Evaluation of the Bactec serum gentamicin assay

Abstract

A radiometric assay for gentamicin was compared with an established radioimmunoassay protocol. The coefficients of variation for within-run, day-to-day, and overall runs were consistently higher for the radiometric method as compared with the radioimmunoassay. The coefficient of correlation for 84 patient sera tested by the two methods for gentamicin levels was 0.82. Though less precise, the radiometric method was felt to be an acceptable means of determining gentamicin levels in laboratories where the radioimmunoassay is unavailable.Gentamicin has established itself as an excellent antimicrobial agent for the treatment of lifethreatening gram-negative infections. The range between therapeutic efficacy and toxicity for gentamicin, however, is narrow, and serum levels must be carefully monitored. The classical procedures for determining adequate antibiotic therapy require 1 to 2 days for results and are fraught with significant error. During the past several years a variety of methods have been developed to determine gentamicin levels in serum and other body fluids by more rapid and sensitive methods. Such methods include microbiological assays (1,7,11,14,16,17,21), enzymatic assays (4,5,13,19,20; R. V. Case, Natl. Meet. Am. Assoc. Clin. Chem., 28th, Houston, Tex., abstr no. 000, 1976), and radioimmunoassays (RIAs) (10,12,13).A rapid radiometric technique for determining serum gentamicin levels has been developed by Johnston Laboratories with their Bactec instrument. The system was originally developed for routine monitoring of blood cultures and has been described elsewhere (2,3,6,9,15,18). The instrument has also been adapted for definitive identification of Neisseria gonorrhoeae and N. meningitidis and is currently being developed for rapid identification and antibiotic susceptibility patterns of Mycobacterium sp. and the determination of certain aminoglycoside levels in patient sera.The principle of the Bactec procedure is based on the fact that gentamicin inhibits the synthesis of urease, an inducible enzyme produced by a species of Proteus. The amount of urease produced is measured indirectly by the amount of "4CO2 released. The quantity of gentamicin in a sample is inversely proportional to the volume of 04CO2 detected by the instrument. The sensitivity range for this assay, according to the manufacturer, is from <1 to 14 ,ug of gentamicin per mi.This study evaluated Johnston Laboratories' gentamicin kit by using the Bactec model 460 to determine gentamicin levels in patient sera as compared with the Monitor Science RIA procedure used routinely in our laboratory. MATERIALS AND METHODSGentamicin test solutions. Two gentamicin test solutions were prepared, containing a low level of gentamicin (3.5 1g/ml) and a high level of gentamicin (10.5 ,ug/ml). The two gentamicin test solutions were prepared by diluting with sterile deionized water a gentamicin (14.0,ug/ml) standard in water provided by the manufacturer. The two test solutions were then aliquoted into 10 vials each and stored frozen at -20°C till assaye...