such a specimen is often difficult t o obtain.

Transcription

1 Assays of Antimicrobial Agents in Serum by Josephine A. Morello, Ph.D. Under certain clinical circumstances, it will be necessary to measure the antibiotic levels in the b l o o d of a patient being given a d r u g. A l t h o u g h i n f o r m a t i o n is available on r e c o m m e n d e d dose regimens of antimicrobial agents, the exact amount of a drug that will be present in the bloodstream of a patient is variable. A n t i b i o t i c levels in t h e b l o o d d e p e n d u p o n several host factors, as well as the pharmacologic p r o p e r t i e s of t h e d r u g. n v i t r o s u s c e p t i b i l i t y tests p e r f o r m e d in m o s t l a b o r a t o r i e s i n d i c a t e w h e t h e r an i n f e c t i o n c a u s e d by t h e o r g a n i s m tested is likely to respond to the dose of antibiotic recommended for treatment of that type of infection. However, in cases of life-threatening disease (such as bacterial endocarditis), it is useful to test the ability of the antibiotic in the patient's bloodstream to kill or inhibit the causative organism. n addition, when antibiotics that may have a toxic effect on body tissues are being administered, a physician may want quantitative information about the actual concentration of antibiotics achieved in the patient. A number of tests are available to perform such serum assays, but they have not been standardized to the same extent as bacterial susceptibility determinations. n these tests specimens obtained from patients w h o have received the drug for at least 24 hours are used. The blood usually is drawn just before adfore administration of a dose of antibiotic (trough level) and also at the time the highest concentration of antibiotic is expected in the bloodstream (peak level). For most antibiotics, peak levels occur one Josephine A. Morello, Ph.D., is Associate Professor of Pathology and Medicine and Director, Clinical Microbiology Laboratories, The University of Chicago. 30 LABORATORY MEDCNE VOL 7, NO. 4 APRL 1976 hour after an intramuscular injection or directly after an intravenously administered dose. Because the serum of some patients has bactericidal activity against certain microorganisms, it is recommended that a control serum obtained prior to administration of an antibiotic be tested as w e l l. n practice, such a specimen is often difficult t o obtain. Schlichter and his colleagues were the first to determine serum inhibitory levels, and thus this test is c o m m o n l y referred to as the Schlichter test. 1 Although it has been used primarily to monitor therapy of patients w i t h bacterial endocarditis, it may, under some circumstances, be of value as a guide in the treatment of other bacterial infections. 2 Quantitative assays are performed primarily when toxic antibiotics such as the aminoglycosides (e.g., kanamycin and gentamicin) are administered. Some physicians prefer to monitor the serum levels of all patients receiving aminoglycosides to insure that they are obtaining adequate, but not potentially toxic, doses. Since aminoglycoside antibiotics are excreted by the kidneys, patients with impaired renal function may have high blood levels of antibiotic: it is especially important that their therapy be m o n i t o r e d. Three methods are c o m m o n l y used to perform quantitative assays, t w o of them biologic and one enzymatic. The biologic assays can be performed easily in all laboratories, but they have an inherent error of f r o m 10% to 100%. The enzymatic assay is much more accurate ( ± 2-5 % ), but it is also more expensive and technically difficult. f the appropriate standards are used, the assays described in this article can be used to monitor the concentration of a n t i m i c r o b i a l agents in b o d y f l u i d s o t h e r t h a n serum, e.g., urine and cerebrospinal f l u i d.

2 1 ML Serum nhibitory or Bactericidal Levels (Schlichter Test) This test determines the maximum dilution of a patient's serum that will exert a bacteriostatic o r bactericidal effect o n the organism isolated from the patient. The bacteriology laboratory, therefore, should save all isolates f r o m blood cultures f o r at least o n e m o n t h in case such a determination is requested. Most of the organisms f r o m bacteremia and endocarditis will survive this long if they are subcultured t o an enriched broth medium or agar slant and refrigerated after incubation. f a freezer is available, it is preferable that t h e organisms be stored at C or lower in 50% trypticase soy broth and 50% inactivated horse or calf serum. W h e n the patient is receiving intermittent therapy, maximum information will be obtained if both peak and trough levels are d e t e r m i n e d. However, if only one blood sample is provided, the time it is drawn in relation to the administration of the antibiotic should be noted. A 5 ml t o 10 ml sample of the patient's blood usually is sufficient t o perform the test. The blood is allowed t o clot, and the serum is removed aseptically. f necessary, serum may be stored in the refrigerator f o r a few hours or at C f o r several days before testing. Twofold serial dilutions of the serum are made in either Mueller-Hinton broth or sterile, pooled normal human serum inactivated at 56 C f o r 30 minutes. Dilutions usually range f r o m undiluted in the first tube through 1:8 in t h e eighth tube. t may be necessary to prepare higher dilutions to reach an end point w h e n bacteria that are very susceptible to antibiotics, such as t h e a-hemolytic streptococci, are tested. A control tube consisting of normal human serum and/or broth is always included. The final volume in all tubes is 1.0 m l. After all of the dilutions have been prepared, each tube is inoculated with 0.05 ml of a 1:100 dilution of a six-hour broth culture of the patient's organism (approximately 5 x 105 colony-forming units per milliliter). A colony count should be made t o determine t h e exact number of bacteria in the i n oculum. 1 ML bbbbb t) PATENT'S UNOL. 1:2 1=4 1:11 1:1 CONTROL ^ ^ ^ ^ - M ^. ^ ^ 1 ML D L U E N T P A T E N T ' S O R G A N S M \\ (-10*/ML ) \^J \ f-«a D D O.OSML * TO EACH TUBE NCUBATE b {jti{j UNOL OVERNGHT b > :1 b b 1; «CONTROL Fig. 1. Procedure for determining serum inhibitory and bactericidal levels. Patient's serum is bacteriostatic and 99.9% bactericidal at 1:8 dilution; it is 100% bactericidal at 1:4 dilution. inoculum is reduced by 99.9%; that is, when no more than 50 colonies are growing on the subculture. This technique is illustrated in Fig. 1. Schlichter regarded a m i n i m u m inhibitory level of 1:2 as consistent with effective therapy; however, most physicians prefer to achieve a bactericidal level of at least 1:8. Quantitative Assays n biologic assays, the concentration of antibiotic in the patient's bloodstream is measured by comparing growth inhibition of a test organism by the patient's serum to inhibition of the same organism by a known amount of the antibiotic administered to the patient. A serious error in the results of these tests can occur if the patient is receiving more than one antimicrobial agent. Therefore, it is important t o know before the assay is performed if multiple antibiotics have been administered. f so, the activity of the penicillins and most cephalosporins can be enzymatically degraded o r, alternatively, a micro- d ORGANSM l O- S N O C U L A T E NTO S E R U M D L U T O N S OF PATENT Following overnight incubation at C, all tubes are examined. The bacteriostatic end point is considered t o be the highest dilution of serum at which there is no visible turbidity. To determine the bactericidal end point, 0.1 ml is subcultured f r o m each tube having no visible turbidity onto a blood agar plate. The 100% bactericidal level corresponds to the highest dilution at which no organisms are recovered from subculture after incubation. The 99.9% bactericidal level is the dilution at which the H T\ b bm- K N O W N TO BE S U S C E P T B L E TO A N T B O T C X TUBES WTH S E R A L D L U T O N S OF A N T B O T C X RECEVNG ANTBOTC X NCUBATE NCUBATE b [j\j^u UNOL. 1:2 1:4 1^8 D L U T O N OF P A T E N T ' S 1=16 bbt) bb UO. ANTBOTC X/ML. Fig. 2. Procedure for broth dilution antimicrobial assay. The concentration of the drug in the patient's serum is the MC x the highest inhibitory serum dilution, or 3. x 4 =.48 fig/ml. LABORATORY MEDCNE VOL 7, NO 4 APRL

3 DSK W T H ANTBOTC STANDARD ZONE OF GROWTH NHBTON L A W N OF ORGANSM D S K Fig. 3. Diagram of disk diffusion assay plate. organism susceptible to the assay antibiotic resistant to the others, can be used. but All antibiotics used in these tests should be standard or reference preparations obtained f r o m the various pharmaceutical manufacturers or f r o m U.S.P. Standards, nc. (Rockville, M D ). Standard organisms for performing these assays are available f r o m the American Type Culture Collection (Rockville, M D ), but many laboratories instead use clinical isolates with appropriate antimicrobial susceptibilities. A c o m m o n method for performing quantitative serum assays is the turbidimetric or broth dilution assay. This is perhaps one of the least accurate tests (±100%) because of the errors inherent in the preparation of serial dilutions. Two sets of dilutions are made in broth (usually Mueller-Hinton); one set consists of k n o w n concentrations of the antibiotic in question, and the second, of t w o f o l d serial dilutions of the patient's serum, as in the Schlichter test. The final volume in each tube is 1.0 m l. All tubes are then inoculated w i t h approximately 105 bacteria (0.1 ml of a 1:1000 dilution of an overnight broth culture). After overnight incubation at 35 C, the tubes are examined for the presence or absence of visible turbidity. The least amount of antibiotic that inhibits growth of the test organism is the minimal inhibitory concentration (MC). The highest dilution of the patient's serum at which there is no turbidity corresponds to the M C. By multiplying the M C by the highest inhibitory serum d i l u t i o n, the level of antibiotic present in the patient's serum can be det e r m i n e d. For example, in Fig. 2 the organism was inhibited by 3. /xg of the antibiotic per milliliter and by the 1:4 dilution of the patient's serum. The 1:4 dilution t u b e is therefore presumed to contain 3. /u,g of antibiotic per milliliter, and the concentration of the drug in the patient's serum is 3. x 4 or.48 Mg/ml. Broth dilution tests are not sufficiently accurate to assay serum levels of nephrotoxic antibiotics. 3 2 from LABORATORY MEDCNE VOL. 7, NO. 4 APRL 1976 Downloaded nstead, a disk diffusion technique developed by Sabath et al. 3 is widely used for this purpose. n addition to its greater accuracy (±10%), this test utilizes only small quantities of serum, takes less time to set up, and can be read w i t h i n t w o to five hours. The major components of the test are commercially available either individually (Difco Laboratories, Detroit, M l ) or, for gentamicin assays, as a kit (Serassay, Schering Corporation, Bloomfield, NJ). Basically, this test compares growth inhibition of Bacillus subtilis 6633 by the patient's serum and by standard concentrations of antibiotic. f the patient is receiving a penicillin or cephalosporin antibiotic, the serum is treated before testing with a " b r o a d s p e c t r u m " penicillinase, /^-lactamase. Agar plates containing 5 ml of Difco Antibiotic M e d i u m No. 5 and approximately 107 B. subtilis spores per milliliter are prepared and then stored in plastic bags in the refrigerator for as long as four weeks. Two plates are used for each assay. Twenty microliters each of the patient's serum and of three standard concentrations of antibiotic are pipetted in quadruplicate o n t o V4-inch filter-paper disks. Two sets of disks are placed in clockwise order on each agar plate, w i t h the disk containing the most concentrated antibiotic standard first and the serum disk last. After t w o to three hours of incubation at C, clear zones of growth inhibition appear around the disks (Fig. 3). The diameters of these zones are measured to w i t h i n 0.02 mm w i t h vernier calipers, and the duplicate values for the antibiotic standards and the unknown on each plate are averaged. Two standard curves (one f r o m each plate) are constructed on semilogarithmic graph paper by plotting the concentrations of the antibiotic standards (on the logarithmic scale) against their zones of inhibit i o n. f the zones of inhibition of the serum sample are projected f r o m the abscissa to the standard curve for each plate, the corresponding points on the ordinate will indicate the concentration of antibiotic in the specimen. The average of these values from each curve is the concentration reported to the physician. For example, Fig. 4 represents the curve for a gentamicin assay in which duplicate standards of, 6, and 1.5 /ng of gentamicin per milliliter produced average zone diameters of 15.88,13.89, and m m, respectively, on one plate, and 15.64,13.74, and 11.6 m m, respectively, on the other. The average sizes of the diameters of the zones of inhibition around the serum-containing disks were and 13.1 m m. These correspond to gentamicin concentrations of 3.9 and 4.5 / i g / m l. The mean of these values, 4.2 ^tg/ml, is reported as the serum concentration of the antibiotic. (continued on page 35)

4 (continued from page 32) Although this test has been used primarily to assay gentamicin, at least four other antibiotics can be assayed in the same manner w h e n the appropriate standards are employed. Sabath et al. 3 recommend the f o l l o w i n g concentrations of standards: gentam i c i n,, 6, and 1.5 jug/ml; kanamycin, 27, 9, and 3 /Ag/ml; streptomycin, 25,.5, and 3.1 /xg/ml; neomycin,, 6, and 1.5 /ug/ml; vancomycin, 40, 20, and 10/ug/ml. Some laboratories currently determine the concentration of antibiotics in serum by a similar disk diffusion p r o c e d u r e, except that clinical isolates having appropriate antimicrobial susceptibilities are substituted for B. subtilis Such tests have not been standardized to the same extent as the Sabath assay, but they probably provide more accurate information than broth dilution determinations. The enzymatic assay for gentamicin developed by Smith et al. 4 is more rapid, accurate, and specific than biologic assays. t requires small amounts of serum, can be completed w i t h i n one to two hours, and is unaffected by the presence of other antimicrobial agents, except possibly some of the other aminoglycosides. O n the other h a n d, the enzyme is not commercially available, and the test requires the use of radioactive material and a scintillation counter, to which many laboratories may not have access Uf) o OOO o > STANDARDS 500 _1_ -L G E N T A M C N, JUG/ML Fig. 5. Standard curve for assay of gentamicin by enzymatic technique. 6 < STANDARDS 1.5 ± 1 L 14 J. 16 ZONE DAMETER, M M Fig. 4. Standard curve for assay of gentamicin by disk diffusion technique. in serum in serum n this assay, adenosine monophosphate is transferred f r o m radioactively labeled adenosine t r i phosphate (ATP) to gentamicin by an enzyme f r o m Escherichia coli W677/HJR66, an organism that carries an R factor. The labeled gentamicin has a positive charge that enables it to adsorb onto negatively charged phosphocellulose paper. Excess labeled ATP w i l l not adsorb and can be washed away. Thus any radioactivity measured on the paper will be proportional to the amount of gentamicin in the sample. The test is set up by mixing a ml aliquot of the patient's serum with a " c o c k t a i l " consisting of [ 14 C]ATP, tris buffer, magnesium chloride, d i t h i o t h r e i t o l, and the enzyme gentamicin adenylase. Four or more standards prepared in heat-inactivated, pooled normal human serum are set up in the same manner. The concentration of the standards used is generally 2.5, 5, 7.5, and 10 /xg of gentamicin per milliliter, but higher concentrations can be used if necessary. A negative control of the pooled human serum also is included. After 30 minutes incubation in a 37 C water bath, an aliquot of each reaction LABORATORY MEDCNE VOL. 7, NO. 4 APRL

5 mixture is pipetted o n t o a 2 x 2 cm square of phosphocellulose paper and allowed to be adsorbed. Thepapersquaresare rinsed well in b u f f e r o r running water and dried completely (an infrared lamp speeds up this step). The dried squares are then placed in a vial w i t h scintillation f l u i d, and the radioactivity in each vial is c o u n t e d. The counts per minute of the gentamicin standards are plotted against their concentrations; the concentrations in the serum samples can then be determined f r o m this standard curve (Fig. 5). As m e n t i o n e d, this test is extremely accurate, w i t h an error of less than 5%: w i t h appropriate standards it can be used to assay kanamycin and tobramycin as w e l l. This technique is preferable to the biologic assays, provided that a scintillation counter and the expertise for preparing and standardizing the enzyme are available. Two additional gentamicin assays, a radioimmunoassay5 and a radiometric test, 6 have been described. The radioimmunoassay, in w h i c h tritiated gentamicin competes f o r antibody b i n d i n g sites w i t h unlabeled gentamicin, is said to be rapid (three to four hours) and specific. The radiometric test, which is still under development, is based upon inhibition by gentamicin of urease synthesis in Proteus species. Using [ 14 C]urea as the substrate for the enzyme, the 1 4 C 0 2 released in both the presence and the absence of gentamicin New (2nd) is measured by the BACTEC instrument. f perfected, this technique could be useful in those laboratories that already use the BACTEC for detection of positive b l o o d cultures. Antimicrobial therapy is becoming increasingly complex as more resistant organisms emerge as significant pathogens. Proper treatment often necessitates close m o n i t o r i n g of b l o o d levels of antibiotics in the patient. Although not all laboratories have the capability to perform the more complex tests described here, the Schlichter test and the disk diffusion assay are sufficiently simple to warrant their implementation. References 1. Schlichter J G, MacLean H: A method of determining the effective therapeutic level in the treatment of subacute bacterial endocarditis with penicillin. Am Heart J 34: Klastersky J et al: Antibacterial activity in serum and urine as a therapeutic guide in bacterial infections. J nfect Dis 9:187, Sabath L D, et al: Rapid microassay of gentamicin, kanamycin, neomycin, streptomycin, and vancomycin in serum or plasma. J Lab Clin Med 78:457, Smith D H, VanOtto B, Smith A L: A rapid chemical assay for gentamicin, N Engl J Med 286:583, Lewis J E, Nelson J C, Elder H A: Radioimmunoassay of an antibiotic: Gentamicin. Nature [New Biol] 239:214, Buda D, Broman R: Radiometric assay of gentamicin in blood by measurement of bacterial evolution of 14 C0 2 from 14C-Urea, Abstracts of the Annual Meeting of the American Society of Microbiology, p. 31 Edition!f TECHNCAL HEMATOLOGY By ARTHUR SMMONS, L.C.S.L.T. The major changes occurring in laboratory hematology since Technical Hematology was first published have necessitated a New (2nd) Edition of this combination text and laboratory manual. Ranging from classical procedures to the very latest in automated methods, the text covers all technical procedures used in a hematology and blood banking laboratory b o t h routine and specialized. J. B. LPPNCOTT COMPANY East Washington Square, Phi a., Pa Please send me: For this edition the author has a d d e d the new tests currently in use. He has expanded the blood bank section to five chapters, completely revising and updating the methods described and adding theoretical aspects to make the technique more understandable. You'll find step-by-step descriptions of techniques, the rationale of the technique for each test, the pitfalls and limitations. All alternate techniques currently in use are described for each test. You'll find all the latest automated equipment used in hematologic determinations fully described and illustrated. 476 pages 67 illustrations Second Edition, tables $21.50 $21.50 Name Address. City State_ At the conclusion of each chapter a brief clinical section helps your understanding of the what and why in addition to the how of each procedure. Technical Hematology Zip. Payment Enclosed (Save postage & handling) D Charge and Bill me (Plus postage & handling) Also available at your medical bookstore