a Janssen Research Foundation, B2340 Beerse, Belgium; b Hôpital de l' Institut Pasteur, 75724 Paris, France; c Fungus Testing Laboratory, The University of Texas Health Science Center, San Antonio, TX 78284-7750; d Division of Infectious Diseases, Santa Clara Valley Medical Center and Stanford University Medical School, San Jose, CA 95128-2699, USA; e Mycology Reference Laboratory, Public Health Laboratory Service, Bristol BS2 8EL, UK
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Abstract |
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Introduction |
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No standardized method for itraconazole bioassays exists, and laboratories differ in the experimental conditions and indicator organisms they use. The considerable variation in reported ratios of bioassay:HPLC levels suggests that interlaboratory reproducibility in bioassay test results may be less than ideal. We therefore undertook a collaborative comparison of itraconazole plus hydroxy-itraconazole level determinations in bioassays done with a standard panel of serum samples.
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Materials and methods |
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The design of the second phase of the study matched that of the first, except that 15 new serum samples were all spiked with itraconazole, hydroxy-itraconazole, or both, and the total (itraconazole + hydroxy-itraconazole) concentrations were chosen to cover the range 0.38- 9.5 mg/L. Each spiked serum pool was again divided into two randomly coded aliquots. The bioassays were run as before except that results were generated with both itraconazole and hydroxy-itraconazole standard curves.
The bioassay methods used in four of the laboratories were as previously described 1,4,6,9 with, respectively, the following indicator organisms: Candida kefyr ATCC 46764, Candida albicans 3153A, C. kefyr isolate SA, and C. albicans NCPF 3281. In the fifth laboratory, the bioassay was performed as follows. A solution containing 3.0 g Bacto agar (Difco, Basingstoke, UK) in 200 mL Eagle' s minimal essential medium (EMEM cat. No. 14-100-49; Flow Laboratories, Irvine, UK) was autoclaved, cooled to approximately 60°C and poured into square plastic dishes, 23 cm x 23 cm (Nunc Biologicals, Roskilde, Denmark). To a solution containing 1.5 g Bacto agar in 30 mL EMEM, autoclaved and cooled to 56°C in a water bath, was added 3 mL of a suspension of C. albicans isolate RV4688 containing 4 x 10 7 cells/mL. This mixture was poured as a seed layer on top of the EMEM agar. The test serum samples and standard solutions in pooled human serum were pipetted in 50 µL volumes on to sterile 1.3 cm filter paper discs which were placed on the surface of the seed layer. The plate was left to stand at 4°C for 24 h then incubated at 30°C for 24 h and the inhibition zone diameters were measured.
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Results |
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The HPLC measurements (all from phase 1 of the study) were accurate to within 10% of the nominal itraconazole and hydroxy-itraconazole concentrations in the seven spiked serum samples. Concentrations as low as 0.005 mg/L were measurable by HPLC and the mean coefficient of variation for all 15 duplicate samples was 2.3%. The four serum samples containing no active compounds were correctly reported as not detectable .
Bioassay first round of tests
The lowest detection limits reported in the bioassays ranged from 0.156 to 0.63 mg/L, and 13 of the 30 samples were reported as below the level of detectability in all five laboratories (Table I). Only seven of the 13 positive samples contained sufficiently high concentrations of azoles for levels to be determined in both duplicates of the samples in all five laboratories. For two of these samples the result was reported as >10 mg/L by one laboratory. The degree of correlation and interlaboratory agreement for the seven measurable samples was good. Mean coefficients of variation for duplicate assays ranged from 5.1% to 12.5%. The measurements, expressed as bioassay activity, indicated an antifungal concentration in the samples that was an average of 2.25 times higher than the total azole level as determined by HPLC.
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The concentrations of itraconazole and hydroxy-itraconazole spiked in the samples in the second phase were chosen to give on-scale bioassay results. Nevertheless, some laboratories still reported results for these samples that were above or below the range of bioassay measurements (Table II). The measured levels of antifungal activity by bioassay varied considerably with the relative content of itraconazole and hydroxy-itraconazole in the samples. With an itraconazole standard curve, laboratories A, B, D and E all obtained results close to the spiked concentrations for the samples containing only itraconazole (Table II), but when hydroxy-itraconazole was present, alone or combined with itraconazole, the bioassay result from these laboratories was several-fold higher than the total spiked azole concentration. In laboratory C, the ratio of bioassay level:spiked level decreased with increasing spiked level, regardless of whether the sample contained itraconazole alone, hydroxy-itraconazole alone, or both azoles.
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The results from laboratory A, which showed the highest variation between duplicate samples in both phases of the study, nevertheless gave the closest estimate of total spiked azole concentrations in phase 2, regardless of the relative spiked concentrations of itraconazole and hydroxy- itraconazole in the sample.
The average coefficients of variation for the bioassay results with the 15 paired test samples ranged from 5.6% to 21.6% with the itraconazole standard and from 5.7% to 16.7% with the hydroxy-itraconazole standard (Table II).
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Discussion |
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Our study has reconfirmed that bioassay methods for estimation of serum levels of itraconazole and its hydroxy- metabolite tend both to overestimate and underestimate the total concentrations of these components in a sample, depending on the experimental conditions used. Despite the interlaboratory variations, the correlative trend between bioassay results and total (itraconazole + hydroxy-itraconazole) concentrations was unequivocal (Tables I and II), showing that bioassay can indicate the relative level of antifungal azole present in a serum sample even when different laboratories use different bioassays rather than a single, standardized assay. Differences in susceptibility of marker isolates to the two agents 1,2 might account for some of the discrepancies between bioassay and HPLC results.
Use of multiplication constants for converting bioassay data to match HPLC results 3 is a valid approach only if the ratio of bioassay result to total (HPLC) azole concentration is constant or nearly so. The results of the present study show that bioassay:HPLC ratios vary between laboratories and must therefore first be established locally if attempts are to be made to express bioassay results as equivalent to a total azole concentration. In practice it is probably better to quote results as bioactivity equivalent to a concentration of azole, rather than to suggest that bioassay directly measures azole concentrations.
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Acknowledgments |
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Notes |
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References |
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Received 20 May 1998; returned 8 July 1998; revised 26 October 1998; accepted 24 December 1998