Multicenter Comparison of the ISO Standard and the Serial 2- Fold Dilution Procedures to Dilute Hydrophilic and Hydrophobic

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1 JCM Accepts, published online ahead of print on 10 March 2010 J. Clin. Microbiol. doi: /jcm Copyright 2010, American Society for Microbiology and/or the Listed Authors/Institutions. All Rights Reserved. Running title: Dilution of antifungal agents for AST Multicenter Comparison of the ISO Standard and the Serial 2- Fold Dilution Procedures to Dilute Hydrophilic and Hydrophobic Antifungal Agents for Susceptibility Testing Alicia Gomez-Lopez 1, Maiken Cavling Arendrup 2, Cornelia Lass-Floerl 3, Juan- Luis Rodriguez-Tudela 1, Manuel Cuenca-Estrella 1* 1 Servicio de Micología. Centro Nacional de Microbiología. Instituto de Salud Carlos III. Majadahonda. Spain. 2 Unit of Mycology and Parasitology, Statens Serum Institut, Copenhagen, Denmark. 3 Department of Hygiene and Medical Microbiology. Innsbruck Medical University, Innsbruck, Austria. Key words: Itraconazole, fluconazole, EUCAST *Corresponding author: Servicio de Micología, Centro Nacional de Microbiología, Instituto de Salud Carlos III, Ctra Majadahonda-Pozuelo Km Majadahonda (Madrid), Spain. Phone: Fax: mcuenca-estrella@isciii.es

2 ABSTRACT A multicenter study was conducted to assess the accuracy of the ISO standard and the serial two-fold dilution procedures for antifungal susceptibility testing. Fluconazole trays can be accurately prepared following ISO and serial dilution schemes. However, itraconazole trays showed significant lack of reproducibility independently of which method was followed. 7 Downloaded from on April 12, 2019 by guest

3 Susceptibility testing of fungi has been standardised since 1997 with recommendations on micro broth dilution MIC determinations from both the CLSI (Clinical Laboratory Standards Institute) and the EUCAST (European Committee for Antimicrobial Susceptibility Testing) antifungal subcommittees (3,4,16,17). According to those standards and the ISO recommendations the 2-fold dilutions of compounds should be performed in a multi-step, rather complex and time-consuming fashion avoiding serial dilutions in order to prevent the risk of a systematic error induced for instance by an imprecise pipette (9). While this is the gold standard for preparation of dilutions series, the question has risen if modern pipettes are sufficiently precise to allow dilution series to be prepared as serial dilutions without loss of accuracy. In this multicenter study we address this issue by comparing the concentrations of hydrophilic and hydrophobic antifungal agents represented by fluconazole (FLC) and itraconazole (ITC) respectively, in micro broth plates prepared according to the ISO standard as well as by serial dilution procedures. Stock solutions of FLC (Pfizer SA, Madrid, Spain) and ITC (Janssen SA, Madrid, Spain) were prepared centrally in 1ml volumes of dimethyl-sulphoxide, DMSO (Sigma-Aldrich, Madrid, Spain) at 12,800 µg/ml and 1,600 µg/ml respectively, and distributed to the participating laboratories: Statens Serum Institut, Copenhagen, Denmark (SSI Denmark); Innsbruck Medical University, Innsbruck, Austria (Inn Austria); and Servicio de Micología of the Spanish National Center for Microbiology, Madrid, Spain (CNM Spain). Stocks were kept at -70 C until use. Microtitre plates were prepared according to the ISO standard and by serial dilution procedures recommended for preparation of trays of both hydrophilic and hydrophobic drugs (9,16). Trays were done in

4 duplicate at separate days at each centre and then, shipped frozen by special delivery to Spain for further chromatographic concentration determinations. Stock solutions and microdilution wells were tested for concentration of antifungal using HPLC methods previously described (1, 2, 5). Samples for analysis were prepared by pipetting 75 µl of the content of selected wells and mixing them with the same volume of distilled water (diluted ½). Fifty µl of the resultant mix was directly injected into the HPLC system. The presence and concentration of FLC or ITC in each well was determined by comparing the chromatographic signal (peak area, retention time and UV spectral pattern between 210 and 300 nm) from sample and from standard solutions. FLC eluted at 4.2 min and ITC at 4.1 min. The limit of detection (LOD), defined as the lowest amount of antifungal reliably differentiated from background noise (21), was found to be 1 µg/ml for FLC and 0.5 µg/ml for ITC respectively. A linear calibration curve was obtained in the range of µg/ml for FLC and µg/ml for ITC using peak area. The method was validated by performing three calibration curves on consecutive days. Comparisons between results obtained by method of tray preparation were done by the ANOVA test. A P<0.05 was taken as statistically significant. All statistical analyses were done with the statistical package PAWS Statistics 18.0 (SPSS, S.L., Madrid, Spain). In order to determine and compare the accuracy of each method described for microdilution two consecutive HPLC determinations of azole concentration were calculated from different wells of trays prepared according ISO recommendations, serial dilutions for hydrophobic drugs and serial dilutions for hydrophilic compounds. Results are shown on tables 1 (FLC) and 2 (ITC).

5 Taking into account data from FLC plates, precision and accuracy studies revealed that average coefficients of variation (calculated as %CV= [standard deviation/mean] x100) and relative errors (calculated as % Error= [(Theorical value-calculated value)/theorical value)] x100) of HPLC determinations ranging between 0.2% and 39.5 % and between 0.3% and 58.4% respectively. HPLC is a sensitive and reproducible method for determining levels of azole components in a great variety of matrix as has been confirmed in previous reports (1,2,5,10-12,15,18-20,22). The detection limit of our HPLC-based technique (1 µg/ml) did not permit us to know what happened for lower concentrations in the trays. Overall, no significant statistical differences (P>0.01) were found between the three methods for FLC quantification. Notably, although relative errors were higher for the lowest concentrations, we did not observe an increasing inaccuracy when performing serial dilutions as compared to dilutions performed following the ISO procedure as would have been expected if the precision of modern pipettes had been insufficient. Thus, serial dilution, which is attractive due to its simplicity, may be appropriate. Regarding serial two-fold dilutions procedures, better results were detected when FLC plates were prepared according to the hydrophilic than the hydrophobic way. Our data showed similar precision and accuracy within each laboratory of the three procedures used for FLC microplate preparation. However, for itraconazole significant differences were found per centre, repetition and method by the ANOVA test analysis (P<0.05). Greatest discrepancy was observed for the lowest concentrations and for the dilutions prepared outside Spain and shipped for concentration determinations. Thus, ITC data obtained from trays prepared by the Inn Austria participant were excluded from the analysis since tray wrappings showed evidences of defrosting and spillage, but also concentra-

6 tions of dilutions prepared in Denmark were unacceptably different from the target concentrations. ITC precipitation after thaw-freeze cycles during transport is the most probable explanation. Further more previous studies have demonstrated a much higher interlaboratory variation in MIC results for quality control strains for itraconazole than for flucytosine and fluconazole (6-8). Considering only data for dilution series prepared in Spain no systematic difference was detected between ISO dilution and serial dilution. ITC is a weakly basic drug (pka ca. 3.7) with very low water-solubility (RPMI is a hydrophilic medium) (14). This azole compound can only be ionized and solubilised in water at very low ph and also earlier studies have indicated that poor solubilisation can have a significant influence on generated MIC values (13). Since the hydrophilic growth medium RPMI-G at ph 7 has been selected for testing in vitro antifungal susceptibility it was important to describe the lack of proper solubility of this compound. In summary, our data suggest that microdilution trays for hydrophilic drugs such as FLC can be accurately prepared following the ISO recommendations or the serial two-fold dilution schemes. However, preparation of ITC trays for AST microdilution tests is associated with significant lack of reproducibility particularly after freezing and shipping independently of which dilution method was followed. Quality control procedures of AST should be implemented in order to minimize the inaccuracy of dilution methods and lack of reproducibility when hydrophobic compounds such as ITC are tested. Conflict of interest: Nothing to declare

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10 Table 1: Precision of fluconazole dilution series by dilution procedure and laboratory. Theor cc Lab ISO standard Serial dilutions (hydrophilic way) Serial dilutions (hydrophobic way) Cc (m±sd) CV% %E Cc (m±sd) CV% %E Cc (m±sd) CV% %E 2 SSI Denmark 1.4± ± ± SSI Denmark 7.1± ± ± SSI Denmark 58.8± ± ± SSI Denmark 128.5± ± Inn Austria 3.2± ± ND Inn Austria 11.8± ± ND Inn Austria 44.6± ± ND Inn Austria 90.5± ± ND CNM Spain 1.6± ± ± CNM Spain 7.0± ± ± CNM Spain 63.8± ± ± CNM Spain 123.5± ± ± Theor cc : Theorical concentration in µg/ml. Cc: Concentration of antifungal agent. m + sd: mean +/- standard deviation. CV: Coefficient of variation. %E: %Rate of error=[(theorical value-calculated value)/theorical value )]x100) from two repetitions. SSI Denmark: Statens Serum Institut, Copenhagen, Denmark. Inn Austria: Innsbruck Medical University, Innsbruck, Austria: CNM Spain: Servicio de Micología. Spanish National Center for Microbiology, Madrid, Spain. ND: Not done

11 Table 2: Precision of itraconazole dilution series by procedure and laboratory. Theor cc Lab ISO standard Serial dilutions (hydrophilic way) Serial dilutions (hydrophobic way) Cc (m±sd) CV% %E Cc (m±sd) CV% %E Cc (m±sd) CV% %E 2 SSI Denmark 0.26± ± ± SSI Denmark 4.23± ± ± SSI Denmark ± ± ± Inn Austria ND - - ND - - ND Inn Austria ND - - ND - - ND Inn Austria ND - - ND - - ND CNM Spain 1.64± ± ± CNM Spain 7.84± ± ± CNM Spain 15.51± ± ± Theor cc : Theorical concentration in µg/ml. Cc: Concentration of antifungal agent. m + sd: mean +/- standard deviation. CV: Coefficient of variation. %E: %Rate of error=[(theorical value-calculated value)/theorical value )]x100) from two repetitions. SSI Denmark: Statens Serum Institut, Copenhagen, Denmark. Inn Austria: Innsbruck Medical University, Innsbruck, Austria: CNM Spain: Servicio de Micología. Spanish National Center for Microbiology, Madrid, Spain. ND: Not done