Immunoassay Analysis of Kanamycin in Serum Using the Tobramycin Kit.

Kanamycin is one of the aminoglycosides used in the treatment of multidrug-resistant tuberculosis. Blood concentrations of kanamycin are predictive for the treatment efficacy and the occurrence of side effects, and dose adjustments can be needed to optimize therapy. However, an immunoassay method for the quantification of kanamycin is not commercially available. We modified the existing tobramycin immunoassay to analyze kanamycin. This modified method was tested in a concentration range of 0.3 to 80.0 mg/liter for inaccuracy and imprecision. In addition, the analytical results of the immunoassay method were compared to those obtained by a liquid chromatography-tandem mass spectrometry (LC-MS/MS) analytical method using Passing and Bablok regression. Within-day imprecision varied from 2.3 to 13.3%, and between-day imprecision ranged from 0.0 to 11.3%. The inaccuracy ranged from -5.2 to 7.6%. No significant cross-reactivity with other antimicrobials and antiviral agents was observed. The results of the modified immunoassay method were comparable with the LC-MS/MS analytical outcome. This new immunoassay method enables laboratories to perform therapeutic drug monitoring of kanamycin without the need for complex and expensive LC-MS/MS equipment.

Fixed exanthema from systemic tobramycin.

Eye drops contain several ophthalmic medications which can produce allergic reactions. We report the case of a patient with contact dermatitis from neomycin and a probable fixed exanthema after parenteral administration of tobramycin who tolerated topical tobramycin and other aminoglycosides.

Aminoglycoside detection using a universal ELISA binding procedure onto polystyrene microtiter plates in comparison with HPLC analysis and microbiological agar-diffusion assay.

The use of enzyme-linked immunosorbent assay for the detection of aminoglycosides has been hindered due to low molecular weight compound adsorption to solid phases. Here, we describe an enzyme-linked immunosorbent assay based on the treatment of polystyrene microtiter plates with Alcian blue prepared in acetic acid prior to coating with the antibiotic. Whereas no detection of tobramycin was possible on commercially treated or untreated enzyme-linked immunosorbent assay plates, the Alcian blue treatment permitted detection of 0.025 and 0.05 microg ml(-1) of tobramycin respectively using 0.05 and 0.1% of Alcian blue with a coefficient of variation of 1.85 and 7.69%, respectively. Comparative studies of five tobramycin samples of unknown quantity using enzyme-linked immunosorbent assay and high-performance liquid chromatography gave equivalent results while those done via microbiological agar-diffusion assay were an overestimation of the actual quantity. The use of the Alcian blue pretreatment enzyme-linked immunosorbent assay procedure has permitted, in previous studies, the measure of antibodies against synthetic peptides and phospholipids. Subsequently, our demonstration of the sensitivity and reliability of this method in the quantification of tobramycin strongly suggests that the use of Alcian blue pretreatment in enzyme-linked immunosorbent assay can be applied universally to avert molecule immobilization problems on solid phases.

Sensitization to aztreonam and cross-reactivity with other beta-lactam antibiotics in high-risk patients with cystic fibrosis.

The immunogenicity, allergenicity, and cross-reactivity of aztreonam were investigated in 21 patients with cystic fibrosis (CF) (aged 5 to 39 years) with well-documented histories of allergic systemic reactions (SRs) to penicillin and/or cephalosporin antipseudomonal beta-lactam antibiotics (BLAs). Skin tests (STs) with penicilloyl-polylysine (PPL), penicillin minor determinant mixture, and antipseudomonal BLA were positive in 19 patients (90%). The BLA causing the most recent allergic reaction, minor determinant mixture, or PPL, was positive in 89%, 53%, and 32% of ST-positive patients, respectively. Serum PPL-specific IgE antibodies were not detectable, although PPL-specific IgG antibodies were found in 64% of patients tested. STs to aztreonam reagents were performed and were initially negative in 20 patients. One patient was ST positive to the polylysine conjugate of hydrolyzed aztreonam (SQ 27629), despite no prior exposure to aztreonam, and was not treated. Of 20 patients treated with aztreonam, four were demonstrated to be sensitized by exposure (one had an SR during initial treatment course, two had SRs on reexposure, and one patient was asymptomatic after intravenous desensitization) by positive aztreonam reagent skin responses on repeat testing. Aztreonyl-specific IgE and IgG serum antibodies were not detected in any patients, including patients with allergic reactions to aztreonam. Thus, aztreonam is generally well tolerated in high-risk patients with CF allergic to other BLAs and appears to have reduced immunogenicity by serologic testing. However, caution should be exercised with aztreonam in BLA-allergic patients with CF in light of 5% preexisting ST cross-reactivity and 20% sensitization rates found in this study.

Acute desensitization of a patient with cystic fibrosis allergic to both beta-lactam and aminoglycoside antibiotics.

A 15-year-old patient with cystic fibrosis developed urticarial reactions to tobramycin, gentamicin, and cephoperazone, and an anaphylactic reaction to ticarcillin during therapy for an extensive pulmonary infection with Pseudomonas aeruginosa and Staphylococcus aureus. Immediate wheal-and-flare skin tests were positive with tobramycin and with penicilloylpoly-L-lysine. Desensitization with tobramycin in gradually increasing intravenous doses was accomplished during 8 hours. The procedure was complicated by a macular rash that remitted within minutes without therapy, but no symptoms or signs of an allergic reaction to tobramycin were detected during full dose therapy. Skin test responses to tobramycin became negative by the end of the desensitization procedure, whereas the responses to penicilloylpoly-L-lysine and histamine remained positive. A worsening course led to an unsuccessful attempt to desensitize the patient to beta-lactam determinants. Wheezing appeared during the administration of oral doses. This case demonstrates the feasibility of acute, antigen-specific desensitization of an aminoglycoside-allergic patient and the failure to achieve a second, simultaneous desensitization. This patient experienced the first serious reaction to oral penicillin desensitization.

An evaluation of the homogeneous substrate-labeled fluorescent immunoassay for tobramycin in serum.

The homogeneous substrate-labeled fluorescent immunoassay for tobramycin in serum was evaluated. Standard curves are linear and reproducible. The within-run coefficient of variation for control samples containing low, medium and high concentrations of tobramycin ranged from 1.9 to 6.5%; the between-run coefficient of variation was 11% for a control sample at low concentration. The accuracy measured on spiked samples was good and comparable to the accuracy measured by a microbiological assay; it was somewhat better than for the radioimmunoassay. For patient samples, the results with this method correlated well (r - 0.914) with results obtained by radioimmunoassay.

Comparative evaluation of three methods for measuring gentamicin and tobramycin in serum.

Three procedures (radioimmunoassay, fluorescence immunoassay, and enzyme immunoassay) for the determination of gentamicin and tobramycin levels were compared. These systems were evaluated on the basis of accuracy, reproducibility, specificity, and cost. All three systems showed a high degree of accuracy and precision. The fluorescence immunoassay gave significantly lower values for gentamicin and tobramycin at levels below 5 microgram/ml. Of the three systems, the radioimmunoassay was the least expensive, provided that a minimum of 10 analyses were performed daily; single tests required more technical time and were more costly. Costs of single tests by fluorescence immunoassay and enzyme immunoassay systems were similar. The enzyme immunoassay system performed equally well for both gentamicin and tobramycin in giving rapid and accurate results.

An enzyme immunoassay for serum tobramycin.

A heterogeneous enzyme immunoassay for serum tobramycin was developed using tobramycin-beta-D-galactosidase conjugate as the labelled antigen and 4-methylumbelliferyl-beta-D-galactoside as the fluorescence substrate. B/F separation was carried out using an immobilized second antibody, Immunobead. The minimal detectable level was 10 pg/tube or 0.2 microgram/ml of serum. Assay recovery was excellent and the intra-assay and inter-assay variances were 4.41% and 12.6%, respectively. The values from the enzyme immunoassay agreed well with those from radioimmunoassay and bioassay. This assay system is highly sensitive and can be easily applied to other drugs and hormones.