- Email: [email protected]
The clinical microbiology laboratory
The Clinical Microbiology Laboratory Utilizationand Cost-Effectiveness
JOHN A. WASHINGTON II, M.D.
Utilization of the microbiology laboratory is subject to limitations posed by the diversity of microbial agents causing infection, multiple and often poorly accessible sites of infection, contamination of specimens with indlgenous flora, and failure of communication between clinician and microbiologist. Cost constraints increasingly llmlt the extent of mlcrobiologic services available on-site and lead to decentralization of laboratory senrices and possible loss of quaiity control. Increased attention Is being paid to process control of cultures, rapid screening and diagnostic tests as culture substltutes, and limitations on antibiotic susceptiblllty testing.
Rochester, Minnesota
The question of what services the microbiology laboratory should provide has preoccupied clinicians and microbiologists alike for many years and has been accentuated by recent legislation aimed at cost-containment in medical care. In a symposium sponsored by the National Institute of Allergy and Infectious Diseases in 1978 on the impact of infections on medical care in the United States, Neu [l] discussed what the clinician should expect from the microbiology laboratory (Table I). Although these expectations remain valid today, they have been but partially fulfilled. Afthough it should not be difficult for a group of experts to decide what infonation is required for a clinical decision, and although current kits and devices provide the microbiology laboratory with the capability of identifying and susceptibility testing bacteria with a degree of accuracy unavailable a decade ago, there remain many unresolved problems in the entire process, extending from specimen collection to interpretation of the results reported by the laboratory. SPECIMEN COLLECTION
From the Mayo Clinic and Mayo Medical School, Rochester, Minnesota. Requests for reprints should bs addrassad to Dr. John A. Washington II, Sactbn of Clinical Microbiology, Mayo Clinic, 200 SW Firal Street, Rochaatar, Minnesota 5!5905.
8
June
28,1965
The Amdcan
Journal
The laboratory diagnosis of any infectious disease depends on the establishment of a differential diagnosis based on the history and physical examination, a consideration of the most probable cause of the disease, and the selection of those tests most likely to detect the most probable cause of the disease. Complicating this decision process is the fact that infectious diseases may involve virtually any system or organ in the body and may be due to a wide variety of microorganisms, including bacteria, mycobacteria, fungi, chlamydiae, mycoplasmas, viruses, and parasites. For these reasons, specimen collection represents the most important part of the process leading to information required for a clinical decision. Guidelines for specimen collection must be designed to provide the laboratory with a sufficient amount and number of specimens for complete examination. All too often a small or even invisible amount of specimen on a swab or a small sample of tissue or fluid is submitted to the
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MANAGEMENT
laboratory with a request for multiple examinations and cultures. In such instances, the likelihood of negative microscopic examinations and cultures is great, and the credibility of negative results is low. Sampling difficulties often complicate the specimen collection process in that some specimens are simply inaccessible except by invasive techniques or are associated with a high likelihood of becoming contaminated with indigenous flora during the collection process. An added problem is the partial replacement of indigenous flora of the skin and mucous membranes by gram-negative bacilli in immunocompromised and other seriously ill, debilitated patients. Prior administration of antimicrobial agents may inhibit recovery of a causative agent from cultures. Finally, the causative agent suspected may not be detected because of lack of communication or dialogue between the clinician and the microbiologist or because, as the result of cost-containment, the procedures necessary for its detection are not available in the laboratory.
AND PREVEEiTlON
TABLE I
June 28,198!5
DISEASES-WASHINGTON
What Should the Clinician Expect from the Laboratory? information required for clinical decision Guidelines for specimen collection Rapid specimen transport Identification of microorganisms Antimicrobial susceptibilities Rapid transmission of results Dialogue
TABLE II
College of American Pathologists: Extents of Services Provided
Exlanl Definitive identification to extent required for diagnosis and assistance in selection of therapy Limited identification with definitive identification by reference laboratory Direct examination (arid inoculation) with identification by reference laboratory All specimens referred to another laboratory
EXTENT OF SERVICES Today, more than ever, it is unreasonable to expect equal capabilities and availabilities of microbiologic services of all laboratories. Indeed, varying extents of services have been recognized by laboratory accreditation agencies for many years. A laboratory that, for example, offers Extent 4 service in mycobacteriology provides definitive identification and antimicrobial susceptibility testing of mycobacteria, whereas a laboratory that offers Extent 1 service simply collects and refers specimens to a more specialized laboratory for isolation, identification, and susceptibility testing of mycobacteria. Although the specific requirements by extent of service vary somewhat for bacteriology, mycobacteriology, mycology, parasitology, and virology, their general definitions are listed in Table II. It should, therefore, be possible to devise a hypothetical scheme of extents of microbiology services by type of hospital (Tdble Ill). The logic of such a scheme has, however, been profoundly altered by the advent of prospective payment based on diagnosis-related groups. The impact of this legislation has been to change the hospital-based laboratory from a profit center to a cost center, which, in turn, is leading in all but Federal hospitals to curtailment in laboratory services. Hospital-based laboratories are reacting to these changes in different ways. Some have contracted with local, regional, or national commercial vendors for laboratory management. Others have decided to utilize their assets and compete with the commercial vendors on a local or regional basis through joint ventures with other hospitals or by acquisitions or affiliations with other hospital, local, or regional laboratories. The ultimate consequences of all of this activity remain uncertain. Extent of service on-site may be reduced, and referral of specimens and tests to other laboratories increased. Low cost and rapid turnaround time have already assumed utmost importance. Screening tests to obviate or replace
OF INFECTIOUS
TABLE Ill
Hypothetlcal Distribution of Extents of Service In Hospitals
SonrIce Bacterblogy Mycobactertolcgy MY~lwY Parasitology Virology
CommunftyCommunity Teachlng Unlvsnity Refsrrmce 3 1 2 2 1
4 2 3 3 2
4 3 4 3 3
4 4 4 4 4
cultures are being implemented. Perhaps of greatest concern is the decentralization of laboratory services and the potential loss of quality control. One pertinent question to ask under current circumstances is what the costs of microbiologic services actually are. In a study at the Massachusetts General Hospital, Ferraro [2] determined that the average cost per case was less than 1 percent and that the average cost per case in the most frequent diagnosis-related groups was also less than 1 percent. Thus, curtailment of microbiology services cannot be expected to have a major impact on reducing costs of medical care. TRENDS IN CUNICAL MICROBIOLOGY The major trends in clinical microbiology laboratory practice today are summarized in Table IV and are designed generally to limit processing of inappropriate specimens and to provide rapid detection of pathogenic microorganisms. By rejecting specimens that were improperly identiThe American Journal of Medlclne
Volume 78 (suppl8B)
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TABLE
IV
AND PREVENTION
Clinical
OF INFECTIOUS
Mlcrobiology
DISEASES-WASHINGTON
Laboratory
Trends
commercial availability of a variety of screening tests for bacteriuria and to immunologic tests or immunoassays for microbial detection (such as group A Streptococcus, Neisseria gonorrhoeae, Chlamydia trachomatis). Work is also progressing with genetic probes based on DNA hybrfdization techniques for rapid detection of a variety of microorganisms.
Screening tests to Determine specimen acceptability Obviate culture Rapid diagnostic methods by Immunologic techniques DNA hybridization Process control limiting Number of isolates undergoing identffiiation and susceptibility tests Number of antimicrobial agents tested
SCREENING
fied or in faulty containers, were delayed in transit, dupiicated another received on the same day from the same site (except blood, spinal fluid, feces), were from sites or sources known to provide little useful information (such as vomitus, loch& Foley catheter tips), or were microscopically unsatisfactory and by limiting identification and susceptibility testing of multiple organisms in mixed cultures of lower respiratory secretions, wound exudates, cervicovaginal secretions, and urine, Bartlett and Rutz [3] minimized reporting of potentially misleading information and reduced the costs of processing these categories of specimens by approximately $42,000 a year. Screening tests to determine specimen acceptability are widely used for sputum [4] and can influence the quality of specimen collected when implemented with the support of the medical specialty groups involved and with the cooperation of nursing services. in certain instances, to be discussed later, the screening test assumes diagnostic importance as well. The management of infectious diseases has often been hampered by the frequently retrospective nature of microbiologic diagnoses imposed by traditional culture methods. Thus, the results of culture have often been largely confirmatory in nature and have, therefore, had relatively little effect on empiric therapy. In a study of ordering patterns and utilization of bacteriologic culture reports, Edwards et al [5] found that therapy was changed with 20.9 percent of positive culture reports, 1.4 percent of negative culture reports, and only 7 percent of ail culture reports. This problem has provided considerable impetus to the deveioprhent of rapid diagnostic tests and has led to the
TABLE
V
Urine Screening
Methods
Tssl
Time
Gram-stained smear Leukocyte esterase/nitrite (LN) strip Fiftrationcolorfmetry (Bat-T-Screen) Bioluminescence (Monolight, Lumac) AlJtobac MS2 AMS
10
Juno
28,1885
The American
15-35 l-6 0.54 4-13
Journal
4 minutes 2 minutes 2 minutes minutes hours hours hours
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TESTS
A variety of rapid screening tests to rule out the presence of lo5 or more colony-forming units/ml of urine have become commercially available (Table V) and are being used in some hospitals to obviate the need for culture or as culture substitutes. Bartlett [6] has estimated a cost saving of about $30,000 a year if a chemical screening test for bacteriuria (Chemstrip LN, Boehringer Mannheim, Indianapolis, Indiana) were used by nursing personnel in clinics and inpatient care units immediately after specimen collection. Although the predictive negative value of this test is approximately 98 percent, its false-negative rate of 17 percent for specimens in our laboratory yielding lo5 colony-forming units/ml or greater is reason for concern [7l and raises doubts about the suitability of the test as a culture substitute unless additional criteria, such as the appearance of the urine and the presence of blood or protein, are used [8]. IMMUNOASSAYS
lmmunoassays have been available for several years for detecting Hemophiius infiuenzae, Neisseria meningitidis, Streptococcus agalactiae (group B), Streptococcus pneumoniae, and Cryptococcus neoformans antigens in cerebrospinal fluid and herpes simplex virus in brain tissue. Because of their technical complexity, counterimmunoelectrophoretic and fluorescent antibody procedures for the common bacterial pathogens were largely restricted in their use to reference laboratories staffed by skilled personnel. Thus, the advent of coaggiutination and latex agglutination tests has offered antigen testing of spinal fluid from children with suspected meningitis to iaboratories with lesser skilled personnel. Use of antigen detection tests might also help to resolve problems with correct interpretation of gram-stained smears of spinal fluid. A latex agglutination test for cryptococcal antigen has been available for several years and provides greater sensitivity and specificity than that of the traditional India ink wet preparation. In our experience, microscopic examination and latex agglutination have detected 42 and 89 percent, respectively, of cryptococci in culturally positive spinal fluid samples, raising the question as to whether the India ink or nigrosin wet preparation tests should be performed at all. A variety of rapid immunodiagnostic methods have become available for detection of group A streptococci in throat swabs. in general, these tests have proved to be specific; however, their sensitivity is directly correlated 78 (auppl85)
MANAGEMENT
TABLE
with the number of group A streptococcal colonies isolated in culture. Although some investigators have considered small numbers of colonies of group A streptococci as being clinically insignificant [9], others have found that although patients with true streptococcal infection are likely to have strongly positive culture results from the upper respiratory tract, there is sufficient overlap that does not allow clinical differentiation of the carrier from the truly infected patient by degree of positivity alone [lo]. A related problem is that the number of colonies isolated is directly related to the vigor with which the throat is swabbed. Although this last variable may be reasonably controlled in any single physician’s practice, the usual clinical laboratory receives specimens obtained by many different physicians whose vigor in swabbing the throat varies considerably. Hence, whatever relationship may exist between disease and degree of positivity of the culture result becomes even more tenuous and compromises, to some extent, the value of immunodiagnostic tests as a culture substitute. In our experience with one commercially available latex agglutination test for detection of group A streptococci, the sensitivity varied from 83 to 87 percent, depending on whether the swab was tested within two hours or after storage overnight, and the false-negative rate was 43 percent when fewer than 50 colonies were isolated by culture [ll]. Coagglutination and counterimmunoelectrophoresis have also been used to test sputum for pneumococcal antigen in patients with pneumonia. As may be seen in Table VI, Edwards and Coonrod [12] found coagglutination to be sensitive; however, specificity was compromised by the high rate of positivity of sputum from patients with bronchitis. Coagglutination was more sensitive than counterimmunoelectrophoresis in detecting pneumococcal antigen in the sputum during therapy of patients with proved or probable pneumococcal pneumonia [12]. The level of sensitivity provided by the coagglutination test of sputum in patients with proved or probable pneumococcal pneumonia substantially exceeds that (48 percent) of microscopic examination of gram-stained smears of sputum for the preponderance of gram-positive lancet-shaped diplococci [13]. Methods have been developed for the diagnosis of C. trachomatis urethritis and cervicitis by immunofluorescent antibody staining of secretions and of gonorrhea by enzyme immunoassay. Tam et al [14] demonstrated 93 percent sensitivity and 98 percent specificity of the fluorescent antibody test for C. trachomatis using monoclonal antibodies on smears from urethral and cervical secretions. This test is now commercially available (Direct Specimen Test, MicroTrak, Syva Company, Palo Alto, California). An enzyme immunoassay method for C. trachomatis (Chlamydiazyme, Abbott Laboratories, North Chicago, Illinois) is under development and investigation. An enzyme immunoassay method for detecting N. gonorrhoeae (Gonozyme, Abbott Laboratories, North Chicago, June z&1985
AND PREVENTION
VI
OF INFECTIOUS
Pneumococcal
DISEASES-WASHINGTON
Antigens
In Sputum Number Positive (percent)
Number of Samples Pneumonia Pneumococcal Proved Probable Nonpneumococcal Bronchitis Pneumococci Present Absent
CoapSlutination
CIE
8 (80)
10 34 11
9 W) 28 (82) 2
29 (85) 3 (27)
8 15
6 (75) 3 (20)
6 (75) 3 (20)
Adapted from [ll]. CIE = counterimmunoelectrophoresis.
Illinois) is available in modified form. Most published evaluations to date of the Gonozyme test relate to its original one-hour version, which lacked sensitivity and specificity. The modified version is a four-hour test. In tests of cervical swabs from a low-risk population of 300 women seen as outpatients at the Mayo Clinic for evaluation of infertility or vaginitis, the modified Gonozyme test yielded three falsepositive resultsand one false-negative result. In testing mailed-in specimens, the Minnesota Department of Health has found the modified Gonozyme test to provide 97 percent specificity; however, sensitivity was 98.5 percent and 75.4 percent for detecting gonorrhea in men and women, respectively [15]. An enzyme immunoassay test for rotavirus (Rotazyme, Abbott Laboratories, North Chicago, Illinois) is also available and has been shown to be sensitive and specific. Accurate detection of cytomegalovirus and herpes virus still requires tissue culture methods, but sensitivity and speed of detection can be substantially increased by enhancing tissue culture infectivity by centrifugation and immunofluorescent staining with monoclonal antibody. Enzyme immunoassay and latex agglutination tests for Clostridium difficile toxin are under investigation. Thus, a variety of immunodiagnostic tests for a variety of microbial antigens have become available commercially. Enzyme immunoassay tests have largely replaced radioimmunoassays for hepatitis A and B virus antigens and antibodies, thereby offering decreasing test turnaround time and obviating the use of radioisotopic probes for these usually high-volume tests. Other immunodiagnostic tests will undoubtedly follow. Despite this flurry of activity in developing immunodiagnostic techniques, problems remain with regard to the availability of specific antibodies, the sensitivity and specificity of antibodies, the diversity of pathogens, and the frequency of polymicrobial infections. The fixed affinity and specificity of monoclonal antibodies may be insufficient for diagnostic purposes and may provide too limited a funcThe
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tional capacity relative to that of polyclonal antibodies. Moreover, unrelated microorganisms may share antigenic specificities. Some of these problems may be resolved by the development of genetic probes based on DNA hybridization. In either case, however, the ultimate questions to be answered are whether a four- to five-hour test is rapid enough to provide information needed for a clinical decision, what the test will cost, and whether the test can replace an existing culture method. If, because of lack of sensitivity, specificity, and other statistical parameters, or if determination of antimicrobial resistance can be made only with colonies isolated by culture methods, the test supplements rather than replaces existing methods, then the costs become supplementary and are unlikely to be accepted by already cost-sensitive hospitals and physicians. Moreover, what is the ultimate medical cost of the bacteriuric patient with a false-negative result from a bacteriuria screening test or of a false-negative enzyme immunoassay in a patient with gonorrhea? What is the legal cost of a false-positive enzyme immunoassay test for gonorrhea? In sum, it appears that the clinical microbiology laboratory will continue to use culture methods for many types of specimens in the foreseeable future and that nonculture (immunologic or genetic) methods will be used for examination of those specimens in which specific microorganisms are sought (such as group A Streptococcus, N. gonorrhoeae, C. trachomatis, enteric pathogens, viruses), provided their sensitivity and specificity are equivalent to those of culture methods and depending on the possibility of developing simple and economic approaches to predicting antimicrobial resistance. Certainly, accurate nonculture methods would provide opportunities not currently available for diagnosis in small hospital and office. laboratories. ANTIBIOTIC
CONTROL
Since antibiotics constitute a major proportion of hospital pharmacy expenditures, considerable thought has been given to the role of the laboratory in controlling antibiotic use. Obviously, the quality of specimen received, the extent of isolation and identification of isolates, the criteria established for susceptibility testing of isolates, and the antibiotics selected for testing affect antibiotic use. A specific antibiotic is less likely to be used if it is not tested and reported by the laboratory. Hence, in discussing the putative merits of a new antibiotic with the clinical staff, pharmaceutical company representatives try to ensure that the laboratory plans to test the new compound and has sufficient disks or powder to begin testing it as soon as FDA approval occurs. The decision as to whether or not to test a new antibiotic or to continue testing an older antibiotic has been greatly facilitated by the current focus of attention on antibiotic costs and, as a consequence, by limitations on antibiotics approved for hospital formularies. Decisions as to which antibiotics should be included in the 12
June
28,1985
The American
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Volume
formulary and tested by the laboratory are not easy and require close communication between formulary committee members and laboratory personnel. In certain instances, antibiotics listed in the formulary may be tested but not reported by the laboratory unless resistance to a less expensive related antibiotic occurs. Of the cephalosporins currently tested in our laboratory (Table VII), only the cefazolin results are reported for Staphylococcus aureus and for susceptible gram-negative bacilli. Reports of the results of testing moxalactam and cefoperazone are restricted to those gram-negative bacilli that are resistant to cefazolin, cefuroxime, and cefoxitin. The selection of bacteria for’ susceptibility testing is a complex process involving a series of decisions based on specimen source, the type of organism isolated, whether the organism was isolated in pure or mixed culture, and how predictable the susceptibilityof the organism is to the drug of choice. The practice of “routine” susceptibility testing results in unnecessary expense to the patient and is potentially misleading to the physician by its connotation of the clinical significance of any isolates tested. As a general rule, isolates from normally sterile body fluids undergo susceptibility testing, although the extent of testing may vary according to the type of organism present. It would, for example, ordiharily be unnecessary for a ’ pneumococcus from spinal fluid to be tested against antibiotics other than penicillin. In contrast, an isolate of Escherichia coli from this site would be tested against aminoglycosides, chloramphenicol, a variety of beta-lactam antibiotics, and perhaps trimethoprim/sulfamethoxazole. For H. influenzae, the laboratory. should be prepared to test for beta-lactamase and, because of the emergence of non-beta-lactamase-producing ampicillin-resistant strains, ampicillin susceptibility. Chloramphenicol-resistant isolates of H. influenzae have been reported but remain as yet rare in the United States. Tests of other antibi- 1 otics for the most frequent bacterial isolates from children with meningitis (H. influenzae, N. meningitidis, and S. pneumoniae) should probably be performed on an ad hoc i basis and could involve a third-generation cephalosporin. The group A Streptococcus remains susceptible to penicillin, and there is no need for this fact to be confirmed by testing individual isolates in the laboratory. Persistence of group A streptococci in throat culture samples from patients treated with penicillin is not due to the development of resistance to penicillin and has not been related to the concurrent presence in the pharynx of peniciilinase-producing staphylococci [16]. Susceptibility testing of group A streptococci to erythromycin may be indicated because of resistance in up to 3 percent of isolates in this country. Susceptibility testing of other bacteria isolated from throat culture samples is not indicated and in fact may be misleading if reported. Testing of isolates of S. aureus, Enterobacteriaceae, Pseudomonas, and Acinetobacter from acceptable sputum specimens is indicated, whereas testing of isolates of Hemophilus from such specimens is ordi78 (suppl8B)
MANAGEMENT
TABLE Vii
AND PREVENTION
OF INFECTIOUS
DISEASES-WASHINGTON
Reporting Protocol for Antimicrobiai Susceptibiiity Testing at the Mayo Clinic
simfltocOcci Ampicillin Chloramphenicol’ Erythromycin Nitrofurantoin+ Penicillin
siaphylococci
Gram-Negattvs
Cefazolin* Chlorarnphenicol’ Clindamycin Erythromycin Oxacillin Penicillin Trimethoprim/ sulfamethoxazole+
Gmm-Porttlva Gacllll
Amikacin Ampicillin Cefazolin Cefoperazone5 Cefoxitin§ Cefuroxime§ Chloramphenicol’ Gentamicin Mezlocillin Moxalactams Nalidixic acid+ Nitrofurantoin+ Piperacillin Tetracycline Trimethoprimkutfamethoxazolet*’ Trimethoprim+
Ampicillin Cefazolin Clindarnycin Erythromycin Oxacillin Penicillin
l Chloramphenicol +Reported *If oxacillin reported. *Reported **Reported
is reported only for isolates from blood, cerebrospinal fluid, eye, and stool cultures. only for urinary isolates. minimal inhibtry concentration is greater than 2 &ml, cefazolin minimal inhibitory concentration
only when cefazolin for Shigella isolates
minimal inhibitory from stools.
concentration
is 16 @ml
!28,19S!i
isolates
is not
or higher.
transport systems) has provided reasonably accurate documentation of clinical significance and a reasonable guideline for performing susceptibility tests. Problems do arise with organisms in mixed cultures, which are probably best reported initially as such and not subjected to susceptibility testing unless special circumstances require it to be performed. Because of the diverse nature of indigenous vaginal and cervical flora, the requirements for antimicrobial susceptibility of isolates from culture samples of these sites would ordinarily be limited to N. gonorrhoeae. identification of organisms other than N. gonorrhoeae, Hemophilus ducreyi, beta-hemoiytic streptococci, S. aureus (in instances of suspected toxic shock syndrome), Listeria monocytogenes, C. trachomatis, herpes viruses, Trichomonas vaginalis, and perhaps yeasts and ureaplasmas from cervical and vaginal specimens is seldom necessary and usually misleading. Bacteria isolated from mixed cultures of abscesses and wounds pose a considerable problem because of the frequent polymicrobial nature of such infections. identification and antimicrobial susceptibility testing of each isolate from such polymicrobial infections is time-consuming for the microbiologist, expensive for the patient, and often bewildering to the clinician because of the diversity ofand seemingly mutually exclusive-antimicrobial susceptibility patterns of each isolate. Therefore, there would seem to be very little useful purpose sewed by reporting the identification and antimicrobial susceptibility of each of four or more aerobic bacterial and an equal number of anaerobic bacterial species isolated from an intra-abdominal abscess, peritoneal fluid, pelvic site, decubitus ulcer, perianal abscess or fistula, or intestinal drainage. A report
narily not required unlessthe gram-stained smear demonstrates a preponderance of small, pleomorphic bacilli resembling Hemophiius. In such cases, testing for betalactamase is indicated; however, the selection of other antimicrobial agents to be tested in problematical. For exampie, cefaclor and cefamandole appear active with standard disk diffusion and dilution tests against beta-lactamase-positive isolates of H. influenzae. However, since the activity of both of these cephalosporins against such isolates is highly inoculum-dependent [17,18] (Figure l), and cases of meningitis have occurred during or shortly after treatment with cefamandoie, [18-201, requests for susceptibility testing of Hemophiius against cefacior or cefamandole should probably prompt a consultation between the clinician and the microbiologist. Although urine culture and antimicrobial susceptibility testing of isolates from urine of patients with unresolved or recurrent bacteriuria appear indicated, questions have been raised about the cost-effectiveness of urine culture and particularly of antimicrobial susceptibility tests of isolates from initial pretreatment urine specimens from women with acute dysuria [21,22]. This question is particularly relevant since Stamm et al [28] have found that acute dysuria in women is often associated with C. trachomatis, which can be detected only by the more technically complex tissue culture or immunofluorescent antibody staining techniques, or bacteriuria of as low as 1O2colonyforming units/ml, which would not be detected by screening tests or by conventional quantitative urine culture techniques. in cases in which urine culture is indicated, the growth in pure culture of l@ colony-forming units/ml or greater (or lo4 or greater with proper collection and June
for staphylococcal
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MANAGEMENT
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Cephalothin cfu/ml cl lo5 m 107
Cefoxitin Cefamandole Cefaclor
34
66
87
05
05
2
4
8
16
32
Moxalactam Zefoperazone ,125
.25
5
1
Concentration
64
128
@g/ml) 7.200” 2
Cefamandole
Moxalactam
.125
.25
.5
1
2
4
Concentration
8
16
32
June
28,1985
The American
Journal
128
&g/ml)
of “mixed fecal flora” in such instances should readily convey the information required for a clinical decision. Susceptibility testing of anaerobic bacteria remains a problem for many laboratories because of the lag time required for growth and testing of isolates and the polymicrobial nature of most anaerobic bacterial infections. Clinicians seem to be generally aware of the types of infections associated with anaerobic bacteria and of the antimicrobial agents active against anaerobic bacteria. For these reasons, Bourgault et al [24] found that suscep tibility test reports for anaerobic bacteria were infrequently utilized and recommended that testing be limited to anaerobic bacteria isolated from bacteremias, orthopedic infections, and central nervous system infections. Their recommendation appears reasonable in view of the continued high levels of activity of chloramphenicol, clindamycin, and metronidazole against the Bacteroides fragiiis group [25,26]. Cefoxitin remains the most active beta-lactam against the B. fragilis group; however, considerable heterogeneity of resistance rates exists to beta-lactam antibiotics among isolates of Bacteroides and Clostridium [25,26], 14
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figure 1. Cumulative percent of betalactamase-positive isolates of Hemophi/us influenzae inhibited (top) and killed (bottom) by cephalosporins. 7.110” Adapted from [ 171.
and susceptibility of anaerobic bacteria to the beta-lactams should never be assumed. Susceptibility testing of tubercle bacilli from previously untreated patients is ordinarily not necessary; however, susceptibility testing is indicated for tubercle bacilli from patients who have acquired the disease outside the United States, as well as those from patients with relapse after a full course of chemotherapy or with persistent positive acid-fast organisms in sputum smears for two to three months or in cultures for five tosix months. Mycobacteria other than Mycobacterium tuberculosis probably do warrant susceptibility testing because of their highly variable rates of resistance to antimycobacterial agents. Fungal susceptibility testing methods remain unstandardized, and the results are highly method-dependent. Since amphotericin has a broad range of activity against yeasts, dimorphic fungi, and filamentous fungi, susceptibility testing with this agent is rarely indicated. Because of the possibility of acquired resistance during therapy, susceptibility testing of flucytosine is warranted. Susceptibility testing of mycobacteria and fungi is tech78 (suppl6B)
MANAGEMENT
nically complex and should probably be performed in reference laboratories. As already discussed, the selection of antibacterial agents to be tested should be guided by the type of organism isolated and its source, as well as by close communication between the laboratory and the formulary committee. The antimicrobial agents currently being reported at the Mayo Clinic are listed in Table VII; however, it must be emphasized that these agents are under active review by formulary committees so that those listed are subject to change at any time. Strategies for testing beta-lactams to represent others are listed in Tables VIII and IX.
AND PREVENTION
TABLE VIII
OF INFECTIOUS
Recommendations for Laboratory Testing of Penicillins
Olpanitm
TestDrop
Enterococcus
Other
gram-positive
Penicillin Oxacillin
cocci
June 28,1985
I-
Enterobacteriaceae
Ampicillin
Pseudomonas
Either of mezlocillin piperacillin Mezlocillin Either of piperacilfin azlocillin
Adapted
from
To Represent
Penicillin or ampicillin
ANTIBIOTIC MONITORING A portion of the cost of administering potentially toxic antimicrobial agents is that of monitoring concentrations to ensure that peak levels are within therapeutic range or that trough levels are not increased due to decreased clearance and drug accumulation. The majority of assays performed for this purpose are for aminoglycosides, vancomycin, and chloramphenicol. Commercially prepared nonradioisotopic immunoassays have largely superseded bioassay, chromatography, radioenzymatic assay, and radioimmunoassay for aminoglycosides and vancomycin and offer speed, simplicity, and accuracy, provided specimens are correctly collected. Chloramphenicol is monitored by bioassay, radioenzymatic assay, or chromatography (high-performance liquid chromatography). In general, blood for determination of peak levels is drawn 30 minutes after completion of an intravenous infusion or one hour after an intramuscular dose, and blood for determination of the trough level is drawn just before the next dose. Blood drawn at inappropriate times and failure to document the timing of collection relative to dose are frequent problems that render the results of assays uninterpretable and potentially misleading. Dosing times recorded on patients’ charts usually reflect dosage schedules rather than actual times of administration of antibiotic, so that blood drawn at the anticipated peak level may have preceded administration of the dose and may thereby actually reflect a trough level. As a consequence, a serum level of gentamicin of 2 pglml could prompt an increase, rather than a decrease, in dose. On the other hand, a serum level of 4 pg/ml, erroneously assumed to represent an elevated trough level and causing a reduction in dosage, could actually represent a satisfactory peak level. In either case, failure to record accurately the times of administration of the dose and of collection of the blood sample lead to improper dosage adjustments. Less subtle discrepancies between anticipated peak and trough concentrations usually prompt repeated assays, adding unnecessary cost and delaying administration of appropriate dosages. It is, therefore, prudent and cost-effective to have the drug to be assayed administered and the blood for assay collected by one or more nurses specially trained for this purpose.
DISEASES-WASHINGTON
l-
Ampicillin analogs klocillin Mezlocillin Piperacillin Methicillin Nafcillin Cloxacillin Dicloxacillin Ampicillin analogs the other Carbenicillin Ticarctllin
f
the other
[271.
TABLE IX
Recommendations for Laboratory Testing of Cephalosporin and Cephem Antibiotics
Organism
Test Drug
To Represent
Flret generation Staphylococcus
Cephalothin
Enterobacteriaceae
Cephalothin or cefazolin None
Cefaclor Cefadroxil Cephalexin Cephaloglycin Cephaloridine Cephapirin Cephradine -
Cefoxitin None None
Cefamandole Cefonicid Ceforanide Cefotetan? -
Enterococcus
Ctecond generation Enterobacteriaceae
Staphylccoccus Enterococcus
Third gene&ion Enterobacteriaceae
Cefoperazone Any of cefotaxime ceftazidime ceftizoxime ceftriaxone moxalactam Any of cefotaxime ceftriaxone moxalactam Cefoperazone
Pseudomonas
Enterococci Adapted
None from
-
the others k
the others k Cefsulodin Ceftazidime -
[271.
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REFERENCES 1. 2.
3. 4.
5.
8. 7.
8.
9.
10.
11.
12.
13.
14.
18
Neu HC: What should the clinician expect from the microbiology laboratory. Ann Intern Med 1978; 89 (part 2): 781-784. Ferraro MJB: How should a laboratory arrive at costs? Twentythird Interscience Conference on Antimicrobial Agents and Chemotherapy. American Society for Microbiology, Las Vegas, 1983. Bartlett RC, Ruk C: Processing control and cost in bacteriology. Am J Clin Pathol 1980; 74: 287-296. Washington JA II: Noninvaslve diagnostic techniques for lower respiratory infections. In: Pennington JE, ed. Respiratory infections: diagnosis and management. New York: Raven Press, 1983; 41-54. Edwards LD, Levin S, Balagtas R, Lowe P, Landau W, Lepper MH: Ordering patterns and utilization of bacteriologic culture reports. Arch Intern Med 1973; 132: 878-882. Bartlett RC: Urine screening-to screen or not to screen. Clin Microbial Newsletter 1984; 8: 29-30. Hughes JG, Snyder RJ, Washington JA II: An evaluation of a leukocyte esterase/nitrite test strip and a bioluminescence assay for detection of bacteriuria. Diagn Mkxobiol Infect Dis 1985; 3: 139-142. Loo SYT, Scottolini AG, Luangphinith S, Adam AL, Jacobs LO, Mariani AJ: Urine screening strategy employing dipstick analysis and selective culture: an evaluation. Am J Clin Path01 1984; 81: 834-842. Breese BB: Bacterioiogy and serology. In: Breese BB, Hall CB, eds. Streptococcal disease. Boston: Houghton Mifflin 1978; Q-33. Kaplan EL, Couser R, Huwe BB, McKay C, Wannamaker LW: Significance of quantitative saliiary cultures for group A and non-group A /3-hemolytic streptococd in patter& with pharyngitis and in their family contacts. Pediatrics 1979; 84: QO4912. Kjos PA, Anhalt JP: Evaluation of Directigen” for detection of group A streptococci in throat swabs (abstr 13). Twenty-fourth Interscience Conference on Antimicrobial Agents and Chemotherapy. American Society for MicrobioloW, 1984. Edwards EA, Coonrod JD: Coagglutination and counterimantigens in the spumunoelectrophoresis for pneu mococcal tum of pneumonia patients. J Clin Microbii 1980; 11: 488491. Rein MF, Gwaltney JM, O’Brien WM, Jennings RH, Mandell GL: Accuracy of Gram’s stain in identifying pneumococci in sputum. JAMA 1978; 239: 2871-2873. Tam MR, Stamm WE, Handsfield HH, et al: Culture-independent
June
26,lS8!S
The Amwlcan
Jour~l
of Modlclna
Votuma
15. 18.
17.
18.
19.
20.
21. 22.
23. 24.
25.
28.
27.
diagnosis of Chlamydia trachomatis using monoclonal antibodies. N Engl J Med 1984; 310: 11461150. Morse CD: An enzyme immunoassay for detection of gonorrhea. Med Lab Forum 1984; 12: 3-4. Eickhoff TC: Management of streptococca I pharyngitis: choice of antibiotics with regard to adverse reactions and bactertological failures. In: Shulman ST, ed. Pharyngitis: management in an era of deciining rheumatic fever. New York: Praeger, 1984; 153-182. Bulger RR, Washington JA II: In vitro activity of cephalospotins against Haemophilus species. In: Current chemotherapy and infectious disease. Washington: American Society for Microbiology, 1980; 10261027. Azimi PH, Chase PA: The role of cefamandole in the treatment of Haemophilus influenzae infections in infants and children. J Pediatr 1981; 98: 995-l 000. Aronoff SC, Thomford W, Bertino JS, Speck WT: Development of meningitis during therapy with cefamandole. Pediatrics 1981; 87: 727-728. Chartrand SA, Marks MI, Roberts R, Jubelirer D, Plunket DC: Development of Haemophilus influenxae meningitis in patients treated with cefamandole. J Pediatr 1981; 98: 10031005. . Komarofl AL: Acute dysuria in women. N Engl J Med 1984; 310: 368-375. Schultz HJ, McCaffrey LA, Keys TF, Nobrega FT: Acute cystitis: a prospective study of laboratory tests and duration of therapy. Mayo Clin Proc 1984; 59: 391-397. Starnm WE, Wagner KF, Amsel R, et al: Causes of acute urethral syndrome in women. N Engl J Med 1980; 303: 409-415. Bourgault A-M, Harkness JL, Rosenblatt JE: Clinical usefulness of susceptibility testing of anaerobes. Arch Intern Med 1978; 138: 1825-1827. Edson RS, Rosenblatt JE, Lee DT, McVey EA Ill: Recent experience with antimicrobial susceptibility of anaerobic bacteria: increasing resistance to penicillin. Mayo Clin Proc 1982; 57; 737-741. Cucheral GJ, Tailey FP, Jacobus NV, et al: Antimicrobial susceptibiliies of 1,292 isolates of the Bactemides fragilis group in the United States: comparison of 1981 and 1982. Antimicrob Agents Chemother 1984; 28: 145-148. Jones RN: Antimicrobial susceptibility testing (AST): a review of changing trends, quality control guidelines, test accuracy, and recommendation for the testing of 8-lactam drugs. Diagn Microbiol Infect Dis 1983; 1: l-24.
78 (wppt
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JOHN A. WASHINGTON II, M.D.
Utilization of the microbiology laboratory is subject to limitations posed by the diversity of microbial agents causing infection, multiple and often poorly accessible sites of infection, contamination of specimens with indlgenous flora, and failure of communication between clinician and microbiologist. Cost constraints increasingly llmlt the extent of mlcrobiologic services available on-site and lead to decentralization of laboratory senrices and possible loss of quaiity control. Increased attention Is being paid to process control of cultures, rapid screening and diagnostic tests as culture substltutes, and limitations on antibiotic susceptiblllty testing.
Rochester, Minnesota
The question of what services the microbiology laboratory should provide has preoccupied clinicians and microbiologists alike for many years and has been accentuated by recent legislation aimed at cost-containment in medical care. In a symposium sponsored by the National Institute of Allergy and Infectious Diseases in 1978 on the impact of infections on medical care in the United States, Neu [l] discussed what the clinician should expect from the microbiology laboratory (Table I). Although these expectations remain valid today, they have been but partially fulfilled. Afthough it should not be difficult for a group of experts to decide what infonation is required for a clinical decision, and although current kits and devices provide the microbiology laboratory with the capability of identifying and susceptibility testing bacteria with a degree of accuracy unavailable a decade ago, there remain many unresolved problems in the entire process, extending from specimen collection to interpretation of the results reported by the laboratory. SPECIMEN COLLECTION
From the Mayo Clinic and Mayo Medical School, Rochester, Minnesota. Requests for reprints should bs addrassad to Dr. John A. Washington II, Sactbn of Clinical Microbiology, Mayo Clinic, 200 SW Firal Street, Rochaatar, Minnesota 5!5905.
8
June
28,1965
The Amdcan
Journal
The laboratory diagnosis of any infectious disease depends on the establishment of a differential diagnosis based on the history and physical examination, a consideration of the most probable cause of the disease, and the selection of those tests most likely to detect the most probable cause of the disease. Complicating this decision process is the fact that infectious diseases may involve virtually any system or organ in the body and may be due to a wide variety of microorganisms, including bacteria, mycobacteria, fungi, chlamydiae, mycoplasmas, viruses, and parasites. For these reasons, specimen collection represents the most important part of the process leading to information required for a clinical decision. Guidelines for specimen collection must be designed to provide the laboratory with a sufficient amount and number of specimens for complete examination. All too often a small or even invisible amount of specimen on a swab or a small sample of tissue or fluid is submitted to the
of MeddWna
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MANAGEMENT
laboratory with a request for multiple examinations and cultures. In such instances, the likelihood of negative microscopic examinations and cultures is great, and the credibility of negative results is low. Sampling difficulties often complicate the specimen collection process in that some specimens are simply inaccessible except by invasive techniques or are associated with a high likelihood of becoming contaminated with indigenous flora during the collection process. An added problem is the partial replacement of indigenous flora of the skin and mucous membranes by gram-negative bacilli in immunocompromised and other seriously ill, debilitated patients. Prior administration of antimicrobial agents may inhibit recovery of a causative agent from cultures. Finally, the causative agent suspected may not be detected because of lack of communication or dialogue between the clinician and the microbiologist or because, as the result of cost-containment, the procedures necessary for its detection are not available in the laboratory.
AND PREVEEiTlON
TABLE I
June 28,198!5
DISEASES-WASHINGTON
What Should the Clinician Expect from the Laboratory? information required for clinical decision Guidelines for specimen collection Rapid specimen transport Identification of microorganisms Antimicrobial susceptibilities Rapid transmission of results Dialogue
TABLE II
College of American Pathologists: Extents of Services Provided
Exlanl Definitive identification to extent required for diagnosis and assistance in selection of therapy Limited identification with definitive identification by reference laboratory Direct examination (arid inoculation) with identification by reference laboratory All specimens referred to another laboratory
EXTENT OF SERVICES Today, more than ever, it is unreasonable to expect equal capabilities and availabilities of microbiologic services of all laboratories. Indeed, varying extents of services have been recognized by laboratory accreditation agencies for many years. A laboratory that, for example, offers Extent 4 service in mycobacteriology provides definitive identification and antimicrobial susceptibility testing of mycobacteria, whereas a laboratory that offers Extent 1 service simply collects and refers specimens to a more specialized laboratory for isolation, identification, and susceptibility testing of mycobacteria. Although the specific requirements by extent of service vary somewhat for bacteriology, mycobacteriology, mycology, parasitology, and virology, their general definitions are listed in Table II. It should, therefore, be possible to devise a hypothetical scheme of extents of microbiology services by type of hospital (Tdble Ill). The logic of such a scheme has, however, been profoundly altered by the advent of prospective payment based on diagnosis-related groups. The impact of this legislation has been to change the hospital-based laboratory from a profit center to a cost center, which, in turn, is leading in all but Federal hospitals to curtailment in laboratory services. Hospital-based laboratories are reacting to these changes in different ways. Some have contracted with local, regional, or national commercial vendors for laboratory management. Others have decided to utilize their assets and compete with the commercial vendors on a local or regional basis through joint ventures with other hospitals or by acquisitions or affiliations with other hospital, local, or regional laboratories. The ultimate consequences of all of this activity remain uncertain. Extent of service on-site may be reduced, and referral of specimens and tests to other laboratories increased. Low cost and rapid turnaround time have already assumed utmost importance. Screening tests to obviate or replace
OF INFECTIOUS
TABLE Ill
Hypothetlcal Distribution of Extents of Service In Hospitals
SonrIce Bacterblogy Mycobactertolcgy MY~lwY Parasitology Virology
CommunftyCommunity Teachlng Unlvsnity Refsrrmce 3 1 2 2 1
4 2 3 3 2
4 3 4 3 3
4 4 4 4 4
cultures are being implemented. Perhaps of greatest concern is the decentralization of laboratory services and the potential loss of quality control. One pertinent question to ask under current circumstances is what the costs of microbiologic services actually are. In a study at the Massachusetts General Hospital, Ferraro [2] determined that the average cost per case was less than 1 percent and that the average cost per case in the most frequent diagnosis-related groups was also less than 1 percent. Thus, curtailment of microbiology services cannot be expected to have a major impact on reducing costs of medical care. TRENDS IN CUNICAL MICROBIOLOGY The major trends in clinical microbiology laboratory practice today are summarized in Table IV and are designed generally to limit processing of inappropriate specimens and to provide rapid detection of pathogenic microorganisms. By rejecting specimens that were improperly identiThe American Journal of Medlclne
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TABLE
IV
AND PREVENTION
Clinical
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Mlcrobiology
DISEASES-WASHINGTON
Laboratory
Trends
commercial availability of a variety of screening tests for bacteriuria and to immunologic tests or immunoassays for microbial detection (such as group A Streptococcus, Neisseria gonorrhoeae, Chlamydia trachomatis). Work is also progressing with genetic probes based on DNA hybrfdization techniques for rapid detection of a variety of microorganisms.
Screening tests to Determine specimen acceptability Obviate culture Rapid diagnostic methods by Immunologic techniques DNA hybridization Process control limiting Number of isolates undergoing identffiiation and susceptibility tests Number of antimicrobial agents tested
SCREENING
fied or in faulty containers, were delayed in transit, dupiicated another received on the same day from the same site (except blood, spinal fluid, feces), were from sites or sources known to provide little useful information (such as vomitus, loch& Foley catheter tips), or were microscopically unsatisfactory and by limiting identification and susceptibility testing of multiple organisms in mixed cultures of lower respiratory secretions, wound exudates, cervicovaginal secretions, and urine, Bartlett and Rutz [3] minimized reporting of potentially misleading information and reduced the costs of processing these categories of specimens by approximately $42,000 a year. Screening tests to determine specimen acceptability are widely used for sputum [4] and can influence the quality of specimen collected when implemented with the support of the medical specialty groups involved and with the cooperation of nursing services. in certain instances, to be discussed later, the screening test assumes diagnostic importance as well. The management of infectious diseases has often been hampered by the frequently retrospective nature of microbiologic diagnoses imposed by traditional culture methods. Thus, the results of culture have often been largely confirmatory in nature and have, therefore, had relatively little effect on empiric therapy. In a study of ordering patterns and utilization of bacteriologic culture reports, Edwards et al [5] found that therapy was changed with 20.9 percent of positive culture reports, 1.4 percent of negative culture reports, and only 7 percent of ail culture reports. This problem has provided considerable impetus to the deveioprhent of rapid diagnostic tests and has led to the
TABLE
V
Urine Screening
Methods
Tssl
Time
Gram-stained smear Leukocyte esterase/nitrite (LN) strip Fiftrationcolorfmetry (Bat-T-Screen) Bioluminescence (Monolight, Lumac) AlJtobac MS2 AMS
10
Juno
28,1885
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Journal
4 minutes 2 minutes 2 minutes minutes hours hours hours
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TESTS
A variety of rapid screening tests to rule out the presence of lo5 or more colony-forming units/ml of urine have become commercially available (Table V) and are being used in some hospitals to obviate the need for culture or as culture substitutes. Bartlett [6] has estimated a cost saving of about $30,000 a year if a chemical screening test for bacteriuria (Chemstrip LN, Boehringer Mannheim, Indianapolis, Indiana) were used by nursing personnel in clinics and inpatient care units immediately after specimen collection. Although the predictive negative value of this test is approximately 98 percent, its false-negative rate of 17 percent for specimens in our laboratory yielding lo5 colony-forming units/ml or greater is reason for concern [7l and raises doubts about the suitability of the test as a culture substitute unless additional criteria, such as the appearance of the urine and the presence of blood or protein, are used [8]. IMMUNOASSAYS
lmmunoassays have been available for several years for detecting Hemophiius infiuenzae, Neisseria meningitidis, Streptococcus agalactiae (group B), Streptococcus pneumoniae, and Cryptococcus neoformans antigens in cerebrospinal fluid and herpes simplex virus in brain tissue. Because of their technical complexity, counterimmunoelectrophoretic and fluorescent antibody procedures for the common bacterial pathogens were largely restricted in their use to reference laboratories staffed by skilled personnel. Thus, the advent of coaggiutination and latex agglutination tests has offered antigen testing of spinal fluid from children with suspected meningitis to iaboratories with lesser skilled personnel. Use of antigen detection tests might also help to resolve problems with correct interpretation of gram-stained smears of spinal fluid. A latex agglutination test for cryptococcal antigen has been available for several years and provides greater sensitivity and specificity than that of the traditional India ink wet preparation. In our experience, microscopic examination and latex agglutination have detected 42 and 89 percent, respectively, of cryptococci in culturally positive spinal fluid samples, raising the question as to whether the India ink or nigrosin wet preparation tests should be performed at all. A variety of rapid immunodiagnostic methods have become available for detection of group A streptococci in throat swabs. in general, these tests have proved to be specific; however, their sensitivity is directly correlated 78 (auppl85)
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with the number of group A streptococcal colonies isolated in culture. Although some investigators have considered small numbers of colonies of group A streptococci as being clinically insignificant [9], others have found that although patients with true streptococcal infection are likely to have strongly positive culture results from the upper respiratory tract, there is sufficient overlap that does not allow clinical differentiation of the carrier from the truly infected patient by degree of positivity alone [lo]. A related problem is that the number of colonies isolated is directly related to the vigor with which the throat is swabbed. Although this last variable may be reasonably controlled in any single physician’s practice, the usual clinical laboratory receives specimens obtained by many different physicians whose vigor in swabbing the throat varies considerably. Hence, whatever relationship may exist between disease and degree of positivity of the culture result becomes even more tenuous and compromises, to some extent, the value of immunodiagnostic tests as a culture substitute. In our experience with one commercially available latex agglutination test for detection of group A streptococci, the sensitivity varied from 83 to 87 percent, depending on whether the swab was tested within two hours or after storage overnight, and the false-negative rate was 43 percent when fewer than 50 colonies were isolated by culture [ll]. Coagglutination and counterimmunoelectrophoresis have also been used to test sputum for pneumococcal antigen in patients with pneumonia. As may be seen in Table VI, Edwards and Coonrod [12] found coagglutination to be sensitive; however, specificity was compromised by the high rate of positivity of sputum from patients with bronchitis. Coagglutination was more sensitive than counterimmunoelectrophoresis in detecting pneumococcal antigen in the sputum during therapy of patients with proved or probable pneumococcal pneumonia [12]. The level of sensitivity provided by the coagglutination test of sputum in patients with proved or probable pneumococcal pneumonia substantially exceeds that (48 percent) of microscopic examination of gram-stained smears of sputum for the preponderance of gram-positive lancet-shaped diplococci [13]. Methods have been developed for the diagnosis of C. trachomatis urethritis and cervicitis by immunofluorescent antibody staining of secretions and of gonorrhea by enzyme immunoassay. Tam et al [14] demonstrated 93 percent sensitivity and 98 percent specificity of the fluorescent antibody test for C. trachomatis using monoclonal antibodies on smears from urethral and cervical secretions. This test is now commercially available (Direct Specimen Test, MicroTrak, Syva Company, Palo Alto, California). An enzyme immunoassay method for C. trachomatis (Chlamydiazyme, Abbott Laboratories, North Chicago, Illinois) is under development and investigation. An enzyme immunoassay method for detecting N. gonorrhoeae (Gonozyme, Abbott Laboratories, North Chicago, June z&1985
AND PREVENTION
VI
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Pneumococcal
DISEASES-WASHINGTON
Antigens
In Sputum Number Positive (percent)
Number of Samples Pneumonia Pneumococcal Proved Probable Nonpneumococcal Bronchitis Pneumococci Present Absent
CoapSlutination
CIE
8 (80)
10 34 11
9 W) 28 (82) 2
29 (85) 3 (27)
8 15
6 (75) 3 (20)
6 (75) 3 (20)
Adapted from [ll]. CIE = counterimmunoelectrophoresis.
Illinois) is available in modified form. Most published evaluations to date of the Gonozyme test relate to its original one-hour version, which lacked sensitivity and specificity. The modified version is a four-hour test. In tests of cervical swabs from a low-risk population of 300 women seen as outpatients at the Mayo Clinic for evaluation of infertility or vaginitis, the modified Gonozyme test yielded three falsepositive resultsand one false-negative result. In testing mailed-in specimens, the Minnesota Department of Health has found the modified Gonozyme test to provide 97 percent specificity; however, sensitivity was 98.5 percent and 75.4 percent for detecting gonorrhea in men and women, respectively [15]. An enzyme immunoassay test for rotavirus (Rotazyme, Abbott Laboratories, North Chicago, Illinois) is also available and has been shown to be sensitive and specific. Accurate detection of cytomegalovirus and herpes virus still requires tissue culture methods, but sensitivity and speed of detection can be substantially increased by enhancing tissue culture infectivity by centrifugation and immunofluorescent staining with monoclonal antibody. Enzyme immunoassay and latex agglutination tests for Clostridium difficile toxin are under investigation. Thus, a variety of immunodiagnostic tests for a variety of microbial antigens have become available commercially. Enzyme immunoassay tests have largely replaced radioimmunoassays for hepatitis A and B virus antigens and antibodies, thereby offering decreasing test turnaround time and obviating the use of radioisotopic probes for these usually high-volume tests. Other immunodiagnostic tests will undoubtedly follow. Despite this flurry of activity in developing immunodiagnostic techniques, problems remain with regard to the availability of specific antibodies, the sensitivity and specificity of antibodies, the diversity of pathogens, and the frequency of polymicrobial infections. The fixed affinity and specificity of monoclonal antibodies may be insufficient for diagnostic purposes and may provide too limited a funcThe
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tional capacity relative to that of polyclonal antibodies. Moreover, unrelated microorganisms may share antigenic specificities. Some of these problems may be resolved by the development of genetic probes based on DNA hybridization. In either case, however, the ultimate questions to be answered are whether a four- to five-hour test is rapid enough to provide information needed for a clinical decision, what the test will cost, and whether the test can replace an existing culture method. If, because of lack of sensitivity, specificity, and other statistical parameters, or if determination of antimicrobial resistance can be made only with colonies isolated by culture methods, the test supplements rather than replaces existing methods, then the costs become supplementary and are unlikely to be accepted by already cost-sensitive hospitals and physicians. Moreover, what is the ultimate medical cost of the bacteriuric patient with a false-negative result from a bacteriuria screening test or of a false-negative enzyme immunoassay in a patient with gonorrhea? What is the legal cost of a false-positive enzyme immunoassay test for gonorrhea? In sum, it appears that the clinical microbiology laboratory will continue to use culture methods for many types of specimens in the foreseeable future and that nonculture (immunologic or genetic) methods will be used for examination of those specimens in which specific microorganisms are sought (such as group A Streptococcus, N. gonorrhoeae, C. trachomatis, enteric pathogens, viruses), provided their sensitivity and specificity are equivalent to those of culture methods and depending on the possibility of developing simple and economic approaches to predicting antimicrobial resistance. Certainly, accurate nonculture methods would provide opportunities not currently available for diagnosis in small hospital and office. laboratories. ANTIBIOTIC
CONTROL
Since antibiotics constitute a major proportion of hospital pharmacy expenditures, considerable thought has been given to the role of the laboratory in controlling antibiotic use. Obviously, the quality of specimen received, the extent of isolation and identification of isolates, the criteria established for susceptibility testing of isolates, and the antibiotics selected for testing affect antibiotic use. A specific antibiotic is less likely to be used if it is not tested and reported by the laboratory. Hence, in discussing the putative merits of a new antibiotic with the clinical staff, pharmaceutical company representatives try to ensure that the laboratory plans to test the new compound and has sufficient disks or powder to begin testing it as soon as FDA approval occurs. The decision as to whether or not to test a new antibiotic or to continue testing an older antibiotic has been greatly facilitated by the current focus of attention on antibiotic costs and, as a consequence, by limitations on antibiotics approved for hospital formularies. Decisions as to which antibiotics should be included in the 12
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formulary and tested by the laboratory are not easy and require close communication between formulary committee members and laboratory personnel. In certain instances, antibiotics listed in the formulary may be tested but not reported by the laboratory unless resistance to a less expensive related antibiotic occurs. Of the cephalosporins currently tested in our laboratory (Table VII), only the cefazolin results are reported for Staphylococcus aureus and for susceptible gram-negative bacilli. Reports of the results of testing moxalactam and cefoperazone are restricted to those gram-negative bacilli that are resistant to cefazolin, cefuroxime, and cefoxitin. The selection of bacteria for’ susceptibility testing is a complex process involving a series of decisions based on specimen source, the type of organism isolated, whether the organism was isolated in pure or mixed culture, and how predictable the susceptibilityof the organism is to the drug of choice. The practice of “routine” susceptibility testing results in unnecessary expense to the patient and is potentially misleading to the physician by its connotation of the clinical significance of any isolates tested. As a general rule, isolates from normally sterile body fluids undergo susceptibility testing, although the extent of testing may vary according to the type of organism present. It would, for example, ordiharily be unnecessary for a ’ pneumococcus from spinal fluid to be tested against antibiotics other than penicillin. In contrast, an isolate of Escherichia coli from this site would be tested against aminoglycosides, chloramphenicol, a variety of beta-lactam antibiotics, and perhaps trimethoprim/sulfamethoxazole. For H. influenzae, the laboratory. should be prepared to test for beta-lactamase and, because of the emergence of non-beta-lactamase-producing ampicillin-resistant strains, ampicillin susceptibility. Chloramphenicol-resistant isolates of H. influenzae have been reported but remain as yet rare in the United States. Tests of other antibi- 1 otics for the most frequent bacterial isolates from children with meningitis (H. influenzae, N. meningitidis, and S. pneumoniae) should probably be performed on an ad hoc i basis and could involve a third-generation cephalosporin. The group A Streptococcus remains susceptible to penicillin, and there is no need for this fact to be confirmed by testing individual isolates in the laboratory. Persistence of group A streptococci in throat culture samples from patients treated with penicillin is not due to the development of resistance to penicillin and has not been related to the concurrent presence in the pharynx of peniciilinase-producing staphylococci [16]. Susceptibility testing of group A streptococci to erythromycin may be indicated because of resistance in up to 3 percent of isolates in this country. Susceptibility testing of other bacteria isolated from throat culture samples is not indicated and in fact may be misleading if reported. Testing of isolates of S. aureus, Enterobacteriaceae, Pseudomonas, and Acinetobacter from acceptable sputum specimens is indicated, whereas testing of isolates of Hemophilus from such specimens is ordi78 (suppl8B)
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Reporting Protocol for Antimicrobiai Susceptibiiity Testing at the Mayo Clinic
simfltocOcci Ampicillin Chloramphenicol’ Erythromycin Nitrofurantoin+ Penicillin
siaphylococci
Gram-Negattvs
Cefazolin* Chlorarnphenicol’ Clindamycin Erythromycin Oxacillin Penicillin Trimethoprim/ sulfamethoxazole+
Gmm-Porttlva Gacllll
Amikacin Ampicillin Cefazolin Cefoperazone5 Cefoxitin§ Cefuroxime§ Chloramphenicol’ Gentamicin Mezlocillin Moxalactams Nalidixic acid+ Nitrofurantoin+ Piperacillin Tetracycline Trimethoprimkutfamethoxazolet*’ Trimethoprim+
Ampicillin Cefazolin Clindarnycin Erythromycin Oxacillin Penicillin
l Chloramphenicol +Reported *If oxacillin reported. *Reported **Reported
is reported only for isolates from blood, cerebrospinal fluid, eye, and stool cultures. only for urinary isolates. minimal inhibtry concentration is greater than 2 &ml, cefazolin minimal inhibitory concentration
only when cefazolin for Shigella isolates
minimal inhibitory from stools.
concentration
is 16 @ml
!28,19S!i
isolates
is not
or higher.
transport systems) has provided reasonably accurate documentation of clinical significance and a reasonable guideline for performing susceptibility tests. Problems do arise with organisms in mixed cultures, which are probably best reported initially as such and not subjected to susceptibility testing unless special circumstances require it to be performed. Because of the diverse nature of indigenous vaginal and cervical flora, the requirements for antimicrobial susceptibility of isolates from culture samples of these sites would ordinarily be limited to N. gonorrhoeae. identification of organisms other than N. gonorrhoeae, Hemophilus ducreyi, beta-hemoiytic streptococci, S. aureus (in instances of suspected toxic shock syndrome), Listeria monocytogenes, C. trachomatis, herpes viruses, Trichomonas vaginalis, and perhaps yeasts and ureaplasmas from cervical and vaginal specimens is seldom necessary and usually misleading. Bacteria isolated from mixed cultures of abscesses and wounds pose a considerable problem because of the frequent polymicrobial nature of such infections. identification and antimicrobial susceptibility testing of each isolate from such polymicrobial infections is time-consuming for the microbiologist, expensive for the patient, and often bewildering to the clinician because of the diversity ofand seemingly mutually exclusive-antimicrobial susceptibility patterns of each isolate. Therefore, there would seem to be very little useful purpose sewed by reporting the identification and antimicrobial susceptibility of each of four or more aerobic bacterial and an equal number of anaerobic bacterial species isolated from an intra-abdominal abscess, peritoneal fluid, pelvic site, decubitus ulcer, perianal abscess or fistula, or intestinal drainage. A report
narily not required unlessthe gram-stained smear demonstrates a preponderance of small, pleomorphic bacilli resembling Hemophiius. In such cases, testing for betalactamase is indicated; however, the selection of other antimicrobial agents to be tested in problematical. For exampie, cefaclor and cefamandole appear active with standard disk diffusion and dilution tests against beta-lactamase-positive isolates of H. influenzae. However, since the activity of both of these cephalosporins against such isolates is highly inoculum-dependent [17,18] (Figure l), and cases of meningitis have occurred during or shortly after treatment with cefamandoie, [18-201, requests for susceptibility testing of Hemophiius against cefacior or cefamandole should probably prompt a consultation between the clinician and the microbiologist. Although urine culture and antimicrobial susceptibility testing of isolates from urine of patients with unresolved or recurrent bacteriuria appear indicated, questions have been raised about the cost-effectiveness of urine culture and particularly of antimicrobial susceptibility tests of isolates from initial pretreatment urine specimens from women with acute dysuria [21,22]. This question is particularly relevant since Stamm et al [28] have found that acute dysuria in women is often associated with C. trachomatis, which can be detected only by the more technically complex tissue culture or immunofluorescent antibody staining techniques, or bacteriuria of as low as 1O2colonyforming units/ml, which would not be detected by screening tests or by conventional quantitative urine culture techniques. in cases in which urine culture is indicated, the growth in pure culture of l@ colony-forming units/ml or greater (or lo4 or greater with proper collection and June
for staphylococcal
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Cephalothin cfu/ml cl lo5 m 107
Cefoxitin Cefamandole Cefaclor
34
66
87
05
05
2
4
8
16
32
Moxalactam Zefoperazone ,125
.25
5
1
Concentration
64
128
@g/ml) 7.200” 2
Cefamandole
Moxalactam
.125
.25
.5
1
2
4
Concentration
8
16
32
June
28,1985
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Journal
128
&g/ml)
of “mixed fecal flora” in such instances should readily convey the information required for a clinical decision. Susceptibility testing of anaerobic bacteria remains a problem for many laboratories because of the lag time required for growth and testing of isolates and the polymicrobial nature of most anaerobic bacterial infections. Clinicians seem to be generally aware of the types of infections associated with anaerobic bacteria and of the antimicrobial agents active against anaerobic bacteria. For these reasons, Bourgault et al [24] found that suscep tibility test reports for anaerobic bacteria were infrequently utilized and recommended that testing be limited to anaerobic bacteria isolated from bacteremias, orthopedic infections, and central nervous system infections. Their recommendation appears reasonable in view of the continued high levels of activity of chloramphenicol, clindamycin, and metronidazole against the Bacteroides fragiiis group [25,26]. Cefoxitin remains the most active beta-lactam against the B. fragilis group; however, considerable heterogeneity of resistance rates exists to beta-lactam antibiotics among isolates of Bacteroides and Clostridium [25,26], 14
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Volume
figure 1. Cumulative percent of betalactamase-positive isolates of Hemophi/us influenzae inhibited (top) and killed (bottom) by cephalosporins. 7.110” Adapted from [ 171.
and susceptibility of anaerobic bacteria to the beta-lactams should never be assumed. Susceptibility testing of tubercle bacilli from previously untreated patients is ordinarily not necessary; however, susceptibility testing is indicated for tubercle bacilli from patients who have acquired the disease outside the United States, as well as those from patients with relapse after a full course of chemotherapy or with persistent positive acid-fast organisms in sputum smears for two to three months or in cultures for five tosix months. Mycobacteria other than Mycobacterium tuberculosis probably do warrant susceptibility testing because of their highly variable rates of resistance to antimycobacterial agents. Fungal susceptibility testing methods remain unstandardized, and the results are highly method-dependent. Since amphotericin has a broad range of activity against yeasts, dimorphic fungi, and filamentous fungi, susceptibility testing with this agent is rarely indicated. Because of the possibility of acquired resistance during therapy, susceptibility testing of flucytosine is warranted. Susceptibility testing of mycobacteria and fungi is tech78 (suppl6B)
MANAGEMENT
nically complex and should probably be performed in reference laboratories. As already discussed, the selection of antibacterial agents to be tested should be guided by the type of organism isolated and its source, as well as by close communication between the laboratory and the formulary committee. The antimicrobial agents currently being reported at the Mayo Clinic are listed in Table VII; however, it must be emphasized that these agents are under active review by formulary committees so that those listed are subject to change at any time. Strategies for testing beta-lactams to represent others are listed in Tables VIII and IX.
AND PREVENTION
TABLE VIII
OF INFECTIOUS
Recommendations for Laboratory Testing of Penicillins
Olpanitm
TestDrop
Enterococcus
Other
gram-positive
Penicillin Oxacillin
cocci
June 28,1985
I-
Enterobacteriaceae
Ampicillin
Pseudomonas
Either of mezlocillin piperacillin Mezlocillin Either of piperacilfin azlocillin
Adapted
from
To Represent
Penicillin or ampicillin
ANTIBIOTIC MONITORING A portion of the cost of administering potentially toxic antimicrobial agents is that of monitoring concentrations to ensure that peak levels are within therapeutic range or that trough levels are not increased due to decreased clearance and drug accumulation. The majority of assays performed for this purpose are for aminoglycosides, vancomycin, and chloramphenicol. Commercially prepared nonradioisotopic immunoassays have largely superseded bioassay, chromatography, radioenzymatic assay, and radioimmunoassay for aminoglycosides and vancomycin and offer speed, simplicity, and accuracy, provided specimens are correctly collected. Chloramphenicol is monitored by bioassay, radioenzymatic assay, or chromatography (high-performance liquid chromatography). In general, blood for determination of peak levels is drawn 30 minutes after completion of an intravenous infusion or one hour after an intramuscular dose, and blood for determination of the trough level is drawn just before the next dose. Blood drawn at inappropriate times and failure to document the timing of collection relative to dose are frequent problems that render the results of assays uninterpretable and potentially misleading. Dosing times recorded on patients’ charts usually reflect dosage schedules rather than actual times of administration of antibiotic, so that blood drawn at the anticipated peak level may have preceded administration of the dose and may thereby actually reflect a trough level. As a consequence, a serum level of gentamicin of 2 pglml could prompt an increase, rather than a decrease, in dose. On the other hand, a serum level of 4 pg/ml, erroneously assumed to represent an elevated trough level and causing a reduction in dosage, could actually represent a satisfactory peak level. In either case, failure to record accurately the times of administration of the dose and of collection of the blood sample lead to improper dosage adjustments. Less subtle discrepancies between anticipated peak and trough concentrations usually prompt repeated assays, adding unnecessary cost and delaying administration of appropriate dosages. It is, therefore, prudent and cost-effective to have the drug to be assayed administered and the blood for assay collected by one or more nurses specially trained for this purpose.
DISEASES-WASHINGTON
l-
Ampicillin analogs klocillin Mezlocillin Piperacillin Methicillin Nafcillin Cloxacillin Dicloxacillin Ampicillin analogs the other Carbenicillin Ticarctllin
f
the other
[271.
TABLE IX
Recommendations for Laboratory Testing of Cephalosporin and Cephem Antibiotics
Organism
Test Drug
To Represent
Flret generation Staphylococcus
Cephalothin
Enterobacteriaceae
Cephalothin or cefazolin None
Cefaclor Cefadroxil Cephalexin Cephaloglycin Cephaloridine Cephapirin Cephradine -
Cefoxitin None None
Cefamandole Cefonicid Ceforanide Cefotetan? -
Enterococcus
Ctecond generation Enterobacteriaceae
Staphylccoccus Enterococcus
Third gene&ion Enterobacteriaceae
Cefoperazone Any of cefotaxime ceftazidime ceftizoxime ceftriaxone moxalactam Any of cefotaxime ceftriaxone moxalactam Cefoperazone
Pseudomonas
Enterococci Adapted
None from
-
the others k
the others k Cefsulodin Ceftazidime -
[271.
The Amerlcen Journal ot Medicine
Volume 78 (suppl8B)
15
MANAGEMENT
AND PREVENTION
OF INFECTIOUS
DISEASES-WASHINGTON
REFERENCES 1. 2.
3. 4.
5.
8. 7.
8.
9.
10.
11.
12.
13.
14.
18
Neu HC: What should the clinician expect from the microbiology laboratory. Ann Intern Med 1978; 89 (part 2): 781-784. Ferraro MJB: How should a laboratory arrive at costs? Twentythird Interscience Conference on Antimicrobial Agents and Chemotherapy. American Society for Microbiology, Las Vegas, 1983. Bartlett RC, Ruk C: Processing control and cost in bacteriology. Am J Clin Pathol 1980; 74: 287-296. Washington JA II: Noninvaslve diagnostic techniques for lower respiratory infections. In: Pennington JE, ed. Respiratory infections: diagnosis and management. New York: Raven Press, 1983; 41-54. Edwards LD, Levin S, Balagtas R, Lowe P, Landau W, Lepper MH: Ordering patterns and utilization of bacteriologic culture reports. Arch Intern Med 1973; 132: 878-882. Bartlett RC: Urine screening-to screen or not to screen. Clin Microbial Newsletter 1984; 8: 29-30. Hughes JG, Snyder RJ, Washington JA II: An evaluation of a leukocyte esterase/nitrite test strip and a bioluminescence assay for detection of bacteriuria. Diagn Mkxobiol Infect Dis 1985; 3: 139-142. Loo SYT, Scottolini AG, Luangphinith S, Adam AL, Jacobs LO, Mariani AJ: Urine screening strategy employing dipstick analysis and selective culture: an evaluation. Am J Clin Path01 1984; 81: 834-842. Breese BB: Bacterioiogy and serology. In: Breese BB, Hall CB, eds. Streptococcal disease. Boston: Houghton Mifflin 1978; Q-33. Kaplan EL, Couser R, Huwe BB, McKay C, Wannamaker LW: Significance of quantitative saliiary cultures for group A and non-group A /3-hemolytic streptococd in patter& with pharyngitis and in their family contacts. Pediatrics 1979; 84: QO4912. Kjos PA, Anhalt JP: Evaluation of Directigen” for detection of group A streptococci in throat swabs (abstr 13). Twenty-fourth Interscience Conference on Antimicrobial Agents and Chemotherapy. American Society for MicrobioloW, 1984. Edwards EA, Coonrod JD: Coagglutination and counterimantigens in the spumunoelectrophoresis for pneu mococcal tum of pneumonia patients. J Clin Microbii 1980; 11: 488491. Rein MF, Gwaltney JM, O’Brien WM, Jennings RH, Mandell GL: Accuracy of Gram’s stain in identifying pneumococci in sputum. JAMA 1978; 239: 2871-2873. Tam MR, Stamm WE, Handsfield HH, et al: Culture-independent
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diagnosis of Chlamydia trachomatis using monoclonal antibodies. N Engl J Med 1984; 310: 11461150. Morse CD: An enzyme immunoassay for detection of gonorrhea. Med Lab Forum 1984; 12: 3-4. Eickhoff TC: Management of streptococca I pharyngitis: choice of antibiotics with regard to adverse reactions and bactertological failures. In: Shulman ST, ed. Pharyngitis: management in an era of deciining rheumatic fever. New York: Praeger, 1984; 153-182. Bulger RR, Washington JA II: In vitro activity of cephalospotins against Haemophilus species. In: Current chemotherapy and infectious disease. Washington: American Society for Microbiology, 1980; 10261027. Azimi PH, Chase PA: The role of cefamandole in the treatment of Haemophilus influenzae infections in infants and children. J Pediatr 1981; 98: 995-l 000. Aronoff SC, Thomford W, Bertino JS, Speck WT: Development of meningitis during therapy with cefamandole. Pediatrics 1981; 87: 727-728. Chartrand SA, Marks MI, Roberts R, Jubelirer D, Plunket DC: Development of Haemophilus influenxae meningitis in patients treated with cefamandole. J Pediatr 1981; 98: 10031005. . Komarofl AL: Acute dysuria in women. N Engl J Med 1984; 310: 368-375. Schultz HJ, McCaffrey LA, Keys TF, Nobrega FT: Acute cystitis: a prospective study of laboratory tests and duration of therapy. Mayo Clin Proc 1984; 59: 391-397. Starnm WE, Wagner KF, Amsel R, et al: Causes of acute urethral syndrome in women. N Engl J Med 1980; 303: 409-415. Bourgault A-M, Harkness JL, Rosenblatt JE: Clinical usefulness of susceptibility testing of anaerobes. Arch Intern Med 1978; 138: 1825-1827. Edson RS, Rosenblatt JE, Lee DT, McVey EA Ill: Recent experience with antimicrobial susceptibility of anaerobic bacteria: increasing resistance to penicillin. Mayo Clin Proc 1982; 57; 737-741. Cucheral GJ, Tailey FP, Jacobus NV, et al: Antimicrobial susceptibiliies of 1,292 isolates of the Bactemides fragilis group in the United States: comparison of 1981 and 1982. Antimicrob Agents Chemother 1984; 28: 145-148. Jones RN: Antimicrobial susceptibility testing (AST): a review of changing trends, quality control guidelines, test accuracy, and recommendation for the testing of 8-lactam drugs. Diagn Microbiol Infect Dis 1983; 1: l-24.
78 (wppt
6B)