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GENERAL GUIDELINES FOR CLINICAL BACTERIOLOGY

General Guidelines for Clinical Bacteriology Lauri D. Thrupp, Roy Cleeland, Ronald N. Jones, William J. Novick, Jr., L. Barth Reller, Clyde L. Thornsberry, and John A. Washington

From the Departments of Medicine and Pathology, University of California at Irvine, Irvine, California; the Department ofMicrobiology, Hoffman LaRoche, Inc.. Nutley, New Jersey; the Department of Pathology, University of Iowa, Iowa City, Iowa; Hoechst Roussel Pharmaceuticals. Inc.. Somerville, New Jersey; the Departments of Pathology and Medicine, Duke University, Durham, North Carolina; the Institute for Microbiology Research. Franklin, Tennessee; and the Department ofMicrobiology. Cleveland Clinic Foundation, Cleveland, Ohio

This guideline summarizes recommendations for (1) developing cogent procedures for diagnosis and antimicrobial susceptibility testing; (2) developing quality-control parameters for the microbiological components of clinical trials; (3) continually updating U.S. Food and Drug Administration (FDA) guidelines; (4) reviewing microbiological recommendations from other groups, such as Microbiology Subcommittees of the National Committee for Clinical Laboratory Standards; and (5) improving the microbiological aspects of FDA package inserts for antimicrobial drugs. Sensitive and specific methods for isolation and identification of pathogens are essential to the proper conduct of clinical trials. Susceptibility tests should be performed in an accurate and reproducible fashion. Verification of results in a reference laboratory is encouraged to monitor quality control.

I. INTRODUCTION II. MICROBIOLOGICAL ASPECTS OF PRECLINICAL STUDIES A. Anti-Infective Activity

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S340 S340 I. Methodology S340 2. Spectrum of Activity S340 3. Mechanism of Action S340 4. Resistance.................................... S340 5. Cross-Resistance S341 6. Bactericidal Activity S341 7. Inoculum Effect S341 B. Quality-Control Standards S341 C. Preclinical Pharmacology and Experimental Infections S341

III. DETERMINATION OF INTERPRETIVE STANDARDS FOR ANTIMICROBIAL SUSCEPTIBILITY TESTING A. Estimates of Breakpoints from Preclinical Studies . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. B. Phase 1 Studies C. Phase 2 and Early Phase 3 Studies. . . . . . . . . . . .. D. Late Phase 3 Trials

IV. CLINICAL MICROBIOLOGY GUIDELINES FOR TREATMENT STUDIES A. Laboratory Expertise B. Standard Guidelines for Diagnosis and Case Definitions

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4. Standards for Bacterial Identification to the Species Level

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Clinical Infectious Diseases 1992;15(Suppl 1):S339-46 © 1992 by The University of Chicago. All rights reserved.

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C. Standardization of Antimicrobial Susceptibility I. Disk Diffusion 2. Dilution Methods 3. Testing of Anaerobes 4. Automated and Semiautomated Rapid Testing Systems . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . ..

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D. Criteria for Microbiological Eradication or Persistence . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. S345 I. Follow-Up Specimens 2. Role of Immunologic Detection Procedures in Follow-Up

Financial support: This work was supported by a contract to the Infectious Diseases Society of America from the U.S. Food and Drug Administration (no. HHS 223-88-130 I). Correspondence: Dr. Lauri D. Thrupp, Infectious Diseases Service, College of Medicine. University of California at Irvine, 10 I City Drive South, Orange, California 92668.

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I. Timing of Specimen Collection S343 2. Techniques for Collection of Microbiological Samples S343 3. Quality of Specimens. . . . . . . . . . . . . . . . . . . . . . . . . .. S343

Testing. .. .. . .. . .. . . .. . . . .. . .. . . . .. . . .. . . .. . S341

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S345 S345 4. Emergence of Resistance. . . . . . . . . . . . . . . . . . . . . . .. S345 3. Superinfection and Reinfection

E. Standards for the Design of Studies Evaluating S345 Efficacy I. Grouping of Pathogenic Species and Special Strain Subsets 2. Clinical Syndromes

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V. RECOMMENDATIONS FOR FDAAPPROVED PACKAGE INSERTS S346 VI. MECHANISM FOR UPDATING OF CLINICAL BACTERIOLOGY GUIDELINES .. S346 A. References and Resources . . . . . . . . . . . . . . . . . . . . .. S346 B. Review and Update Procedures S346 C. New Studies S346

I. INTRODUCTION This is one of a series of guidelines that have been prepared to assist sponsors and investigators in the development, conduct, and analysis of studies of new anti-infective drugs. These guidelines deal with the conduct of phase I through phase 4 clinical trials and are subsets of the General Guidelines for the Clinical Evaluation of Anti-Infective Drug Products, which should be consulted for the prerequisites to the conduct of studies in humans. This document describes microbiological aspects of staged trials in humans, the design of such studies, and methods of presenting and analyzing microbiological data. It summarizes recommendations for the following: ( I ) developing cogent procedures for diagnosis and antimicrobial susceptibility testing (AST); (2) developing quality-control parameters for the microbiological components ofclinical trials; (3) continually updating the guidelines set forth by the U.S. Food and Drug Administration (FDA); (4) reviewing microbiological recommendations from other groups, such as Microbiology Subcommittees of the National Committee for Clinical laboratory Standards (NCClS); and (5) improving the microbiological aspects of FDA package inserts for antimicrobial drugs. The comments on AST cover both aerobic and anaerobic bacteria but not other microbial pathogens (e.g., mycobacteria, fungi, mycoplasmas, chlamydiae, and viruses). The AST procedures used with the latter organisms are reviewed in the guidelines dealing with infections caused by those specific pathogens.

II. MICROBIOLOGICAL ASPECTS OF PRECLINICAL STUDIES See General Guidelines, section II. In vitro studies of susceptibility to anti-infective drugs require standardized bioassay techniques and (when applicable) specific chemical assay procedures. The assay method should take into consideration the effects of diluents, body fluids, and various culture media. Drug interactions that may affect the assay methods must be determined, including the effects ofother antimicrobial drugs or common intravenous supplements (for drugs administered parenterally).

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1. Methodology

A standard data base of representative clinical isolates needs to be developed. Additional strains should be included for comparative studies, as discussed in section II.A.2. MICs should be determined by broth dilution or agar dilution procedures. Standard methodology, media, and culture conditions should be employed. Publications such as NCClS series M7 (e.g., see [I]) delineate standard systems for MICs. Whichever primary method is chosen, a representative sample of clinical test and quality-control strains should be tested in parallel to establish equivalence of methods. This measure will minimize later potential problems with the interpretation of unexpected discrepancies between agar and broth dilution procedures. For example, differences between broth and agar dilution results have been encountered for aminoglycosides, largely because of differences in divalent cation content of media. Disk and drug diffusion characteristics should be defined for the new anti-infective drug. Again, standard methodology, media, and culture conditions, such as those specified in the publications ofNCClS series M2 [2] and M7 [I], should be used. The optimal content of standard disks for susceptibility testing should then be determined by means of regression correlation and population distribution analyses, as outlined in NCClS series M23 [3]. 2. Spectrum of Actiyity

Data bases should be developed to define the general and species-specific spectrum of activity for the new anti-infective drug. The sources and clinical significance of the strains studied should be included. The data bases should comprise cohorts ofstored selected strains representing (a) appropriate numbers of each relevant species, including uncommon but clinically significant species; (b) partially resistant or multiresistant species and strains; and (c) routine clinical isolates. This last category should be the largest (preferably ;?;500 strains) and should reflect the relevant spectrum and prevalence of current clinical isolates. 3. Mechanism of Action

Studies of the mechanism of action ofa new anti-infective drug need not precede early clinical trials if the data from studies of in vitro activity and from experimental pharmacokinetic and toxicologic investigations are sufficient to allow the drug's use in humans. 4. Resistance

A. Anti-Infective Activity See General Guidelines, section II.D. The microbiological issues that should be addressed in the required systematic investigations of anti-infective activity include the following.

The stability of anti-infective activity against representative strains of appropriate species should be assessed by means of cell population analyses. These analyses include serial transfer to media containing increasing levels of drug

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after exposure to subinhibitory concentrations as a screen for the development of resistance. There is no need to identify mechanisms of resistance before beginning clinical trials.

5. Cross-Resistance

Preclinical studies ofantimicrobial activity by dilution and disk diffusion procedures should assess cross-resistance, both among representative drugs of the same family and among other licensed drugs used for similar indications. The samples tested should include selected cohorts of stored strains and representative current clinical isolates.

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tion, two lots ofdisks from different manufacturers should be evaluated to assess consistency between manufacturers. As an aid in monitoring the results of in vitro dilution tests, expected performance ranges should be derived from a study (at five or more laboratories) of MIC distributions for the appropriate quality-control strains from ~20 separate tests per participant. Sequential twofold dilutions must be used to assure "on-scale" MIC end points (see NCClS document M23 [3]). These quality-control standardization studies should be undertaken for rapidly growing aerobic bacterial pathogens and fastidious pathogens and for anaerobes as needed and appropriate for clinical trials.

6. Bactericidal Activity

C. Preclinical Pharmacology and Experimental Infections

The relevance of routine testing of in vitro bactericidal activity as a guide to clinical management is not clear. Such testing need not precede clinical studies of new anti-infective drugs. However, the microbiological data obtained before a drug is approved for early clinical trials should include information on general bactericidal activity against an appropriate sample of stored and current clinical strains tested by a standardized procedure, such as that outlined in NCClS documen t M26 [4). The new drug should be tested in parallel with a representative licensed agent from the same class of drugs. Inoculum size, end-point definition, media, and culture conditions all require careful standardization. Further investigations of the mechanisms of phenomena that affect end points, such as persisters, the paradoxical (HEagle") effect, or tolerance, are encouraged.

Information on a drug's bioavailability, stability, metabolism, protein binding, tissue distribution, excretion, and other pharmacological characteristics is critical to the interpretation of microbiological data. These data are needed for the planning of initial experimental studies of anti-infective activity and pharmacokinetics in animal models and for the planning of clinical trials. Appropriate animal models of infection should be used for tests of selected inocula and drug-dose ranges in studies designed to establish in vivo efficacy. Should preliminary results in experimental infection show significant discrepancies between the efficacy predicted on the basis of in vitro anti-infective activity and that actually seen in animals, the sources of the discrepancies should be sought in more extensive investigations. An adequate method of predicting in vivo efficacy is needed before trials are initiated in humans. Additional studies should also be conducted if efficacy in animal models exceeds that predicted on the basis of in vitro studies.

7. Inoculum Effect

Standard inoculum sizes-e.g., 5 X 105 cfu/rnl. for broth dilution tests or 104 cfu per spot for agar dilution tests-need to be utilized for most studies. However, as a more comprehensive basis for evaluating subsequent studies, preclinical studies should include an assessment of the inoculum effect on MICs for selected bacterial species (e.g., with inocula ranging from IDO-fold less than to IDO-fold greater than the standard). In such studies, the new compound should be tested in parallel with selected licensed drugs of the same class.

B. Quality-Control Standards As outlined in NCClS document M23 [3], systematic definition ofquality-control standards is required for monitoring of the reproducibility and accuracy of in vitro AST during clinical trials. Quality-control standards for disk diffusion tests using the selected disk concentration should be derived from a study including at least five laboratories. At least five lots of medium from two manufacturers as well as a reference lot of Mueller-Hinton agar medium should be used, and at least 20 separate tests should be performed at each laboratory for appropriate ATCC quality-control strains. In addi-

III. DETERMINATION OF INTERPRETIVE STANDARDS FOR ANTIMICROBIAL SUSCEPTIBILITY TESTING Interpretive criteria for AST are critical to the generation of clinically useful information and to the avoidance of arbitrary categorization, prejudicial evaluation, and poor reproducibility of testing in clinical laboratories. Selection of breakpoints will occasionally be straightforward, based on clearly dichotomous or unimodal strain end points (MICs or zone diameters) from the population distribution. However, even in the absence of clear-cut in vitro results, tentative breakpoints that will guide initial clinical trials must be estimated from early evaluations of in vitro susceptibility, pharmacological investigations, and experimental studies in animal models. It is essential for sponsors and the FDA to maintain communication during this process.

A. Estimates of Breakpoints from Preclinical Studies Distributions of end points must be examined as a means of predicting estimated breakpoints for resistance and suscep-

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tibility whenever feasible. Categories ofintermediate or moderate susceptibility may be used if appropriate. NCCLS document M23 [3] explains this process. Unimodal distributions may not be present. Data from initial studies ofdrug pharmacology and experimental infection in animals may also help define breakpoints.

B. Phase 1 Studies See General Guidelines, section lILA. 1. Susceptibility breakpoints should be derived from MIC/zone scattergrams, error-rate bounding analyses, and regression-line assessments. These studies should include (1) population distributions ofend points determined for samples ofclinical isolates representative of community-acquired as well as nosocomial infections and for focused samples of strains (stored or fresh) representing other selected problematical pathogens and (2) pharmacokinetic data from phase I studies in volunteers, including information on bioavailability, metabolism, excretion, and serum and tissue concentrations. C. Phase 2 and Early Phase 3 Studies See General Guidelines, sections IILA.2 and III.A.3. Data from initial studies of the treatment of infected patients should provide the basis for breakpoint evaluation. The relevant data include those on clinical efficacy and bacteriologic eradication. The estimated breakpoint should be reevaluated from analyses of ( I) new data on zone diameter and MIC distribution from in vitro studies of the clinical trial isolates, (2) data on clinical pharmacokinetics from studies of infected patients, and (3) initial data on clinical efficacy from evaluations of both clinical response and microbiological efficacy (eradication of the organism) in treated patients. Studies should include the following: ( I) in vitro population distributions determined with use of an expanded data base including the results of preclinical studies, with both zone diameter and MIC end points identified to facilitate derivation of correlative breakpoints; (2) pharmacokinetic data from phase 1 volunteers plus those from infected patients in phase 2 studies (these data should include ranges of serum levels and [if available] tissue levels as well as information on the kinetics of different dosages); and (3) evaluation ofclinical response and bacteriologic eradication in specific patients infected with strains whose susceptibility end points have been measured. Data for patients with septicemia, for those with suboptimal results, or for those in whom clinical treatment fails are most useful for this evaluation.

D. Late Phase 3 Trials See General Guidelines, section III.A.3. Expanded data should permit refinement of earlier standards by means

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of similar but broader-based analyses that include the following. (I) Expanded in vitro results will be available, e.g., population distributions of strain end points (with data from a larger number ofinvestigators) and comparative in vitro endpoint distribution data (from parallel tests on the same pathogens with related licensed drugs). (2) Supplemental pharmacological data will be available from expanded phase 2 and phase 3 studies in various infections and special groups of patients (e.g., those with renal failure, those who are elderly). (3) Data on clinical response and bacteriologic eradication will be generated by the expanded clinical studies (including comparative and blinded trials), with assessment correlated with in vitro end points for specific pathogens. (4) Although not usually required, additional laboratory studies may be warranted for selected problematical infections, for the assessment of changes in dosage, or for the resolution of any aspects of the developing data bases that appear discrepant. These tests may include (a) determination of bactericidal end points by standard methods, such as those described in NCCLS document M26 [4]; (b) determination of serum inhibitory and bactericidal titers by standard methods, such as those described in NCCLS document M21 [5]; and (c) assessment of the postantibiotic effect. (While not established as a standard upon which breakpoints and dosage recommendations should be derived, assays of new drugs for a postantibiotic effect may be worthwhile in some settings, e.g., if in vitro and in vivo results are discrepant or if wide dosage ranges or extended dosing intervals are contemplated.) It is strongly recommended that a joint conference be held at this stage between FDA monitors and the pharmaceutical sponsor to review the data and to develop early consensus guidelines for subsequent clinical trials.

IV. CLINICAL MICROBIOLOGY GUIDELINES FOR TREATMENT STUDIES

A. Laboratory Expertise To qualify for participation in clinical trials, the clinical microbiology laboratory at a participating center should be certified by the College of American Pathologists or a similar organization and should be licensed by the Health Care Financing Administration as a high-complexity facility. Such laboratories must participate in a recognized inspection and quality-control or proficiency program. Alternatively, the laboratory may be recognized as having demonstrated expertise in the field under study (e.g., the clinical research laboratory of a principal investigator). The qualifications of the laboratory must be reviewed with and approved by the FDA before clinical trials are initiated.

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B. Standard Guidelines for Diagnosis and Case Definitions Each study protocol should outline specific clinical and microbiological procedures for diagnosis and follow-up as well as criteria for the specific infection(s) under study. Examples of criteria to be considered for optimal diagnosis and case definition include the following. 1. Timing of Specimen Collection

Protocols should designate how long before administration of the study drug a specimen should be collected. Prior therapy needs to be noted since it may distort the evaluation of clinical efficacy and obscure the detection of valid pathogens. Defining an acceptable interval for transport is essential if fastidious pathogens are to be recovered. 2. Techniques for Collection of Microbiological Samples

The techniques used for the collection of samples must be defined for each type of infection studied. It is especially important that specific, uniform criteria be established for sites of infection that are not readily accessible or for circumstances in which the specimen is expected to contain normal commensal flora. Examples of problematical infections include osteomyelitis, in which drainage, aspirate, and/or surgical specimens may be collected; endometritis, in which specimens may be collected (with special double lumen-protected tube swabs or brushes or by aspiration to minimize cervical flora contamination) or endometrial biopsy samples may be assessed; and urinary tract infection, in which defined clean-voided procedures and culture methods (including semiquantitation) and uniform interpretive guides differentiating infection from colonization or contamination must be used. Methods for collection and transport of specimens should be defined. For certain specimens or certain target organisms, the maximal interval allowable from collection to processing in the laboratory should be specified. 3. Quality of Specimens

Methods for collection of specimens should be uniform and based on the patient's age. As was just stated, the methodology used is especially important for sites with a normal colonizing flora that may include potential pathogens. Direct smear and gram staining (as well as other types of staining, ifrelevant) should routinely be used as an aid in evaluating the specimen's quality and the relevance of subsequent growth. Assessment of respiratory tract specimens (especially expectorated sputum) requires careful adherence to strict clinical criteria and uniform clinical microbiology laboratory procedures. Whenever possible, an accepted qualityscoring procedure based on assessment of a direct gram stain should be employed. Consensus is lacking about the interpretation and relevance of different methods for scoring sputum samples. For pediatric patients, criteria for such a scor-

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ing system have not been established. Nevertheless, protocols should take into account semiquantitative estimates of numbers of white blood cells (WBCs) and epithelial cells. Evaluation of sputum should also include the application of defined semiquantitative criteria for identifying specific bacterial morphotypes in smears, for assessing the amount of growth of potential pathogens, and for deciding which isolates warrant full identification to the species level and/or AST. If activity against Mycoplasma. Legionella, or Chlamydia is claimed for the drug being studied, then criteria for consistent diagnosis of infections with these organisms must be established. 4. Standards for Bacterial Identification to the Species Level

In general, identification to the species level should be standard. Procedures such as those outlined in the Manual of Clinical Microbiology [6] or those required for the use of licensed commercial kits would be appropriate for the identification of isolates that are likely to be the cause of infection. Limited or general reports of results (e.g., "staphylococci" or "enteric gram-negative bacilli") are unacceptable. 5. Clinical Relevance of Isolates

Repeated recovery of a potentially pathogenic species from a given body site can be helpful in establishing the pathogenicity of that organism in problematical situations. For example, if recovered repeatedly, coagulase-negative staphylococci might be assessed as a valid cause of septicemia rather than as mere colonizers of the skin or of an access line. Specimens from wounds require systematic assessment (as suggested for sputum in section IV.B.3) to confirm the presence of WBCs on direct smear and the presence in the smear of the bacterial morphotypes subsequently recovered from cultures. Assessment of growth from cultures of these problematical sites should include standardized semiquantitation-e.g., a score of 4+ for a predominant growth of Staphylococcus aureus as opposed to a score of I + or 2+ growth when its presence is part of the normal colonizing flora-to help assess the likelihood of the isolate's pathogenicity. 6. Infections with Mixed Flora

In specific infections caused by mixtures of species, standardized criteria for microbiological evaluation should be established prospectively. For example, in posttraumatic or postsurgical abdominal infections with mixed fecal flora, the relevance, difficulty, and excessive cost ofa complete evaluation of all bacterial strains must be addressed, and reasonable, clinically relevant procedures must be established. However, for late-postoperative, partially treated abdominal sepsis or abscess, definitive speciation of all persisting strains would be warranted.

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7. Serological Diagnosis and Direct Immunologic or DNA-Probe Detection Procedures

A variety of procedures for antibody or antigen detection have been developed, but each procedure may have limitations in sensitivity and specificity as well as in practicality of use. For example, for detection of Mycoplasma pneumoniae in respiratory specimens, culture procedures may not be as sensitive as direct fluorescent antibody or direct geneticprobe procedures. Likewise, the evaluation of nasopharyngeal specimens may be superior to that of sputum samples, depending on the clinical situation and the test used. Each protocol should define the diagnostic criteria and acceptable methodologies to be used when serological or genetic-probe procedures are recommended or required and should indicate when appropriate serological or genetic-probe data will be acceptable as diagnostic alternatives if the agent (e.g., Legionella or Mycoplasma) is not isolated from primary specimens. C. Standardization of Antimicrobial Susceptibility Testing AST procedures should be standardized and should include routine testing of appropriate quality-control strains. When practical, strains considered pathogenic should be saved by the Clinical Investigator. In selected circumstances (e.g., when the patient fails to respond clinically to treatment and/or the presumed pathogen is not eradicated), these microorganisms may be forwarded to the sponsor or to a reference facility for confirmatory speciation and repeat AST. It may be appropriate for a systematic prospective sample of all strains to be retested by the sponsor or by a reference laboratory. Primary AST procedures in the clinical centers may include both dilution and disk diffusion tests, although either procedure alone-properly standardized-should be satisfactory for organisms that grow aerobically. Dilution tests are recommended for anaerobic organisms. 1. Disk Diffusion

Procedures should follow standardized guidelines, such as those in NCClS document M2 [2]. For clinical trials the zone diameters must be recorded. 2. Dilution Methods

Procedures should follow standardized guidelines, such as those in NCClS document M7 [I]. The range of dilutions tested should yield "on-scale" (rather than "off-scale") end points for as many strains as feasible to define the MIC so or MIC 90 for isolates from clinical trials. Commercial systems using limited screening dilutions or breakpoint concentrations rather than full twofold dilution series are not acceptable. 3. Testing of Anaerobes

Standard dilution procedures, such as those outlined in NCClS document Mil [7], should be followed. For clinical

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trials broth-disk elution screening tests are not acceptable. Establishing the clinical significance of each individual anaerobic bacterial isolate in polymicrobial anaerobic infections may be problematical. In mixed infections routine AST is clinically warranted only for occasional selected anaerobic isolates to guide clinical management. In clinical trials more than one anaerobic species from polymicrobial infections should be tested only when the clinical investigator determines that the research value substantiates the need for the individual patient. However, anaerobes should be considered likely pathogens and worthy of AST when they are recovered (usually in pure culture) from specimens such as blood, pleural fluid, or CSF or when they are present as pure or as predominant isolates from tissue or deep-abscess specimens. 4. Automated and Semiautomated Rapid Testing Systems

AST for clinical trials requiring precise estimates of MIC should not routinely be performed by indirect, extrapolated growth curve or automated procedures unless the system has been thoroughly evaluated and found to give MICs comparable to those obtained with standardized agar or broth microdilution tests using a full dilution series. Automated rapidgrowth systems using a limited number of concentrations are not acceptable when precise MIC estimates are needed, since many MICs will be off-scale or fall within a four- or eightfold log2 interval between the test concentrations provided. D. Criteria for Microbiological Eradication or Persistence See General Guidelines, section XIII.C. If more than one organism is considered pathogenic, the investigator should assess the microbiological response of each. Aspects to be considered include the following. 1. Follow-Up Specimens

Each protocol should define the recommended timing for the collection of follow-up specimens. For example, obtaining urine specimens at defined intervals during and after treatment of urinary tract infection is reasonable and straightforward. However, consideration of a special culture procedure (instead of the typical bacteriuria screening tests) may be warranted if the goal is to isolate organisms in low numbers (below the sensitivity of usual tests) and thus to detect suppression of a strain previously present in higher numbers. 2. Role of Immunologic Detection Procedures in Follow-Up

While direct immunologic and genetic-probe detection methods are gaining acceptance as diagnostic procedures in the initial workup for a number of pathogens (e.g., group A Streptococcus, Mycoplasma, and Chlamydia), uniform guidelines for the interpretation of the results of these tests at follow-up evaluation have not yet been established. The persistence of antigen from nonviable organisms and the

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suppression (but not elimination) of viable pathogens to a level below that detectable by a probe procedure are examples of potential false-positive and false-negative results, respectively. Pharyngitis due to group A Streptococcus is a disease in which positive results in direct antigen-detection tests may be an acceptable basis for a patient's enrollment in a trial, but the evaluation of these tests is not yet adequate to permit their use alone for initial diagnosis or for follow-up studies in clinical trials. 3. Superinfection and Reinfection

Documentation of superinfection or reinfection is vital to the overall evaluation of the efficacy of a new agent. These outcomes must be carefully defined. Clinical signs of true superinfection should be distinguished from microbiological colonization without clinical signs ofa new infection or clinical exacerbation of the old infection. See General Guidelines, section XIII.C. 4. Emergence of Resistance

The emergence of resistance should be monitored by means of full species identification and AST of any isolate that appears to represent the same species as the original pathogen. Any apparent change in zone diameter or MIC should be confirmed by retesting ofthe second strain in parallel with the original isolate. The appearance of a new potential pathogen (with clinical signs of new infection) should also prompt full species identification and AST. In general, a fourfold or greater increase in MIC or an equivalent decrease in zone diameter (e.g., more than -., 3-6 mm) suggests a significant change in antimicrobial susceptibility. Such changes should be recorded even if the shift in end point does not represent a change in the proposed interpretive category. Changes in zone diameter or MIC in tests with other drugs should also be recorded in such instances, especially for representative drugs in the same class. The biochemical profile of original and follow-up strains should be recorded when resistant variants or new pathogens emerge; the use of this procedure may aid in distinguishing a resistant variant of the original isolate from a new superinfecting strain. Additional typing techniques, such as plasmid analysis, may be necessary to differentiate such strains definitively. Routine use of these methods should not be required in clinical trials. However, such typing procedures should be available for application to selected pertinent isolates such as via reference laboratories. E. Standards for the Design of Studies Evaluating Efficacy In an evaluation of microbiological results and clinical efficacy, the assessment of clinical relevance can be enhanced by detailed guidelines such as the two that follow. 1. Grouping of Pathogenic Species and Special Strain Subsets

It has been standard practice to collate infections by broad groups of pathogens (e.g., gram-positive and gram-negative)

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and to analyze by species. However, certain microorganisms must be subdivided for relevant assessment of clinical and microbiological response. Examples include the following. (a) Methicillin-resistant Staphylococcus aureus (MRSA) should be analyzed as a subset distinct from methicillin-susceptible S. aureus. (b) Gram-negative species resistant to a basic class of drug (such as ampicillin, mezlocillin, ticarcillin, or piperacillin) should be compared to fully susceptible strains. (c) For Haemophilus infiuenzae, S. aureus, Neisseria gonorrhoeae, and Moraxella catarrhalis, 11-lactamase-positive and 11-lactamase-negative strains warrant separate comparative analysis, especially if the regimen under study is a 11-lactam antimicrobial drug with or without a 11-lactamase inhibitor. (d) New resistant strains or apparent variants of an original pathogen with altered susceptibility patterns should be evaluated for the mechanism of resistance. Such information is more meaningful if microorganisms not fully susceptible to the parent drug are included. For example, in trials of newer cephalosporins for the treatment of infection due to gramnegative bacilli, pathogens that were fully susceptible to the older cephalosporins and those that were not should be compared in terms of rates of clinical response, bacteriologic clearance, and emergence of resistance or superinfection. 2. Clinical Syndromes

Historically, there has been a tendency in clinical trials to require designation of the "pathogen" or "probable pathogen" for all infections studied. However, it is sometimes difficult to determine the etiologic agent. For instance, some clinical infections are at sites not accessible to routine, direct microbiological sampling. Examples include otitis media, sinusitis, and pelvic inflammatory disease. Protocols should define whether diagnostic studies must include cultures for aerobic and anaerobic bacteria or detection procedures for viruses or mycoplasmas from indirect sites such as nasal or pharyngeal swabs (in cases of sinusitis or otitis media) or cervical specimens (in patients with pelvic inflammatory disease). It should also be spelled out whether invasive procedures, such as tympanocentesis, sinus aspiration, or endometrial aspiration or biopsy, are required. In addition to infections at inaccessible sites, those at sites contaminated with mixed flora pose difficulties in terms of etiologic assessment. Bronchitis and aspiration pneumonia are examples. Such infections may be caused by true primary pathogens (aerobic bacteria) that are obscured by commensal flora. Some pathogens (e.g., anaerobes in aspiration pneumonia) may not be recoverable because of contamination with commensal aerobic and anaerobic flora or may not be recognized in the absence of special diagnostic procedures (e.g., those for viral, mycoplasmal, or chlamydial infections). Mixed anaerobic and aerobic enteric flora in primary specimens from abdominal infections following trauma, appendicitis, or diverticulitis are additional examples of polymicro-

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bial populations in which it may be difficult or impossible to define all ofthe primary pathogens and for which a full microbiologic workup ofall organisms may not be warranted. Any prior (even brief) course of anti-infective drug therapy must be recorded, since it may alter the microbial flora recovered in primary specimens. All anaerobic and aerobic organisms recovered from follow-up specimens warrant speciation and AST, especially if the clinical response is poor.

V. RECOMMENDATIONS FOR FDA-APPROVED PACKAGE INSERTS The following limited modifications in FDA package inserts are proposed to enhance clinical relevance and thereby improve patient care. (A) AST procedures included in newly approved package inserts should be updated regularly. (B) It is strongly recommended that mechanisms be developed for early coordination among the FDA, the sponsors, and the investigators to resolve differences in interpretive breakpoints. (C) Package inserts should clearly distinguish in vitro activity against organisms causing infections that are not among the approved clinical indications (i.e., infections for which clinical efficacy data are inadequate for approval) from in vitro activity against organisms causing infections that are among the approved indications. Furthermore, microbiologically relevant restrictions on the reporting of results should be applied; e.g., AST results for cephalosporins against MRSA should not be reported, since the clinical efficacy of these drugs in MRSA infection has not been proven. VI. MECHANISM FOR UPDATING OF CLINICAL BACTERIOLOGY GUIDELINES New and improved methodologies are continuously being developed for diagnostic clinical microbiology, immunoserology, and AST. Therefore, mechanisms are needed for regular review and updating of these guidelines. Procedures for ensuring the continued clinical relevance of microbiological methods should include the following. A. References and Resources Standard multidisciplinary committee reports, professional association position papers, and pertinent articles in medical scientific journals should be reviewed at regular intervals. Included in these references and resources are (1) original papers containing information on significant new methods (including comparisons with older methods) that appear in peer-reviewed journals such as the Journal ofClinical Microbiology. Antimicrobial Agents and Chemotherapy, Clinical Infectious Diseases, Diagnostic Microbiology and Infectious Diseases. and the European Journal ofClinical Microbiology; (2) comprehensive review papers appearing in such journals; (3) position papers such as those appearing in the Cumitech series [8], the Clinical Microbiology Newsletter [9],

and the American Academy of Pediatrics "Red Book" report of the Committee on Infectious Diseases [10]; and (4) consensus guidelines or standards for microbiology laboratory practice developed by multidisciplinary groups, such as the NCeLS [11]. B. Review and Update Procedures Responsibility for the review of references and resources and for the consequent updating of guidelines resides with FDA professionals. Advice and/or consultation can be obtained as needed from multidisciplinary groups through advisory committees. C. New Studies Selected aspects of clinical microbiology procedures for diagnosis or AST and of clinical trials of the efficacy of specific drugs may involve issues not yet resolved in the medical literature. Thus current and planned clinical trials may require studies in these areas to provide reasonable assurance that the methods being used are both accurate and reproducible. References 1. Methods for dilution antimicrobial susceptibility tests for bacteria that grow aerobically. NCCLS document M7-A2. 2nd ed. Tentative standard. Villanova. Pennsylvania: National Committee for Clinical and Laboratory Standards. 1989. 2. Performance standards for antimicrobial disk susceptibility tests. NCCLS document M2-A4. 4th ed. Approved standard. Villanova, Pennsylvania: National Committee for Clinical and Laboratory Standards. 1990. 3. Development of in Vitro susceptibility testing criteria and quality control parameters. NCCLS document M23-T. 2nd ed. Tentative standard. Villanova. Pennsylvania: National Committee for Clinical and Laboratory Standards, 1989. 4. Methods for determining bactericidal activity of antimicrobial agents. NCCLS document M26. Approved standard. Villanova. Pennsylvania: National Committee for Clinical and Laboratory Standards,

1989. 5. Methodology for the serum bactericidal test. NCCLS document M21. Approved standard. Villanova. Pennsylvania: National Committee for Clinical and Laboratory Standards. 1989. 6. Lennette EH. Balows A, Hausler WJ. Shadomy HJ, eds. Manual of Clinical Microbiology. 5th ed. Washington, DC: American Society for Microbiology. 1991. 7. Methods for antimicrobial susceptibility testing of anaerobic bacteria. NCCLS document M 11-T2. 2nd ed. Tentative standard. Villanova, Pennsylvania: National Committee for Clinical and Laboratory Standards, 1989. 8. Spector S, ed. Cumitech. Washington, DC: American Society for Microbiology. 9. Ferraro MJ, Granate PA, Morello JA. Zabrowsky RJ. eds. Clinical microbiology newsletter. New York: Elsevier. 10. Peter G, Giebink GS, Hall CB, Plotkin SA, eds. Reports of the Committee on Infectious Diseases. Elk Grove Village, Illinois: American Academy of Pediatrics. II. Third Informational Supplement. NCCLS document M 100-S3. Villanova, Pennsylvania: National Committee for Clinical and Laboratory Standards, 1991.