and quality control efforts and for which dangerously misleading (false-susceptible)” clinical correlation is still questionable. For (42). Theseguidelinesalsonote that cephalothin these organisms,recommendationsof NCCLS susceptibility test results cannot be relied upon suggestthat routine testing is still a research to detect cephalosporinresistanceamongmethi- procedure except for a very limited number of cillin-resistant staphylococci (42). drug-organismpairs; tests should be limited or A third setting in which organismsshouldnot discontinued in most hospital settings (43). be routinely tested is situations in which the Thus, it would be unusualfor a hospital laborasusceptibility test itself cannot be interpreted or tory to routinely test susceptibility of fungi to hasnot beenstandardized(25). For example, the antifungal agents. updated NCCLS guidelinesfor dilution tests no Retestingof SequentialIsolates longer include ceforanide, cinoxacin, doxycycline, and minocycline in the standardsbecause The clinical responseof a patient is the ultiadequate quality control information has not mate standardfor the validity of in vitro suscepbecomeavailable after publication of the initial tibility testing. Thus, a suspicionof the emer-

6

SAHM ET AL.

CUMITECH

TABLE Testing method

Agar dilution Broth macrodilution Broth microdilution In-housee Commercialfrozen Commerciallyophilized Agar disk diffusion Automated systems Broth disk elution

25

3. Relative comparison of in vitro susceptibility testing methods

Accuracy

of results em

Reporting TAT”

Reporting format b

Versatility

of:

Drug selection

Standard organismsc

Other organism&

Work flow impact

Reference method Good Reference method Good

Flexible Flexible

Excellent Excellent

Excellent Excellent

Excellent Excellent

Poor Poor

Reference method Good >90” Good

Flexible Flexible

Excellent Limited

Excellent Excellent

Excellent Limited

Moderate Good

>90”

Good

Flexible

Limited

Excellent

Excellent

Good

>90” HO” HO”

Good Excellent Good to poor

Limited Flexible Limited

Excellent Limited Limited

Excellent Excellent

Limited Limited

f

f

Good Excellent Moderate

“Time between inoculation and reading of results: excellent, ~16 h; good, 16 to 24 h; poor, >24 h. ‘Flexible, Results may be reported quantitatively (MIC), qualitatively (susceptible, intermediate, moderately susceptible, or resistant), or both. “Standard organisms: staphylococci, enterococci, Enterobacteriaceae, P. aeruginosa, and Acinetobacter species. dOther organisms: fastidious microorganisms, including H. influenzae, N. gonorrhoeae, S. pneumoniae, nonenterococcal streptococci, and anaerobic bacteria. eCorrelates with the reference method at least 90% of the time. fUse is usually limited to the testing of anaerobic bacteria.

gence of resistancein a previously susceptible organismis a valid indication for susceptibility testing of the sameorganismin sequentialcultures. In the absenceof such clinical developments, laboratory personnelshouldconsultwith their clinical colleaguesto define an interval during which repeated isolation of the same organismwill not trigger repeated susceptibility testing. Because the time frames that may be established likely will vary with the patient services available at a given hospital and with the type of specimen,generalrecommendations for the time intervals between susceptibility tests cannot be made. Similarly, specificrecommendationsconcerningthe needto perform susceptibility testing on the sameorganismisolated from two or more sites shouldbe established;if isolatesare thought to be the sameby biochemical characteristics,susceptibility testing of each generally would not be necessary.Consultation by the patient’s physician should make clear special situations where testing both isolates would be necessary.

peutic managementof patients; this interval likely will vary from hospital to hospital (52). Test results must be provided in formats that medicalpersonnelprefer (if reasonable)and can easily interpret. Also, the susceptibility testing system that is selectedmust be capable of providing accurate results for the majority of clinically significantorganismsencounteredwith the antibiotics most commonly used in a particular institution. Second, susceptibility testing protocols shouldmeet the needsof the laboratory in which they are performed. Whenever possible the methods chosen should be cost-effective, they should accommodateand facilitate laboratory work flow, and results should be readily interpretable (6). The susceptibility methodscurrently available for use in clinical microbiology laboratories include agar disk diffusion (5, 37), microdilution and macrodilutionbroth procedures(26,38), agar dilution (38, 39,69), mechanized,automatedsystems (1, 64; M. S. Gradus, C. N. Baker, and C. Thomsberry, Antimicrobic Newsl. 9:65-71, 1985; M. S. Gradus, C. N. Baker, and C. SELECTING METHODS FOR Thornsberry, Antimicrobic Newsl. 10/11:73-82, ROUTINE TESTING 1985),and broth disk elution (41, 62). Because The choice of susceptibility testing proce- extensive technical information about these vardures for routine use must depend upon two ious methodsmay be obtained from the refermajor considerations.First, the selectedmeth- ences or, in the case of commercial products, od(s) must support and meet the needsof the from product information inserts, we will use medical staff being serviced by a laboratory. this text to discussonly the relative qualities of Therefore, the testing protocols must produce each. The relative qualities of each susceptibility accurate, reliable results within a time frame that is helpful to the physicians in their thera- testing methodwith respectto accuracy, report-

CUMITECH

25

ing turnaround time (TAT), reporting format, versatility in drugs and organismsthat may be tested, and effect on laboratory work flow are given in Table 3. With respectto accuracy, agar dilution, broth macrodilution, and broth microdilution (38, 39) produce the most accurate resultsand usually serve asthe reference methods by which other susceptibility systemsare evaluated. The other available susceptibility methods (commercial broth microdilution, agar disk diffusion, automated systems, and broth disk elution) usually demonstrate >90% agreement with the reference methods and show good overall accuracy (Gradus et al., Antimicrobic Newsl. 10/11:73-82, 1985). Therefore, any of the currently available methodsare reliable for producing accurate susceptibility results. For most methods the reporting TAT from inoculation to the availability of results is 16 to 24 h (Table 3). The advantage of substantially decreasingTAT to 3 to 10 h may be gained by usingmechanized,automatedsystems(1). However, each institution must balance the cost of these systemsand problemswith the testing of certain drug-pathogen combinations (71) with the potential patient and laboratory benefitsthat may result from their use. As another possible approach to decreasingthe susceptibility TAT, Coyle et al. (11) have shown that accurate agar disk diffusion resultsmay be obtained at 4 and 6 h after inoculation, insteadof the usual 16 to 18 h (overnight). However, further studies that establishthe accuracy of testing severaldifferent organism-antibioticcombinationsshouldbe performed before laboratories adopt “rapid” agar disk diffusion methods.Currently, the TAT of 48 h associated with most susceptibility testing methodsfor anaerobesseemsunavoidable. Except for agar disk diffusion and broth disk elution, all testing systemsallow a flexible reporting format so that MIC, category interpretations (susceptible,intermediate,moderately susceptible, or resistant), or both may be reported (Table 3). The selectionof a reporting format for a laboratory shouldbe basedon discussionswith the medical personnel who will be using the data. Clearly, if qualitative (i.e., category) resultsare all that are required by clinicians, then the reporting formats of agar disk diffusion and broth disk elution should not be considered limited. A system that has not gained widespreaduse but that allows MIC determinations from inhibition zone diameters may allow a flexible reporting format for the agar disk diffusion method (12). The considerationof drug versatility becomes more complex as more antibiotics are released for therapeutic use. As there are hospitalformulary changes,clinical microbiology laboratories must be capable of adapting to these changes

SUSCEPTIBILITY

TESTING

7

and testing the pertinent drugs. Therefore, the ability of a susceptibility testing system to accommodatechangesin the battery of antibiotics tested is of primary importance. In this regard, agar dilution, broth macrodilution, and in-house broth microdilution methodsdemonstrateexcellent versatility (Table 3). Virtually any antibiotic that is available in powder form may be incorporated in the media and used for testing. For agar disk diffusion the requirement of approval by the Food and Drug Administration doesdelay the availability of antibiotic disks, but the option of selectingany combination of disksfor testing doesmakethis systemversatile. While the broth disk elution method for testing anaerobesalso usesdisks,the versatility of this systemmust be considered limited. Broth disk elution results have not been adequately compared with reference methodsfor all available antibiotics active againstanaerobes,and somestudies have indicated that the disk elution method may be problematic with certain beta-lactamantibiotics such as broad-spectrum cephalosporins and imipenem (57, 75). Most commercial broth microdilution systems,whether they employ frozen or lyophilized reagents, and most automated systems provide their own standard antibiotic batteries that may or may not suit the needs of a particular laboratory. Because users cannot freely manipulatethe antibiotics tested in these systems,their drug versatility has been characterized as limited (Table 3). Most commercial supplierswill provide customized susceptibility panels, but unlessthe testing volume of a laboratory is large, the cost of these panels is often prohibitive. Another approach to this problem has been the provision of panels that contain most, if not all, clinically pertinent antibiotics. However, a disadvantageof these panelsis that somedrugs are not present in full-range doubling-dilution concentrations, but only at breakpoint concentrations, so that the reporting is frequently limited to category results alone. Organismversatility pertains to the variety of organismsthat can and cannot be reliably tested by the various susceptibility testing methods (Table 3). The “standard” organismsfor susceptibility testing include staphylococci, enterococci, Enterobacteriaceae, P. aeruginosa, and Acinetobacter spp. Generally, all methods are capable of accurately testing most of these organisms. However, certain commercial broth microdilution and automated systemsmay have difficulty in reliably detecting methicillinresistant staphylococci, and users should be aware of any precautions needed when testing for these microorganisms. In addition, users should also be aware that some commercial systemshave particular difficulties with certain drug-organismcombinations(Gradus et al., An-

8

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ET AL.

timicrobic Newsl. 9:65-71, 1985; Gradus et al., Antimicrobic Newsl. 10/11:73-82, 1985). The “other organism” column in Table 3 refers to fastidious microorganismssuch as II. infl~enzae, N. gonorrhoeae, Streptococcus pneumoniae, nonenterococcal streptococci, and miscel-

laneousgram-positive and gram-negative aerobic and anaerobic bacteria. For the testing of theseorganisms,agar dilution, broth macrodilution, in-housebroth microdilution, and commercially available lyophilized systemsare excellent in that the media can conveniently be supplemented, or entirely replaced,by a more enriched formula that facilitates the growth of many of thesemicroorganisms.Although the growth media in commercially available frozen broth microdilution panels may be supplemented, their use is considered limited because such supplementationoften dilutes antibiotic concentrations in the wells, resulting in different drug concentration testing ranges. To circumvent such supplementationproblems, somecommercial providers have made special broths available that were developed to support the growth of fastidious microorganisms.Others also provide specific systemsfor the susceptibility testing of anaerobic bacteria. Agar disk diffusion methods may be used to test H. injluenzae, N. gonorrhoeae, S. pneumoniae, Listeria monocytogenes, and nonenterococcal streptococci againsta limited number of antibiotics (37,42). However, the procedure has been characterized as limited becausezone size interpretive standardshave not beenestablished for other miscellaneousor fastidious bacteria; therefore, these organismscannot be tested reliably by this procedure. Additionally, agar disk diffusion is not recommendedfor testing anaerobes. In general, the automated systemsas yet are not reliable for testing mostfastidiousmicroorganisms, and persons using these methods shouldbe aware of the limitations. Finally, in regard to laboratory work flow impact, when factors such as medium preparation, inoculation, and reading of results are considered,the cumbersomeandlabor-intensive nature of agar dilution and broth macrodilution methodsmakesthem prohibitive for usein most laboratories(Table 3). The in-housepreparation of broth microdilution panelsalsois labor intensive and cumbersome,but once the panelsare prepared, they may be conveniently stored and used. However, another significant factor that must be considered is the substantial cost of purchasingthe laboratory hardware required for the in-house preparation of MIC panels. The broth disk elution method is somewhatcumbersome but is manageable,depending upon the number of daily anaerobesusceptibility tests a laboratorv nerforms. The automatedinoculators

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25

and readers supplied with most commercially available broth microdilution panelshave facilitated their use so that these systems may be incorporated into laboratory work flow with the samerelative easeas agar disk diffusion testing. However, the use of the frozen panelsrequires laboratories to have adequate freezer storage space. The automated systems require less hands-ontechnical time than most other susceptibility methods,especiallyfor the interpretation of results, and may actually enhancelaboratory work flow. REPORTING OF SUSCEPTIBILITY TESTING RESULTS

The approach and policy of a laboratory for reporting test results are as important as any other aspect of in vitro susceptibility testing. The reporting format and the drugs that are selectedfor reporting directly affect the usefulness of the information for clinicians and can substantiallyinfluencewhich antibiotics are chosenfor therapeutic use. Reporting

Format

The format chosenfor the reporting of results shouldbe decided through discussionsbetween personsperforming the tests and those who will use the data for patient management.With the exception of agar disk diffusion and disk elution methods (Table 3), most systems allow for a choice betweenquantitative (MIC) and category (susceptible,intermediate, moderately susceptible, or resistant) reporting formats. Some institutions may choose to use one format or the other. However, the versatility of being able to choose the format dependingupon the clinical situationwould seemto be a usefulcompromise. While category reporting may offer sufficient information for the treatment of many infections, there are occasions when quantitative results are helpful (e.g., endocarditis and the oral therapy of osteomyelitis). In addition to their usefulnessfor the therapy of certain infections, MICs can also help to establishthe relative resistance of certain microorganismsthat cannot be ascertainedby category reports (e.g., relatively penicillin-resistant S. pneumoniae, variable penicillin resistanceof viridans group streptococci, non-beta-lactamase-producing ampicillin-resistant H. influenzae, and penicillinresistantN. gonorrhoeae). One hospital laboratory hascombinedsusceptibility and pharmacy data to identify patients who are receiving antibiotics to which their pathogensare not susceptibleor who could be receiving less expensive antibiotics (18). The applicability of this approachfor other hospitals and other settingsdeservesfurther study.

25

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TABLE

SUSCEPTIBILITY 4. Variations in interpretation

Antibiotic

Interpretive breakpoint (p,g/ml of drug)

Organism

Ampicillin

acid Haemophilus Others

species, staphylococci

Enterobacteriaceae

Staphylococci and B. catarrhalis species and L. monocytogenes Enterococci Nonenterococcal streptococci and other gram-positive organisms Gram-negative enteric organisms and staphylococci Haemophilus species Haemophilus

Ampicillin-sulbactam Carbenicillin Penicillin G

Enterobacteriaceae Pseudomonas

spp. Staphylococci and B. catarrhalis

N. gonorrhoeae

Enterococci L. monocytogenes

Nonenterococcal

9

according to bacteria tested in dilution testinga

Sb

Amoxicillin-clavulanic

TESTING

streptococci

S. pneumoniae

Ib

Rb

c8 so. 12 0.25-2

NAC NA NA NA NA NA NA

1814 132116 232 20.5 r4 216 r4

5814

1618

NA

132116

5211 516 32 5128 so. 12 so. 12 0.25-2 58 52 so.12 0.25-2 ~0.06 0.12-l

NA NA NA NA NA NA NA NA

2412 264 ~256 ~0.25 14 216 24 24 22

5412 5814 58 so.25 52

MS6

1618 16

“Adapted from reference 42. %, Susceptible; MS, moderately susceptible ; I, intermediate ; R, resistant. “NA, Not applicable.

Reporting Categories Retailed descriptionsof interpretive standards and reporting categoriesfor agar disk diffusion and dilution techniques have been developed (37, 38,42). Four major aspectsof thesereporting standardsshould be emphasized.First, for the MIC interpretive standards,only three reporting categoriescurrently are defined(susceptible, moderately susceptible, and resistant). The conditionally susceptibledesignationpreviously used as an interpretive category for urinary tract isolates has been discontinued; isolatespreviously placed in this category now are included in the moderately susceptiblecategory. Second, interpretive categoriesfor broth dilution testing (i.e., susceptible, moderately susceptible, and resistant) and interpretive categories for agar disk diffusion testing (i.e., susceptible, moderately susceptible,intermediate, and resistant) for certain penicillin drugs vary with the organismtested (Tables4 and 5). Third, zone diameters obtained when testing enterococci against either ampicillin or penicillin are interpreted as either resistant or moderately susceptible. L. monocytogenes tested against these samedrugsis interpreted aseither susceptibleor resistant but not intermediate. Fourth, the moderately susceptible category, rather than the intermediate category, is reported for zone diameters obtained with a variety of other betalactam antibiotics (Table 6). The difference between these two interpretive categoriesis that moderately susceptibleindicates that organisms

may be inhibited by drug concentrations attainable with maximum dosesindicated in the drug package insert or by drug concentrations achieved in siteswhere the drug is concentrated (e.g., urinary tract). In contrast, the intermediate category simply provides a “buffer zone” that prevents technical artifacts from causing major interpretive discrepancies(37, 38). Most of the drugs for which the moderately susceptible category is recommendedare beta-lactams becausethis group of drugs is safer than many others at high doses. Reporting Precautions Precautionsfor reporting resultsobtained with certain drug-organismcombinationsmust be emphasized.For Enterobacteriaceae,cefazolin may be more active than other first-generationcephalosporinsagainstEscherichiacoli and Klebsiella species.Therefore, susceptibility to all first-generation cephalosporinsshould not be presumed and reported solely on the basis of results obtained with cefazolin. P. aeruginosasusceptibility to ceftizoxime should not be reported for organismsisolatedfrom any site other than the urinary tract; chloramphenicol,tetracycline, and trimethoprim-sulfamethoxazole should not be tested or reported for this organismif a systemic infection is suspected.Regardlessof in vitro test results, methicillin-resistant staphylococci should not be reported as susceptible to any beta-lactam antimicrobial agent, including all cephalosporinsand imipenem. Finally, suscep-

10

SAHM ET AL. TABLE

CUMITECH

5. Variations in interpretation

according to bacteria tested in agar disk diffusion testinga

Antibiotic

Interpretive breakpointb (zone size [mm])

Organism S

Amoxicillin-clavulanic

acid

species, staphylococci

Huemophilus

Other Ampicillin

Enterobacteriaceae

Ampicillin-sulbactam Carbenicillin Oxacillin

18-22

(17

217 213 220 229 220

14-16 11-12

113 510 519 528 519 114 519

Interpretive

~16~ 1121” 515 515 514 112 514 rlOd 514” 513 514 514 519

14-17 12-13

223

spp.

TABLE 6. Susceptibility testing of beta-lactam antimicrobial agents by the agar disk diffusion method: interpretive standards that use the category of moderately susceptible”

Ampicillin Ampicillin Aztreonam Cefoperazone Cefotaxime Cefotetan Cefoxitin Ceftizoxime Ceftizoxime Ceftriaxone Moxalactam Penicillin Gb Penicillin GC

R

119 513 511

Enterobacteriaceae Pseudomonas

tibility testing of cephalosporinsand aminoglycosidesshould not be performed routinely with enterococcal isolates. Results of these drugorganism combinations are unreliable and reporting them could be extremely misleading. However, tests for high-level resistanceto aminoglycosidesmay be done with enterococci to determinethe likelihood of synergy with a betalactam (e‘g., in endocarditis).

Resistant

I

229

“Adapted from reference 42. ‘For abbreviations, see Table 4, footnote b. =For determining the relative resistance of S. pneumoniue

Antibiotic

MS

120 ~18 214

Staphylococci and B. ca tarrhalis Haemophilus species and L. monocytogenes Enterococci Nonenterococcal streptococci Gram-negative enteric organisms and staphylococci Haemophilus species Staphylococci Pneumococci, penicillin G susceptibleC Staphylococci and B. catarrhalis N. gonorrhoeae and L. monwytogenes Enterococci Nonenterococcal streptococci

Penicillin G

25

breakpoint

Moderately susceptible

(mm) Susceptible

a76 22-2Y 16-21 16-20 15-22 13-15 15-17 Illd 15-19e 14-20 15-22 215 20-27

“Adapted from reference 42. ‘For enterococci. ‘For nonenterococcal streptococci. dFor urinary 1‘so 1ates of P. aeruginosa “For all other organisms.

230 222 221 223 116 218 220” 221 223 228

only.

~28

120 230 2 14 120

228

217 22-29 12-13

215 20-27

519 516 121 511 519

to penicillin, not to oxacillin.

SelectiveReporting of Susceptibility Results The selection of antibiotics for testing and reporting should be done in a manner that encouragesthe use of the most effective and least expensive drug whenever possible. In general, drug selectionis basedon the type of organism being tested, the body site from which the organismwas isolated, the drugs available for use in a particular institution, and recent susceptibility summariesfor the most frequently encountered pathogens(34,70). However, the selectionprocesshasbeencomplicatedby the appearanceof a plethora of “me too” antimicrobial agentswith similar spectra and indications (52). As discussed previously, one method for managing this problem is sequential testing (Table 2). Another approach is to institute a protocol for the selective reporting of susceptibility results; several antibiotics are tested againsteach bacterial isolate, but basedon criteria establishedin cooperation with membersof the medical staff, only results of the drugs most likely to be appropriate for therapy are reported. Somerecommendthe selectivereporting of this type as a way to improve the use of antimicrobial agents and as an attempt to decreasethe costs of medical care (6, 52, 59, 68). These authors arguethat when resultsshowthat an organismis susceptible to relatively safeand lessexpensivedrugs, there is rarely a needfor routinely reporting the results for potentially more toxic or expensive agents. However, if microorganismsresistant to these safer, lessexpensivedrugsare encountered,then susceptibility resultsfor the more potent, expen-

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sive, or toxic backup drugs may be released. Some automated systems for susceptibility testing now have the ability to analyze data obtained from a test battery and selectively report the results for the testing of specific drugs based on the results of other drugs (1). If a selective reporting approach is used, the ability to provide results for the backup drugs on request is imperative. Factors not apparent to a clinical laboratory may make use of the more potent or potentially toxic agents necessary. For example, allergy of the patient to routinely reported drugs, the need of the physician to select antibiotics for the treatment of polymicrobial infections (including organisms other than those isolated and tested for susceptibility), or a variety of pharmacologic considerations may invalidate laboratory decisions about what to report and when. Thus, the clinical staff of a hospital must have full input into the algorithms used for selective reporting (70), and the flow of information between the laboratory and medical staff must be such that, when needed, exceptions to the protocol may be instituted promptly. For medicolegal reasons, the format for selective reporting should indicate the time that results were made available to the clinician, so that it will be clear what information was available to the prescriber at the time that clinical decisions were made. PROVIDING SPECIAL SUSCEPTIBILITY TESTS Certain susceptibility test procedures may involve methods other than testing the direct effect of a drug on an isolate, e.g., the production of certain enzymes such as beta-lactamases. Other procedures measure the susceptibility of an organism to agents or a combination of agents in patient serum before or during therapy. Still other in vitro procedures attempt to determine whether combinations of antimicrobial agents achieve a synergistic effect against microorganisms. In terms of time and materials, each of these tests has different costs to laboratories. Some (serumstatic or serumcidal tests and synergy studies) are especially time intensive to perform. Since laboratories must be efficient in today’s hospitals, the resources needed for each of these tests must be weighed against the potential benefit. Such decisions are difficult for hospital microbiologists to make when there is honest disagreement among authorities about the value of certain tests. Some of these tests are pertinent only to certain limited disease processes (e.g., endocarditis). Each laboratory must make its own judgments as to the validity of offering such testing in light of the freauencv with which

SUSCEPTIBILITY

TESTING

11

pertinent types of infection are being treated in the institution (6). The impact of the procedure on the quality of patient care is a paramountconsideration. However, it is often difficult to measurethe impact of such testing on overall costs of patient management. For example, measuringthe serumstatic or serumcidallevel of an antimicrobial agent in the blood of a patient receiving oral therapy for osteomyelitis has become a popular way to monitor patients who receive an initial courseof parenteral therapy in the hospital, followed by an extended period of therapy on an outpatient basis with the oral antimicrobial agent (7, 72). While coststo microbiology laboratoriesmay be high in terms of performing such time-intensive procedures, the costs for hospitals as a whole may be lessif the testing leadsto early discharge of inpatients (35, 53). A major trend today is for third-party insurers to contract with physician groups to set minimum standardsfor testing in certain types of illness. Two examples of this trend are the development of standards for the care of patients with bacteremiaor endocarditisor both by the Society for Researchand Education in Primary Care Internal Medicine, under commission from Blue Cross and Blue Shield (3), and the recommendationsby a College of American Pathologistsstudy group that “testing for synergistic activity should be discouragedin the absence of a standardized consensusmethodology” (59). Guidelinessuchas these will play an increasingrole in assistinglaboratories in determining a reasonablerole for these specialtests. SPECIAL TEST PROCEDURES Some specialtesting procedureswill be considered at greater length becausethey are frequently employed. Beta-LactamaseTests Beta-lactamasetests are specifically useful for detecting the beta-lactamase-mediatedresistance of Staphylococus aureus, H. influenzae, N. gonorrhoeae, and Branhamella ca tarrhalis to penicillin, ampicillin, and other aminopenicillins (e.g., amoxicillin). Detailed descriptionsof how the most commonmethodsfor determiningbetalactamase production (i.e., acidimetric, iodometric, and chromogeniccephalosporin)should be performed are provided elsewhere(55, 65). Becausethese tests do not require 16- to 24-h incubations, they provide laboratories with a relatively rapid method for detecting penicillin or ampicillin resistance.However, certain limitations of beta-lactamasetests must be realized. While all beta-lactamase-positiveisolates may be considered resistant to both penicillin and ampicillin, resistant strainsof H. influenzae and

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N. gonorrhoeae also exist that are beta-lactamasenegative (9, 33,48). Therefore, beta-lactamase-negativestrains may need to be tested by other methods to confirm their susceptibility. The testing of organismsother than those listed in the previous paragraph for beta-lactamase production may be seriously misleading and is not recommended.Positive or negative test results obtained with isolatesof Enterobacteriaceae, P. aeruginosa, or other gram-negative bacilli are not reliable indicators of susceptibility or resistanceto the various beta-lactam antibiotics used to treat infections caused by these bacteria. Beta-lactamasetesting of anaerobesmay offer useful information pertaining to the susceptibility of theseorganismsto penicillin classdrugs; however, little, if any, information pertaining to cephalosporin susceptibility is gained. Susceptibility TestsInoculated Directly from Patient Specimens Several investigators have studied the usefulnessand accuracy of directly inoculating various susceptibility testing systemswith bacteria present in specimensfrom the patient. By using these methods, which have included the inoculation of agar dilution plates (24), agar disk diffusion plates (11, 15), broth microdilution panels(28), and automatedsystems(14,54), the 18 to 24 h usually neededfor the primary subculture is saved and preliminary results are available that much sooner. Because of the therapeutic urgency associatedwith bacteremic patients, most of these direct-inoculum studies have been conducted with blood cultures. In general, studies have shown that results obtained with the direct-inoculum technique compare favorably with those obtained by susceptibility tests performed in the conventional manner. Iiowever, depending upon the type of susceptibility testing systembeing used, certain drug-organismcombinations may yield inaccurate results-for example, staphylococci versus oxacillin (54). In addition, when a direct-inoculum procedure is being used, the presenceof mixed cultures may not be detected, especiallyif automated systems are used, and could give seriously misleadingresults. Thus, specialcare must be taken to detect polymicrobial infections (e.g., Gram stain and culture evaluation). Becausethe direct-inoculum approachto susceptibility testing has not been standardized, subcultures of clinically important isolates should be tested by a conventional method to obtain final, definitive susceptibility data. This repeat testing will obviously increaselaboratory expenses; communicationswill be needed between clinical microbiologists and the medical staff to decide whether patient benefits gained

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from the rapid availability of direct testing outweigh the increasedlaboratory costs (6). ToleranceDeterminationand MBC Generally stated, tolerance refers to the phenomenon in which bacterial isolates are inhibited but are not killed by concentrations of cell wall-active antibiotics, suchasbeta-lactamsand vancomycin, that are usually bactericidal. Tolerance is determined in vitro by comparing the MIC with the MBC of a particular drug tested against the isolate of interest. The most commonly used definition of tolerance is an MBC/ MIC ratio of ~32 (55, 56). The most prominent clinical situation in which the testing for bacterial tolerancehasbeen advocated is for thoseinfections in which the eradication of microorganismsfrom an infected site strongly dependson the bactericidal activity of the drug (e.g., endocarditis, infections in immunocompromisedpatients, meningitis, and osteomyelitis). In vitro tolerance has been observed with strainsof various bacteria, including enterococci, lactobacilli, Listeria spp., S. pneumoniae, and S. aureus (21,67). Although no clear consensusexists as to the clinical implicationsof tolerance (51), experience with experimental endocarditis modelsand treatment of endocarditis in humanssuggeststhat in vitro tolerance among streptococcihasin vivo significance,whereasthe clinical importanceof S. aureus tolerance is not clear (56, 67). One of the major reasonsfor this lack of clarity has been the numeroustechnical artifacts associatedwith in vitro methodsusedfor determiningtolerance (56). Detailed descriptions of methods for determining the MBC essentialfor defining tolerance are given elsewhere(45, 55). The major factors that may influence MBC test results and substantially affect the interpretation of in vitro tolerance determinationsinclude (i) the growth phaseof the organismbeing tested, (ii) the type of culture medium and the type of material or glasswarein which the test is being performed, (iii) the number of organismsin the inoculum, which must be adequate to yield statistically reliable results (and thus the inoculum, the method of inoculation, and the volume of inoculum are all important), (iv) the premixing of tubes or wells 4 h before subculture, (v) antibiotic carry-over, and (vi) the time that the medium is incubated before the enumeration of survivors (e.g., 24 h, 48 h, or longer) (55, 63). If the adopted MBC procedure is carefully followed and the MBC/MIC ratio obtained is ~32, the isolate may be reported as demonstratingin vitro tolerance. Of importance is that even though this laboratory information can be generated for clinically important isolates of S. aureus, the in vivo consequencesof such find-

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ings are, at best, uncertain and controversial (61). At present the indications for the determination of an MBC are “extremely limited” (70); one paper suggests that “MBC testing is an experimental reference laboratory test that should not be done by clinical microbiology laboratories” (49).

SUSCEPTIBILITY

TESTING

13

antibacterial activity of various antibiotics tested in combination is synergistic (i.e., the effect observed with the combination is greater than the sum of the effects observed with the drugs independently), additive or indifferent (i.e., the combined effect is equal to the effects producedby the drugsindependently or equal to the most active drug alone), or antagonistic(i.e., Serumcidal Testing the effect of the combination is lessthan that of Basically the serumcidal assay is an MBC the most effective drug alone) (55). Currently, procedure for testing the activity of serum ob- the most prominentreasonsfor the in vivo useof tained from a patient receiving antimicrobial antibiotic combinationsare to increasethe antitherapy against the bacterial pathogen thought microbial spectrum, to treat polymicrobial infecto be causing the patient’s infection. One can tions, to provide empiric therapy until definitive find various opinions about the clinical usefulpathogensare identified by the laboratory, to ness of serumcidal determination; one author decreasethe dosageof potentially toxic antibiotics such as aminoglycosides,to decreasethe notes that “the clinical data base for interpretation of serum bactericidal titers in the manage- chancesfor the emergenceof resistantbacteria, ment of infective endocarditis is sparse despite and to obtain increasedkilling of bacteria infectthe widespread use of SBT tests” (50). Such ing seriously ill patients. The primary role that lack of clinical correlation leads to various opin- antibiotic combinationsplay in the management ions about the usefulness of the test (2, 50, 51, of infectious diseasesclearly indicates that in 60,74). It remains to be seen whether the recent vitro methodsfor the evaluation of drug interactions are important. Indeed, extensive informapublication of a proposed standard method for such testing (44) will help resolve the question tion concerning in vitro and in vivo activity of (19, 60, 61). Although the clinical relevance of drug combinations has been generated (31). this test is still controversial, in certain situa- However, a major controversy surrounding antions data generated by the test are used by timicrobial combinationsis whether or not clinsome for the therapeutic management of pa- ical microbiology laboratories should establish tients. Most notable uses of the assay are to and perform in vitro proceduresto generatedata monitor the therapy of infections requiring bac- that might be used to guide a physician’s aptericidal activity (e.g., endocarditis, infections proach to combination therapy (16). of immunocompromised patients, osteomyelitis, Numerous methods for synergy testing have and meningitis), to evaluate the activity of anti- beenoutlined and describedpreviously (31, 55), biotic combinations after they have been admin- but to date no standardized method has been istered, and to monitor the antibacterial activity established.Somerelatively simplemethodsfor of serum after a patient has been changed from evaluating the activity of certain drug combinaparenteral to oral therapy (7, 72). tions are available.’ A good example of these Proper procedures must be carefully followed to methods is the use of combination disks for testing the in vitro activity of trimethoprimensure that results are as accurate as possible. NCCLS recently released proposed guidelines sulfamethoxazole, amoxicillin-clavulanic acid, that discuss and describe clinical and technical ticarcillin-clavulanic acid, and sulbactam-ampiaspects of the test (44). The guidelines present cillin combinations. Screening tests for enterodetailed information concerning such important coccal resistanceto aminoglycoside-beta-lactam procedural aspects as proper timing for specimen synergy alsoare readily performedin many clinicollection, the selection of broth or serum di- cal laboratories.However, laboratory evaluation luents, macro- and microdilution test methods, of many clinically relevant combinationsrequires inoculum preparation, test inoculation, the reading the use of cumbersome,time-consuming,laborintensive, and unstandardizedtechniquessuchas and interpretation of results, and quality control. Laboratories that either are currently performing “checkerboard” testing and time-kill studies(31, serumcidal assays or are planning to do so should 55). These techniqueshave been useful for reconsult these proposed guidelines and comment searchpurposesandhave beenused,alongwith in on them to NCCLS so that a standardized ap- vivo studies,to establishour knowledgeconcemproach to this complex susceptibility testing ing drug combinations.Therefore, many therapeumethod may be established. tic decisionsmay be basedon publishedresearch data rather than on resultsproducedby laboratoTesting of Antibiotic Combinations: ries that must perform these sporadically reSynergy Testing questedand technically complicatedprocedures. Synergy testing refers to any in vitro suscepThe primary reasonfor clinical microbiology tibility procedure designedto ascertain if the laboratoriesto perform drug combinationtests is

14

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ET AL.

to determine if the antibiotics selected are antagonistic, as this situation could result in a patient receiving less than adequate therapy. Therefore, rather than use the checkerboard or time-kill studies to produce data for calculating antagonism, the less cumbersome and recently standardized serumcidal assay (44) could be used to test the antibacterial activity of serum obtained from a patient who has received the initial dose of a drug combination. If the serumcidal activity is found to be adequate (44), then any antagonism between the drugs must be negligible with respect to the adequacy of therapy and the use of the drug combination may be continued. If serumcidal activity is not found to be adequate, changes in therapy must be considered; whether or not the inadequacy of the drug combination was due to antagonism is of secondary importance. Breakpoint Testing On occasion, a compromise is possible between the desires of clinicians to have available certain costly tests and the need for laboratories to operate in a cost-efficient fashion. An example of this is the trend toward breakpoint testing methods. By these methods drugs are tested only at the specific concentrations needed to differentiate between interpretive categories of susceptible, moderately susceptible, and resistant rather than at full-range doubling-dilution concentrations used to determine MICs. Although breakpoint methods may bridge the gap between the potential inaccuracies of the agar disk diffusion procedure and the expense of full-range MIC determinations (G. V. Doer-n, Clin. Microbial. Newsl. 9:81-88, 1987), they provide less than optimal information (lo), and quality control techniques have not been standardized. QUALITY CONTROL OF SUSCEPTIBILITY TESTING The results of antimicrobial susceptibility tests may be markedly influenced by a number of technical variables or errors in test interpretation (47, 65). Major controllable, technical variables of susceptibility tests include inoculum density; the stability of the antimicrobial agents tested; the time, temperature, and atmosphere of incubation; differences in constituents, ionic content, and pH of the test medium; and the use of direct susceptibility tests or mixed cultures. The agar disk diffusion test, in particular, is greatly influenced by the growth characteristics of the organism tested and by the type, depth, and concentration of agar used. In regard to medium composition, attempts to standardize Mueller-Hinton agar are currently being evaluated by NCCLS (40). Therefore, there is now reason to believe that in the near future all lots of

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25

Mueller-Hinton agar from any source will yield highly comparable antimicrobial susceptibility test results, given that all other components of the test remain standard. This effort will result in substantial gains for the standardization of antimicrobial susceptibility testing. Because of the large number of variables affecting susceptibility tests, it is impractical to monitor each one. A more rational approach is a quality control program designed to monitor susceptibility test endpoint accuracy and precision to confirm that test results are within acceptable limits. The term “accuracy” applies to the degree of closeness with which an observed result (e.g., disk diffusion zone diameter or MIC) agrees with the true or accepted value; ‘ ‘precision’ ’ is the degree of agreement among test results which should be identical. At present, a test for precision is available only for agar disk diffusion testing and not for the broth dilution procedures (37, 38, 42; J. M. Miller, B. V. Addison, and S. P. Caudill, Clin. Microbiol. Newsl. 8:74-75, 1986). Test variables can usually be controlled by adhering to established standard NCCLS antimicrobial susceptibility test procedures (37-39, 42). By adhering to standard methods, practical quality control can be easily performed by monitoring with appropriate reference control organisms (Table 7). When technical or interpretive problems are encountered, microbiologists may check test variables individually or in groups, depending on the problem (36-39). This provides a practical approach to troubleshooting most out-of-range test results. When quality control criteria set forth by NCCLS (37-39, 42) are not met, the drugs involved should not be reported until the problem is resolved. The tests should be repeated and an investigation of factors influencing the test should be undertaken to determine the source of error. A careful review of quality control data from a large number of clinical laboratories has led to the conclusion that daily quality control testing may not produce more useful information than less frequent testing (27, 37, 38). NCCLS and the College of American Pathologists have recently made a policy change in acceptance of the cost-effective weekly quality control testing of routine antimicrobial susceptibility tests for laboratories that have documented satisfactory performance with daily quality control testing. Whenever weekly quality control tests yield unacceptable results, daily quality control testing must be performed long enough to define the source of aberrant results and to document the resolution of the problem. An interpretive summary for implementing newer quality control frequency guidelines has recently been reported (Miller et al., Clin. Mi-

25

CUMITECH

SUSCEPTIBILITY TABLE

Control Staphylococcus Escherichia Escherichia Escherichia Pseudomonas Pseudomonas Streptococcus Streptococcus Staphylococcus Bacteroides Bacteroides Clostridium

7. Control strains for NCCLS standard antimicrobial

strain

ATCC no.

25923 25922 25922 35218 27853 27853 29212 29212 29213 25285 29741 3124

aureus coli coli coli aeruginosa aeruginosa faecalis faecalis aureus fragilis thetaiotaomicron perfringens

Test for control

Agar disk Agar disk Dilution Agar disk Agar disk Dilution Agar disk Dilution Dilution Anaerobic Anaerobic Anaerobic

diffusion diffusion diffusion” diffusion diffusion’ dilution dlution dilution

TESTING

15

susceptibility tests NCCLS

standard(s)

M2-A3 (37)/MlOO-S2 M2-A3 (37)/MlOO-S2 M7-A (38)/MlOO-S2 M2-A3 (37)/MlOO-S2 M2-A3 (37)/MlOO-S2 M7-A (38)/M lOO-S2 M2-A3 (37)/MlOO-S2 M7-A (38)/M 1O-S2 M7-A (38)/MlOO-S2 Mll-A (39)/MlOO-S2 Mll-A (39)/MlOO-S2 Mll-A (39)/MlOO-S2

(original/update)

(42) (42) (42) (42) (42) (42) (42) (42) (42) (42), M17-P (41) (42), M17-P (41) (42), M17-P (41)

“Designated for the quality control of disks containing combinations of beta-lactams and beta-lactamase inhibitors (e.g., amoxicillin-clavulanic acid and ticarcillin-clavulanic acid). For quality control performance parameters, see references 36 and Miller et al., Clin. Microbial. Newsl. 8:74-75, 1986 (updated in reference 42). bTo determine whether the Mueller-Hinton agar medium has sufficiently low levels of thymidine and thymine, Streptococcus faecalis ATCC 29212 or 33816 may be tested with trimethoprim-sulfamethoxazole. For quality control performance parameters, see the references given in footnote b.

crobiol. Newsl. 8:74-75, 1986). Appropriate quality control testing must also be performed before use of each new lot (or batch) of media, disks, or antibiotic solutions. Participation in external proficiency surveys conducted by the Collegeof American Pathologists has provided clinical laboratories with a meansfor assessing their capability to accurately perform and interpret antimicrobialsusceptibility testingon clinical isolateswith known susceptibility profiles. In turn, the proficiency survey programof the Collegeof American Pathologistshas had a great impact on NCCLS in regardto stimulating review, study, and, where appropriate, revision of criteria so that they are in accordance with contemporarypractice (22). LITERATURE

CITED

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