Evaluation of a Bioassay Method for Serum Amikacin Concentrations

Transcription

1 ANNALS OF CLINICAL AND LABORATORY SCIENCE, Vol. 11, No. 5 Copyright 1981, Institute for Clinical Science, Inc. Evaluation of a Bioassay Method for Serum Amikacin Concentrations JOHN P. MANOS, M.D.,* ARTHUR J. BOTTING, M.D.,f NICHOLAS M. BURDASH, Ph.D.,* META H. MASON, MT(ASCP),* and MARCIA E. WEST, SM(ASCP)* *Department o f Laboratory Medicine, Medical University o f South Carolina, Charleston, SC and \Fish Memorial Hospital, New Smyrna Beach, FL ABSTRACT The bioassay system from American Diagnostic for amikacin was compared to the same company s radioimmunoassay (RIA) technique used in this hospital. The bioassay is performed by adding serum from a patient to wells cut in agar which have been seeded with a susceptible fast growing microorganism (Enterobaeter). After incubation of five hours at 37, the zones of inhibition for four standards are measured and plotted. The amount of amikacin in the serum sample is then determined from the linear graph. Ten replicates of a serum with 8 fx,g per ml of amikacin and 10 replicates of a serum with 20 (jlg per ml of amikacin were assayed on each of four days. Aliquots of the same samples were assayed in a similar manner by RIA. The coefficients of variation (CVs) of the within run assays by the bioassay and RIA methods for the low amikacin test sample ranged from 4.7 to 7.2 percent and 6.2 to 13.0 percent, respectively. The within run CVs for the high amikacin test sample for the bioassay and RIA methods ranged from 5.2 to 6.9 percent and 5.0 to 13.9 percent, respectively. The day to day and overall CVs for the bioassay and RIA methods for the low amikacin test sample were 1.5 and 5.8 percent and 7.4 and 11.8 percent, respectively. F or the high amikacin test sample, they were 0.4 and 6.0 percent and 4.0 and 9.6 percent, respectively. The correlation coefficient for 49 sera from patients on amikacin was 94 percent between the two methods. Introduction The use of aminoglycoside antibiotics in serious or life threatening Gram negative infections has afforded the clinician with a potent tool. Despite their efficacy, however, resistant organisms continue to em erge to the commonly used am inoglycosides, gentamicin and tobramycin. Amikacin, a semisynthetic derivative of kanamycin, has an advantage over the other aminoglycosides in that organisms resistant to gentamicin and tobramycin /81/ $00.90 Institute for Clinical Science, Inc.

2 412 MANOS, BOTTING, BURDASH, MASON, AND WEST are often susceptible to amikacin at system ically obtainable blood levels.3' 9 Amikacin exhibits a high degree of resistance to aminoglycoside inactivating enzymes in that only one of the nine characterized bacterial enzymes is capable of inactivating amikacin.6 Like gentamicin and tobramycin, however, amikacin is potentially ototoxic and nephrotoxic.1,7 Because of the rather narrow range between the therapeutic and toxic blood levels, rapid m ethodologies for m onitoring serum levels is imperative, especially in patients with renal impairment. A variety of methods have been developed for monitoring amikacin including bioassays,5 enzymatic assays,2'8 and radioim m unoassay (RIA).4 The availability of sophisticated instrumentation for RIA is not always available, and simple methodologies within the scope of bacteriology lab o rato ries are often ad vantageous. This paper reports on the evaluation of a bioassay procedure for amikacin* and compared to the same company s amikacin radioimmunoassay procedure. Materials and Methods P r i n c i p l e The bioassay procedure utilizes an agar well diffusion technique. The zones of inhibition of microbial growth around wells containing known amounts of amikacin are measured and plotted on semi-log paper. The linear graph resulting is then used to calculate unknow n serum concentrations. R e a g e n t s The kit supplies the bacterium Enterobacter aerogenes, agar based m i crobial growth medium in screw capped tubes (18 cc per tube), saline for dilutions, * Supplied by American Diagnostics, N ewport Beach, CA. four amikacin standards (5, 10, 20 and 40 fjug per ml), square plates, a zone reader, a well borer, and semi-log work sheets. A p p a r a t u s Additional equipment needed by the laboratory includes an aspirating system, a 20 fil pipet, a 46 to 49 water bath, and an incubator. P r o c e d u r e The bacterial growth medium is melted in boiling water and cooled in a 46 to 49 water bath. Saline (6.5 ml) is added to the vial containing the lyophilized bacteria and swirled to resuspend all of the powder. One ml of the reconstituted bacterial suspension is added to each tube of cooled bacterial medium and mixed thoroughly. The seeded medium is then poured onto a leveled square plate and allowed to solidify. The plate may be used immediately or refrigerated for up to two weeks in a sealed plastic bag containing a moist sponge. The plate is placed over the template provided and 16 wells are cut as indicated. The wells are labeled as shown in the kit brochure, and 20 (jl1 of the standards and test sera are dispensed into the appropriate wells. The standards and test sera are all done in duplicate on each plate. The plate is then incubated at 37 for four to six hrs and the zones of inhibition of the four standards are measured to the nearest 0.1 mm. The zone diameters of each duplicate standard are averaged and plotted on semi-log paper against the known concentrations of the standards. A curve is constructed from the four plots, and the amikacin concentrations of the unknown serum samples are calculated from this curve. To determ ine the precision and repeatability of the bioassay procedure, two test samples were prepared in normal serum containing approximately 8 fig and 20 /Ltg of amikacin per ml. Several aliquots of the two test samples were dispensed

3 BIOASSAY METHOD FOR SERUM AMIKACIN CONCENTRATIONS 413 and stored at -20 until assayed. Each test serum was run 10 times on four different days. These same test sera were also run in an identical manner by the amikacin RIA procedure of American Diagnostics for comparison. The latter methodology employs the principle of competitive protein binding, and the resulting precipitate is counted in an automatic gamma radiation counter according to the manufactu rer s instructions. For com parative studies, 49 serum samples from patients on amikacin therapy were assayed by both procedures. Results In table I are summarized the range of the assays and the coefficients of variation (CVs) calculated from the assays p erformed on the low and high test samples by the bioassay and RIA methods for within runs, day to day, and overall laboratory variations. For the low amikacin test sample, the within run CVs of the bioassay method run on four separate days with 10 replicates each day ranged from 4.7 to 7.2 percent and for the RIA method 6.2 to 13.0 percent. The day to day CV for the bioassay method was 1.5 percent and for RIA 7.4 percent. The overall variation for the bioassay method (40 total replicates) was 5.8 percent and for the RIA method was 11.8 percent. In table I are shown similar data for the high amikacin test sample. The within run CVs for the bioassay method varied from 5.2 to 6.9 percent and for the RIA method from 5.0 to 13.9 percent. The day to day CV for the bioassay method was 0.4 percent and 4.0 percent for RIA. The overall total laboratory variation was 6.0 percent for the bioassay method and 9.6 percent for RIA. In figure 1 is shown the distribution of amikacin levels of 49 serum samples from patients on amikacin therapy as d etermined by the bioassay and RIA procedures. Analysis of the scattergram shows a correlation coefficient (r) of The linear regression line determined by the method of least squares is Y = 1.27X 3.1. Discussion The CVs for both the bioassay and RIA procedures for the within run comparison were all within acceptable limits with one exception. For unexplained reasons, the CVs calculated for the RIA methodology performed on day 2 were elevated somewhat over those run on days 1, 3, and 4, as is shown by the higher CV of 13.0 percent for the low amikacin test sample and 13.9 percent for the high amikacin test sample. If the second day runs by RIA are eliminated, the w ithin run CVs for both methods range from 4.7 to 7.4 percent, all within acceptable ranges. Variations for the day to day and total laboratory were lower by the bioassay method than by the RIA procedure for both the low and high TABLE I Coefficients of Variation of Serum Amikacin Assays Amikacin Level Within Run No. No. of Range of Range of No. of Repli- Assays CVs of Runs cates (ug/ml) (Percent) Runs Day to Day Range of Assays (vg/ml) CV (Percent) Methodology Total Laboratory Total CV Repli- (Percates cent) Low (8.0 yg/ml) Bioassay RIA High (20.0 Bioassay Vig/ml) RIA

4 414 MANOS, BOTTING, BURDASH, MASON, AND WEST amikacin test samples. This is true even if one calculated the CVs with the elimination of the runs on day 2 for RIA. The overall mean for the low amikacin test sample was higher than the expected value by the bioassay method and lower than the expected value by the RIA procedure. The reverse was true for the high amikacin test sample. The overall means by both methodologies, however, fell between 6 and 11 percent of the expected values. Recovery studies performed on combinations of two samples containing 10 and 25 ig of amikacin per ml resulted in recoveries greater than the expected value by both methodologies, although the values calculated by the RIA methodology were closer to the expected values than were those by the bioassay methodology. The design of the experiments for both procedures is such that the precision and repeatability of the procedures can be determined but the accuracy of these procedures cannot be measured since there was no predetermined mechanism by which the true or actual values could be calculated. Thus, one could only relate to expected values. Analysis of the CVs, however, indicates that both procedures are essentially comparable and precise. They are both reliable in determining serum amikacin levels, although the bioassay method was somewhat more precise than the radioimmunoassay method. The precision of the bioassay method, however, depends on the technologist performing very precisely the various steps in the procedures. Specific cautions are noted. It is important to cut the wells as circular and perpendicular as possible with all of the agar from the wells removed from the bottom of the plastic plate. Each plate must have its own duplicate set of four standards. If standards of one plate are used to calculate concentrations of unknowns from another plate, the technique becomes significantly less precise, even if both plates are run simultaneously. Al- B IO -A S S A Y IJ I9 /m l F ig u r e 1. Comparison of am ikacin levels in sera from 49 patients on amikacin therapy by bioassay and radioimmunoassay (RIA). The correlation coefficient (r) is The linear regression line (broken line) determ ined by the m ethod of least squares is Y = 1.27X 3.1. The solid line represents the line of equality. though the kit includes a plastic zone reader, it is better to use a hand lens, as recom m ended by the m anufacturer, which magnifies the zones so that they can be m easured more accurately to the nearest 0.1 mm. Another point of significance is that the tem perature of the melted agar medium should be kept as close as possible to 49. As the medium approaches 46, it tends to solidify and the plate will contain lumps and thus be uneven. On the other hand, allowing the temperature of the medium to go over 49 may destroy the suspended organisms. It is also extremely important to have the plate level so that the agar will be of uniform thickness throughout. Wells should not be cut in plates to be stored but should be cut just prior to use. Finally, the accuracy of the 20 fjd pipet should be within ± 1 percent. In comparison of the bioassay and RIA determ inations of the 49 p atien t sera

5 BIOASSAY METHOD FOR SERUM AMIKACIN CONCENTRATIONS 415 (figure 1), higher concentrations by the one method over the other and vice versa were about equal. In only four instances were toxic concentrations (>25 fig per ml) reported by one m ethod and w ithin therapeutic concentrations by the other. This occurred with two sera for each m ethod. N ine sera had toxic concentrations by both methods and the remaining 36 samples were below toxic concentrations by both methods. Concentrations below 5 fig or above 40 fig per ml, the normal range of the procedure, can be extrapolated with care provided the zones are readable. With concentrations significantly higher than 40 fig per ml, it is probably safer to repeat the bioassay by diluting the serum sample twofold, calculating the am ikacin concentration and then doubling the value. The amikacin concentrations determ in ed by the bioassay method represent only concentrations of amikacin, since the results are unaffected by the presence of other antibiotics in the patient sera. The organism used in the bioassay (E. aerogenes) is susceptible only to the aminoglycosides. The high correlation coefficient (0.94) also suggests this since the bioassay values correlated well with the RIA values; the latter values, by virtue of the methodology, are measures of amikacin even in the presence of other antibiotics. Summary The bioassay method in this study was somewhat more precise than the RIA procedure. The needed additional equipment is available in most microbiology laboratories. The bioassay procedure is somewhat longer than the RIA procedure in that it takes between six and eight hrs to perform including approximately two hrs of technical time. The RIA procedure can be performed within two hrs requiring approximately one hr of technical time. However, the bioassay method does not require expensive equipment, does not use radioactive materials, and has a significantly longer shelf life. Cost per serum specimen is about the same for both procedures. Shorter methodologies, such as RIA, are certainly preferred for determ ining serum antibiotic concentrations. However, in hospital laboratories where such equipm ent is lacking, the longer bioassay method does afford the clinician w ith relevant inform ation that m ight otherwise be totally unavailable. References 1. Cox, C. E.: Amikacin therapy of urinary tract infections. J. Infect. Dis. 134(Suppl.):S362- S368, H a ss, M. J. and D a v ie s, J.: Enzymatic acétylation as a means of determ ining serum aminoglycoside concentrations. Amimicrob. Agents Chemother. 4: , L e F ro c k, J. L., Sze y k o, G. H., Be c h a r d, D. L., and T il l o t s o n, J. R.: Clinical effectiveness of amikacin in Gram-negative infections. Curr. Ther. Res. 25: , L e w is, J. E., N e l s o n, J. C., and E l d e r, H. A.: A m ikacin: a rap id and sen sitiv e rad io im munoassay. Antimicrob. Agents Chem other. 7:42-45, Ma r e n g o, P. B., Wil k in s, J., and O v e r t u r f, G. D.: Rapid, specific microbiological assays for am ikacin (BB-K8). A ntim icrob. A gents Chem other. 6: , SCHIFFMAN, D. O.: E v a lu a tio n o f a m ik a c in s u l fate (am ik in ). J. A m er. M e d. A ssoc. 238: , Sm it h, C. R., Ba u g h m a n, K. L., E d w a r d s, C. Q., R o g e r s, J. F., and L ie t m a n, P. S.: C ontrolled comparison of amikacin and gentamicin. New Eng. J. Med. 296: , St e v e n s, P., Yo u n g, L. S., and H e w it t, W. L.: Im proved acetylating radioenzymatic assay of amikacin, tobramycin, and sisomicin in serum. A ntim icrob. A gents C hem other. 7: , T a l l y, P. F. and G o r b a c h, S. L.: Review of 152 patients w ith bacterem ias treated w ith amikacin. Amer. J. Med. 62: , 1977.