VERIFICATION AND VALIDATION OF SENSITITRE YEASTONE® ANTIFUNGAL SUSCEPTIBILITY TESTING USING VORICONAZOLE FOR PATIENT MANAGEMENT IN A HIGH VOLUME TESTING LABORATORY

Wohlfiel SL, Contezac J, Roberts GD

Author address: 

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Abstract: 

In the year 2002 we performed 1,131 antifungal susceptibility tests (287 quality control and 844 clinical) using Sensititre YeastOne ® , (Trek Diagnostic System, Inc. Westlake, OH) (SYO) for Amphotericin B, Fluconazole, Itraconazole, Ketoconazole and 5-Flucytosine. Since this is considered a “research use only” test, to add an antifungal agent such as Voriconazole, we must perform verification and validation studies. Verification is defined by the NCCLS as a “one-time process completed before the test or system is used for patient testing.” Characteristics used to confirm test performance include sensitivity, specificity, precision and accuracy. To determine sensitivity and specificity of Voriconazole using (VSYO) we obtained 40 yeast isolates from the Fungus Testing Laboratory (University of Texas, San Antonio, TX) (VSG) that had susceptibility patterns ranging from 8 µl/mL determined by the NCCLS criteria in microtiter format. To perform the VSYO a 0.5 McFarland standard suspension was made from a 24-hour subculture. 100 µl of the suspension was inoculated into the appropriate wells, the plate sealed and incubated at 35ºC. for 24-48 hours. Cryptococcus species were incubated up to 72 hours. Plates were read with the aid of a mirror and blue was considered negative and red as positive. The minimal inhibitory concentration (MIC) was read as the lowest concentration that inhibited the growth. To date, 15 yeast isolates have been tested by VSYO and 11 gave similar results compared to those provided by the Fungus Testing Laboratory. Minor discrepancies were noted for four isolates; 3 gave results within 2 dilutions and a single isolate gave results within 3 dilutions. The two quality control organisms, C. parapsilosis (ATCC 22019) and C. krusei (ATCC 6258) tested within guidelines. All discordant results will be resolved by re-testing by VSYO and VSG. Precision studies (10 days of duplicate quality control samples) are pending. Accuracy, (or within run reproducibility determined by running 10 replicates on the same day) must be between 90-99%. Validation is defined by NCCLS as “the action, or process, of proving that a procedure, process, system, equipment, or method works as expected and achieves the intended result.” The components of validation are quality control, proficiency testing, employee competency, and correlation with clinical findings. Since these are already performed for Amphotericin B, Fluconazole, Itraconazole, Ketoconazole and 5-Flucytosine, Voriconazole will be monitored in the same manner. Quality control is recorded and monitored using a web-based event management tool. Proficiency testing is performed using New York State and the College of American Physicians programs. Employee competency is validated by yearly observation and testing. Trends are recorded and discussed with infectious disease division. Because this test is routinely done for Amphotericin, Fluconazole, Itraconazole, Ketoconazole and 5-Flucytosine; our laboratory anxiously awaits Voriconazole testing.
2003

abstract No: 

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Full conference title: 

The 15 th Congress of the International Society for Human and Animal Mycology
    • ISHAM 15th (2003)