The study subjects included 161 HCW and 86 non-atopic healthy controls that were recruited from a single tertiary hospital and the general population. The HCW consisted of 138 nurses and 23 pharmacists. The nurses were divided into the following two groups: group I included 125 nurses working in wards where they were exposed to cephalosporins (current-exposure group) and group II included 13 nurses working in outpatient departments or in offices, who had a prior history of working in general wards at least six months before the study (previously-exposed group). The pharmacists-who were employed in the hospital pharmacy-might have been exposed to cephalosporins. The control subjects were healthy volunteers who had no history of allergic diseases or antibiotic allergy. Informed consent was obtained from each subject, and the study protocol was approved by the Institutional Review Board of the hospital (AJIRB-GEN-SUR-09-195).

The questionnaire was a modification of that used in the International Study on Asthma and Allergies in Childhood (ISAAC),14 which included workplace, use of protective equipment when handling cephalosporins, smoking status, duration of employment, personal history of allergic diseases, presence of work-related symptoms, and duration of the latent period. Personal history of allergic diseases included asthma, rhinitis, atopic dermatitis, or any kind of antibiotic allergy. Work-related symptoms were defined as those that developed after handling any cephalosporin and consisted of upper and/or lower respiratory symptoms, cutaneous symptoms, and anaphylaxis. Upper and/or lower respiratory symptoms included nasal itching, runny nose, sneezing, nasal congestion, cough, sputum, shortness of breath, or wheezing, and cutaneous symptoms included urticaria or dermatitis.

The most commonly prescribed intravenous cephalosporins, i.e., cefotiam, ceftriaxone, and ceftizoxime, were selected for use in this study. Cefotiam was purchased from CJ Pharmaceutical Co., Ltd (Seoul, Korea), ceftriaxone from Hanmi Pharmaceutical Co., Ltd. (Seoul, Korea), and ceftizoxime from Dong-A Pharmaceutical Co., Ltd. (Seoul, Korea).

HCW and healthy controls were tested by SPTs using common aeroallergens and the three cephalosporins. The aeroallergens included Dermatophagoides farinae, tree pollens, weed pollens, Japanese hops, grass pollens, and cat dander (Bencard, Brentford, UK). The concentration of each cephalosporin solution used was 10 mg/mL. Saline and 1 mg/mL histamine were used as negative and positive controls, respectively. Atopy was defined as those subjects who showed positive responses to at least one common inhalant allergen on SPT.

Serum total IgE antibody levels were measured using an ImmunoCAP System (Phadia, Uppsala, Sweden). Serum specific IgE antibodies to cephalosporins were detected by capture enzyme-linked immunosorbent assay (ELISA) using cephalosporin-HSA conjugates. Three cephalosporin-HSA conjugates (cefotiam, ceftriaxone, and ceftizoxime) were prepared according to methods described previously.3,10,15

Cephalosporin-HSA conjugates or mock-HSA conjugate were dissolved in phosphate buffered saline (PBS, pH 7.4) at 10 µg/mL. This solution (100 µL) was coated onto 96-well ELISA microplates (Corning, New York, NY, USA) for two hours at 37℃ and the plates were placed at 4℃ overnight. After washing four times with 350 µL PBS containing 0.05% Tween 20 (PBS-T; Sigma-Aldrich, St. Louis, MO, USA), unoccupied antigen binding sites on the plate were blocked using 200 µL of blocking buffer that contained 10% fetal bovine serum (FBS; GIBCO; Invitrogen, Carlsbad, CA, USA) in PBS at 37℃ for two hours. The plates were again washed four times with PBS-T, and 50 µL of three-fold diluted serum samples in blocking buffer were added to the wells, and the plates were incubated for two hours at room temperature with shaking. The plates were washed four times with PBS-T, and 100 µL of goat anti-human IgE antibody (Kirkegaard and Perry Laboratories, Inc., Gaithersburg, MD, USA), diluted 1:1,000 v/v in 10% FBS-PBS, was added to each well. The plates were then incubated for one hour at room temperature, followed by washing four times with PBS-T. A total of 100 µL of alkaline phosphatase-conjugated rabbit anti-goat IgG antibody (ReserveAP; Kirkegaard and Perry Laboratories, Inc.) diluted 1:500 v/v in 10% FBS-PBS was added to each well, and plates were again incubated for one hour at room temperature. PNPP (100 µL; p-nitro-phenyl phosphate; Sigma-Aldrich) was added as the substrate. The reaction was stopped by the addition of 100 µL of 1 N NaOH. Optical densities were measured at 405 nm on a microplate reader (Synergy HT; Bio Tek Instruments, Inc., Winooski, VT, USA). The final absorbance values of samples tested using cephalosporin-HSA conjugates were determined by subtracting the absorbance values of the same samples tested on mock-HSA conjugate. Values were then multiplied by 1,000. Positive cutoff values for the ELISAs were determined from the mean plus three times the standard deviation of the absorbance values of the non-atopic healthy controls (n=86).

Binding specificities were confirmed by an ELISA inhibition test. Increasing amounts (1-100 µg/mL) of free and conjugated cephalosporins and HSA alone were incubated overnight with 50 µL of three-fold diluted serum samples from subjects who had high levels of specific IgE antibodies to any cephalosporin-HSA conjugate. These mixtures were subjected to ELISAs, as described previously. The percentage of inhibition was calculated as follows: % inhibition=100×(1-[absorbance with inhibitor/absorbance without inhibitor]).

All statistical analyses were performed using SPSS ver. 12.0 (SPSS Inc., Chicago, IL, USA). The χ² test, Fisher's exact test, and one-way analysis of variance (ANOVA) followed by a post-hoc Bonferroni correction, were used to compare data between the three study groups. Cross-tabulation analysis and independent t-tests were used to evaluate associations between the characteristics of study subjects, work-related symptoms, SPT results, and specific IgE antibodies to cephalosporins. Multivariate logistic regression analysis was used to evaluate the risk factors for work-related symptoms, positive SPTs, and presence of serum specific IgE antibody to cephalosporin-HSA conjugates. A value of P<0.05 was considered statistically significant.

The number of male subjects was significantly higher in the healthy controls (25/86; 29.1%) than in HCW (1/161; 0.6%). The mean age was significantly younger in the healthy controls (22.2±3.2 years) than in HCW (27.4±4.3 years). Among HCW, the mean age was significantly greater in group II nurses (32.1±3.5 years) than in group I nurses (26.8±4.2 years) or pharmacists (27.6±3.6 years). No subject used any protective equipment when handling cephalosporins. All subjects were non-smokers. The duration of employment was also significantly longer in group II nurses (298±93 months) than in group I nurses (151±127 months) or pharmacists (133±135 months). The personal histories of allergic diseases or antibiotic allergies in the three groups did not differ significantly (Table 1).

The overall prevalence of work-related symptoms associated with cephalosporin exposure in HCW was 17.4%. The most common symptoms were upper and/or lower respiratory (14.4%), followed by cutaneous (4.3%) and anaphylaxis (0.6%). The prevalence of work-related symptoms and the duration of latent periods of HCW did not differ significantly between the three groups (Table 1). There were no significant associations between the presence of cephalosporin-associated work-related symptoms and the results of SPT or serum specific IgE antibodies to any cephalosporin (data not shown).

There was no significant difference in the atopy rate between the three groups of HCW. SPT using cephalosporins demonstrated that five subjects (3.1%) had positive responses to ceftriaxone, which was the only cephalosporin that elicited positive SPT responses. There was no significant difference in the prevalence of positive SPT responses between the three groups. There was no significant difference in atopy rate between positive and negative groups on SPT to the cephalosporins (60% and 38.5% respectively, Fisher's exact test).

The mean serum total IgE level was 158±433 kU/L, and there was no significant difference in serum total IgE levels among the three groups (Table 1). The serum specific IgE levels to the three cephalosporin-HSA conjugates are shown in Fig. 1. The prevalence of detection of specific IgE antibodies to cephalosporin-HSA conjugates were 17.4% for any cephalosporin, 10.4% for cefotiam, 6.8% for ceftriaxone, and 3.7% for ceftizoxime (Table 1). There was no significant difference in the atopy rate between the positive and negative groups for specific IgE to the cephalosporin-HSA conjugates. The prevalence of specific IgE to cefotiam tended to be higher in group I than in group II, but no significant difference was found in the prevalence of specific IgE to cefotiam and ceftriaxone-HSA conjugates among the three groups. The prevalence of serum specific IgE to ceftizoxime-HSA conjugate was significantly higher in pharmacists (12.0%) than in groups I or II (1.6 and 7.7%, respectively; Fisher's exact test). ELISA inhibition tests of the three cephalosporin-HSA conjugates demonstrated significant dose-dependent inhibition after addition of both free and conjugated forms of the corresponding cephalosporins (Fig. 2).

Based on a multivariate logistic regression analysis, a personal history of antibiotic allergy was a significant risk factor for the presence of cephalosporin-associated work-related symptoms (OR, 24.93; 95% CI, 2.61-238, Table 2), but not for the presence of serum specific IgE antibodies to cephalosporins (OR, 0.9; 95% CI, 0.18-4.53, data not shown). Additionally, a personal history of atopic dermatitis was a significant risk factor for the presence of serum specific IgE antibody to cefotiam-HSA conjugate (OR, 6.30; 95% CI, 1.23-32.3, Table 3), while no risk factor was significantly associated with the other cephalosporin-HSA conjugates or positive cephalosporin SPT results (data not shown).