Istituto di Microbiologia, Facoltà di Medicina e Chirurgia, Università degli Studi di Parma, Via Gramsci 14, 43100 Parma, Italia
Introduction
In recent years, the remarkable increase in the number of infections caused by fungal pathogens, especially in immunocompromised people, has stimulated interest in medical mycology in general and in antifungal susceptibility testing in particular.1,2 In-vitro procedures for determining the activity of drugs against mould and yeast isolates involve clinicians in the choice and monitoring of antifungal chemotherapy. A number of factors, however, may influence the evaluation of the sensitivity of fungi to antifungal drugs, even in the most standardized procedures such as the determination of the MIC and minimal fungicidal concentration (MFC) in optically read microautomated systems. The nature and the form of the involved fungus, the preparation of the inoculum, the solubility and stability of the antifungal drugs tested, the pH and composition of the test medium, the duration and the temperature of incubation and the criteria for determining endpoints are variables that significantly affect the reproducibility of the test. 2,3,4,5,6,7
The NCCLS has proposed reference parameters for antifungal susceptibility testing, which were recently amended.8,9 The original procedure, which was based on macrodilutions of the drug in the synthetic medium RPMI 1640 buffered at pH 7.0 in morpholinopropansulphonic acid (MOPS) 0.165 M, is very laborious. Comparative studies have been carried out to modify the NCCLS's procedure to a miniaturized microtitre plate system that may be effectively automated.10,11,12,13,14,15,16 Despite the equivalency of the achievable results, the micromethod cannot be readily adopted in many laboratories and thus, the commercially available systems have been based on different methods such as the Etest or ATB fungus tests.14,15,16,17,18,19,20,21,22,23,24,25
Major advances in intralaboratory and interlaboratory reproducibility have been achieved, particularly through the NCCLS's guidelines, and commercial kits have rendered antifungal susceptibility testing feasible in every mycological laboratory. Current methods, however, do not fulfil completely the crucial need for correlation between the results of in-vitro susceptibility testing and in-vivo response. If the goal of obtaining reproducible tests has been only partially achieved, the one of providing reliable results to clinicians as a guide for antifungal therapy still poses problems. The next critical step to be taken should be to ensure the clinical relevance of any proposed method for antifungal susceptibility testing by determining the relationship between the MIC and MFC of antifungal agents and clinical response. In addition to the variables associated with the fungal organisms per se and the antifungal agents, other factors may affect how reliably the in-vitro tests predict the clinical outcome; these include the patient's state, such as underlying disease, cellular immune function, antibody titres, complement and pharmacokinetic properties. Such factors might affect the differential plasma protein binding and the metabolic stability of antifungal agents and need to be considered. The development of an in-vitro system that could be more predictive of in-vivo outcome may have to be adjusted and tailored to individual patients.
In this study, we compared the results of antifungal susceptibility testing obtained by the micromethod performed with reference to the original guidelines recommended by the NCCLS and using RPMI, with those obtained by the same procedure but carried out in several human plasma specimens in order to take into account the unique interaction that occurs between the fungus and its host.
The purpose of this study was to determine whether any variation from the reference standard method could be detected using each individual's plasma with the assumption that antifungal susceptibility testing performed in physiological/pathological fluids could provide information that corresponded better with the personal characteristics of the patient and thus be better correlated with clinical effectiveness.
The aim of this study was to determine whether use of an individual's plasma in antifungal susceptibility testing would be more useful than the standard method in terms of correlation with clinical effectiveness.
Materials and methods
Yeast strains
Nine yeasts isolated from clinical materials were used in this study. They included two strains of Candida albicans (UP26 and UP62), one of Candida glabrata (UP1), one of Candida parapsilosis (UP1), one of Candida kefyr (Y0601), one of Candida tropicalis (UP260), and three of Cryptococcus neoformans var. neoformans (UP195, UP238 and UP300).
All of these yeast isolates were identified with the Vitek AutoMicrobic System (bioMérieux Vitek, Hazelwood, MO, USA) and API 20C AUX (bioMérieux, Marcy l' Etoile, France), and maintained in sterile distilled water in our collection.
Antifungal drugs
The antifungal drugs used in this study were amphotericin B (Bristol-Myers Squibb, Rome, Italy), fluconazole (Roerig-Pfizer Italia SpA, Rome, Italy) and 5-fluorocytosine (5-FC) (Roche, Milan, Italy). These three antifungals were used for the preparation of the desired dilutions in a similar way to the recommendation of the NCCLS.8 The stock solutions of 5-FC and fluconazole were prepared at a concentration of 12,800 mg/L in sterile distilled water while amphotericin B was dissolved in 100% dimethyl sulphoxide (DMSO; Sigma Chemical Co., St Louis, MO, USA) at a concentration of 160 mg/L.
Synthetic medium and human plasma
The antifungal drug susceptibility tests were carried out in a comparable manner in the synthetic medium RPMI 1640 (Seromed, Biochrom KG, Berlin, Germany) with the addition of L-glutamine, free of sodium bicarbonate and buffered at pH 7 with MOPS 0.165 M (Sigma) following the recommendations of the NCCLS8 and in the plasma of individual human blood donors. All of the plasma samples were checked for the presence of intrinsic inhibitory activity against the study strains by a conventional spot test on the surface of agar plates.26
Microtitre plate drug dilutions
Ninety-six well microtitre plates (Falcon 3918, Becton-Dickinson Labware, Lincoln Park, NJ, USA) were used for the antifungal drug susceptibility tests. The stock solutions of the three antifungals were diluted either in RPMI or in each of the plasma samples in order to achieve final concentrations of 128 mg/L for fluconazole and 5-FC and 16 mg/L for amphotericin B taking account of the dilution effect of the inoculum. Serial dilutions were prepared from each solution in the two adopted media (plasma and RPMI) in order to obtain final concentrations ranging from 128 mg/L to 0.25 mg/L for fluconazole and 5-FC and from 16 mg/L to 0.0312 mg/L for amphotericin B.
Ninety microlitres of each antifungal dilution in each medium was added to the wells of the microtitre plates numbered from 1 to 10. In the eleventh well of each row, 0.09 mL of medium or plasma without antifungal was added, together with the same amount of the diluents (DMSO or sterile distilled water) used in the first well of each row. In the twelfth well, 0.09 mL of medium or plasma devoid of any antifungal or diluent was added as a control for yeast growth. Once ready for testing, the plates were maintained at -20°C until used.
Preparation of the inoculum
A yeast suspension of 0.5 McFarland optical density was initially prepared in sterile distilled water that corresponded to approximately 5 x 106 cfu/mL. Then, the yeast suspension was diluted 1:100 in sterile distilled water for the antifungal drug susceptibility tests carried out in RPMI and in 103 Yeast Nitrogen Base (YNB) (Difco Laboratories, Detroit, MI, USA) for the tests performed in plasma. For comparative purposes, 103 YNB was also used to dilute a 1:100 yeast suspension for antifungal drug susceptibility testing carried out in RPMI. Ten microlitres of the yeast suspension (equivalent to approximately 5 x 102 cells) were inoculated in each well either containing medium or plasma. The microtitre plates were incubated at 37°C.
Interpretation of the results
The optical density of the cultures was measured at 620 nm with a spectrophotometer (SLT Labinstruments, Austria) after 24 and 48 h for the Candida strains and after 4872 h for the C. neoformans isolates. The MICs were determined as the concentration of each antifungal giving 80% of growth inhibition in comparison with the control wells and according to the NCCLS guidelines.8 The MFC of each antifungal against all of the investigated yeasts was determined by subculturing a calibrated loopful (18 µL) of each microtitre well (after shaking the microtitre plates) on Sabouraud agar (Difco) free of antimycotics. The MFC plates were incubated for 4872 h at 37°C before the results were read. The endpoint for MFC was considered as that showing no visible growth of yeast colonies. All of the tests were carried out in duplicate.
Results
The human plasma samples used in this study did not exert any direct inhibitory activity against the yeast isolates. The MICs and MFCs of each antifungal drug against all of the yeast isolates, tested in the standard medium and in the individual human plasma, are shown in the Table. The antifungal drug susceptibility tests carried out in RPMI with the yeast suspensions diluted with water or 10x YNB gave the same results.
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Significant variable results were also obtained using the individual plasma specimens in the determination of the MFCs of fluconazole and 5-FC against C. parapsilosis UP1, the MFC of amphotericin B and the MICs of fluconazole and 5-FC (at 48 h) against C. tropicalis UP260.
With regard to the C. neoformans isolates investigated, significant variability was observed, particularly in the MICs obtained with some individual plasmas in respect to the reference synthetic medium.
Discussion
Several reports have addressed the question of the need for standardized procedures that could allow intralaboratory and interlaboratory reproducibility for antifungal drug susceptibility testing for yeasts, while avoiding the need to face problems with moulds, which present even more intrinsic technical problems. In this perspective, correlation efforts coordinated by the NCCLS have produced guidelines for the rational performances of macrodilution broth methods to serve as a reference standard that could be adapted to microtitre plates for practical automation procedures.3,8,9
The NCCLS proposed standard reference method with broth dilutions undoubtedly represents a great improvement for the in-vitro standardization of antifungal yeast susceptibility tests. Even though meaningful data may be obtained as a result of this proposed methodology, enabling satisfactory intralaboratory and interlaboratory reproducibility, the clinical relevance of the reference standard method, in respect to the relationship between in-vitro and in-vivo antifungal activity, is still unacceptable.5,27,28,29,30
Various parameters intrinsic to the patient, such as serum concentration, protein binding, mucosal adsorption and immune function might all greatly influence the interpretation of the potential in-vitro activity of antifungal drugs.31 The development of in-vitro susceptibility tests that would correlate more closely with in-vivo results may, therefore, have to be varied for each patient. Since most of the variables are related to the characteristics of an individual's blood, we have speculated that plasma, as well as other biological fluids, would be more pertinent and significant to the individual's clinical response to antifungal therapy.
We have adapted the NCCLS method in order to try to obtain more `physiological' conditions. Since, for some yeasts (e.g. C. neoformans) plasma, as well as RPMI, does not allow suitable growth, both media used were modified by the addition of YNB (which, significantly, replaced RPMI in the revised NCCLS guidelines 9). The addition of YNB to RPMI did not affect the MIC values compared with those obtained with RPMI alone (data not shown).
Our data revealed that the results obtained in the antifungal susceptibility tests carried out with individual plasma specimens may differ substantially from each other and from the results obtained with RPMI. Thus, particularly in the case of the largest divergences, the result obtained with a patient's own plasma could be considered more predictive of the response in vivo to antifungal therapy.
Although the standardized reference procedure performed according to the guidelines recommended by NCCLS represents the key method of intralaboratory reproducibility tests and interlaboratory communication and statistical information, we believe that personalized antifungal susceptibility testing could be carried out and the results prioritized in cases of discrepancies. Further studies, however, are required to verify the supposed closer correlation between the data obtained in vitro using personalized antifungal susceptibility tests in place of the reference standard method and the actual clinical outcome of patients in the course of antifungal treatment.
Acknowledgments
The authors are grateful to Dr Libero Ajello for his critical reading of the manuscript.
Notes
* Corresponding author. Tel:+39-521-988885; Fax:+39-521-993620;
E-mail: lucpol{at}ipruniv.cce.unipr.it
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Received 8 June 1998; accepted 6 October 1998