Med Microbiol Immunol (1992) 181:117-126

�9 Springer-Verlag 1992

Original investigations

Flow cytometric assay for estimating fungicidal activity of Amphotericin B in human serum*

Edith Martin, Ute Schlasius, and Sucharit Bhakdi

Institute of Medical Microbiology, University of Mainz, Augustusplatz, W-6500 Mainz, Federal Republic of Germany

Received March 25, 1992

Abstract. We describe a simple and rapid bioassay for estimating fungicidal activity of Amphotericin B in human serum using flow cytometry. The method exploits the fact that Candida albicans damaged by Amphotericin B show a decrease in size and take up propidium iodide to exhibit a red fluorescence after deoxycholate treatment. These phenomena display characteristic dose dependencies, and their assessment permits serum fungicidal activity to be broadly grouped into three categories: (1) subfungicidal; (2) fungicidal; and (3) strongly fungicidal. In normal human serum, these three categories correspond to Amphotericin B concentra- tions of 0 < 0.5 ~tg/ml, 0.75-1.5 ~tg/ml, and > 2 ~tg/ml, respectively. Pilot analysis of serum samples obtained from four patients undergoing Amphotericin B therapy confirmed the feasibility of using the flow cytometric assay for estimating drug fungicidal activity ex vivo. The method is very simple, generates results within 5 h, and could prove useful for monitoring therapy with this effective but toxic drug.

Introduction

Despite its toxicity, Amphotericin B continues to represent the most widely used drug for treating systemic fungal infections. Proponents of Amphotericin B therapy stress the excellent and broad fungicidal activity of this agent, and the fact that emergence of resistant strains is rare [3, 11, 12, 15, 19, 20, 22, 30, 33]. Amphotericin B interacts with ergosterol molecules in yeast cell membranes, and with lower affinity with cholesterol molecules in mammalian cell membranes [3, 22, 38]. Binding probably invokes alterations of the membrane permeability barrier, which lead to cellular dysfunction. This forms the basis for its toxicity to yeasts as well as to mammalian cells [25, 26, 29, 30, 38]. Systemically applied Amphotericin B binds to serum lipoproteins, proteins and blood cells [1, 6, 7], and

* This work forms part of the thesis of U.S. Correspondence to: S. Bhakdi

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distributes to peripheral organs, where it has been shown to accumulate in the liver, spleen and the kidney [1, 12, 14, 30]. Different therapeutic regimens have been developed empirically, with the aim of minimizing acute toxic effects whilst achieving sufficient therapeutic levels as rapidly as possible [3, 4, 10, 22].

An acceptable assay for routinely monitoring therapy is not available. Achievement of sufficient concentrations of Amphotericin B in tissues is probably critical for successful therapy, but tissue concentrations can, of course, not be directly determined. Therefore, determinations of serum concentrations are the mainstay of all efforts to perform drug monitoring. The slow elimination rate of Amphotericin B, and absence of its metabolization [1, 10, 12, 14, 16, 22, 24, 39], augment the feasibility of this approach. The pharmacokinetics of Amphotericin B apparently show considerable individual variations. Generally, administration of 0.5-1 mg/kg Amphotericin B daily leads to Amphotericin B serum peak levels between 0.5-3.5 gg/ml [1, 9, 10, 16, 20, 22, 39, 40]. Higher doses accelerate development of unacceptable toxic effects [3, 9, 13, 22, 24]. At the other extreme, serum levels of < 0.5 gg/ml are subfungicidal in vitro [32]. Thus, it is reasonable to assume that therapeutic regimens should aim at achieving plasma drug levels of 1- 2 gg/ml. Determination of drug concentrations in serum is possible using HPLC [2, 8, 17, 18, 21, 23, 31, 34], but this method cannot be employed for routine analyses of a large number of samples. An alternative method involving spectrophotometric determination of Amphotericin B concentrations in acetoni- trile serum extracts has been reported [37].

Measurement of absolute Amphotericin B concentrations in serum may have shortcomings. Binding of the drug to serum components probably varies depending on the blood protein and lipoprotein composition. Such variations could represent a significant unknown factor that would influence the amounts of Amphotericin B remaining available for binding to target cells. Accordingly, we felt that a bioassay for assessing serum fungicidal activity might provide advantages over determinations of total serum Amphotericin B concentrations.

In this communication, we report that yeast cells damaged by Amphotericin B diminish in size and take up propidium iodide (PI) when treated with deoxycho- late. This forms the basis of a rapid and simple flow cytometric assay for estimating serum fungicidal activity which is far superior to the conventional bioassay involving determinations of colony-forming units (CFU).

Materials and methods

A Candida albicans isolate was obtained from the diagnostic laboratory of our Institute. The yeasts were cultured overnight in tryptic soy broth for 12-13 h at 37~ The cells were washed thrice in phosphate-buffered saline (PBS), suspended to approximately 3 X 10 7 cells/ml, and kept on ice until use. Human serum was obtained from 15 healthy adults. Amphotericin B (Squibb Pharma, Vienna, Austria) was diluted in PBS immediately prior to experiments and added to serum samples at final concentrations ranging from 0.5-5 lag/ml. PI (Sigma, Munich, Germany) was kept as stock solution of 4 rng/ml in water at --20~ Deoxycholate (Serva, Heidelberg, Germany) was kept as a 250-mM stock solution in water at room temperature.

Experimental protocol

Calibration experiments. C. albicans were added to 200 ~1 serum samples containing Amphoteric- in B in Eppendorf reagent tubes to yield final yeast cell concentrations of 3 X 10 6 cells/ml.

R e s u l t s

Samples were incubated at 37~ with continuous shaking (1100 rpm) in a Thermomixer 5436 (Eppendorf Inc., Hamburg, Germany). After 2 and 4 h, 50 lal samples were removed, added to 50 ~tl water containing 8 lag/ml PI for 3 min, after which each sample received 100 ~tl of 25 mM deoxycholate. After another 3 min, the samples were diluted with 0.4 ml ice-cold PBS and placed on ice until analysis. Flow cytometric analyses were performed using a FACScan flow cytometer (Becton Dickinson, Heidelberg, Germany) with computer-assisted evaluation (FACScan soft- ware).

Determinations of CFU were performed in parallel. Each determination was performed in duplicate after 2 and 4h of incubation. For these assays, samples spiked with 0.5~tg/ml AmphotericinB were employed as negative controls. As reported previously [28, 32] this concentration is subfungicidal, but prevents artefacts that otherwise arise due to germ-tube formation and cell aggregation.

Ex vivo determinations of serum fungicidal activity. Serum samples were obtained 18-22 h post infusion from four patients undergoing Amphotericin B therapy. C. albicans was added to 200-1al aliquots at 3 X 106 CFU/ml and the test was performed as described above. A serum sample obtained before the first Amphotericin B infusion was spiked with 0.5 lag/ml Amphotericin B and employed as the negative control. In all cases, light microscopy was used to check whether germ- tube formation or cell aggregation had taken place after incubation of the yeast for 2-4 h in the sera. The latter processes were reflected by an increase in particle size and granularity that was detectable by flow cytometry.

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Figure 1 depic ts the results of b ioassays ut i l iz ing the "gold s t a n d a r d " of co lony count ing. In accord with Nugen t and Couc ho t [32], we found tha t a 2- to 4-h incuba t ion o f C. albicans in serum con ta in ing 0.5 ~tg/ml A m p h o t e r i c i n B d id no t resul t in r educ t ion o f C F U . Kil l ing was first observed at concen t ra t ions o f a p p r o x i m a t e l y 0.75 pg /ml . Af te r 2- and 4-h incuba t ion , 3 0 % - 6 0 % and 7 0 % - 9 0 % reduc t ion in C F U were obse rved respect ively, at A m p h o t e r i c i n B levels o f 1.25- 2 t.tg/ml.

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Fig. 1. Fungicidal activity of Amphotericin B in human serum. C. albicans were suspended at 3 • 106 (CFU)/ml and determinations of CFU were performed after 2- and 4-h incubation at 37 ~ C. Serum spiked with 0.5 lag/ml AmphotericinB served as the control. Plotted are the mean values + SD of experiments performed with six different donors

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Figure 2 illustrates the principles of the flow cytometric assay. Yeast cells incubated for 2 h in serum containing 0.5 ~tg/ml Amphotericin B were detected as a homogeneous population of particles, the majority of which did not take up PI after treatment with deoxycholate (left panels). As originally reported by Lehrer [27], deoxycholate is not toxic to C. albicans and we have previously shown that yeasts treated with 0.51.tg/ml AmphotericinB also do not lose viability after deoxycholate treatment [28]. The first parameter that was employed to assess activity of Amphotericin B was the analysis of yeast cell size. Increasing concen- trations of Amphotericin B caused a decrease in average cell size and relative granularity during an incubation time of 2 h (right panels). This phenomenon was dose dependent and could be expressed in quantitative terms by the relation

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where lAB 0.5] denotes serum samples spiked with 0.5 ~tg/ml Amphotericin B (FSC, forward scatter; SSC, sideward scatter).

The second parameter that generated information on serum fungicidal activity was the uptake of PI by yeast cells after treatment with deoxycholate. Thus, when yeasts were incubated for 2h with serum samples containing > 0.75 lag/ml Amphotericin B, subsequent incubation with deoxycholate plus PI always revealed a population exhibiting red PI fluorescence (right panels). This phenomenon exhibited a basically similar dose response to that seen in the bioassay employing colony countings, but the maximal percentage of fluorescing cells was in the range of 40 %-70 % rather than 90 %. In the depicted experiment, 48% of the yeast particles exhibited red fluorescence after a 2-h incubation in 2 btg/ml Amphotericin B.

Adding a 4-h analysis improved the performance of the fluorescence assay. Typically, the population of PI-fluorescing cells generated by moderate levels of Amphotericin B (0.75-1.5~tg/ml) increased significantly after 4-h incubation compared with the 2-h values. In contrast, at high Amphotericin B levels (2> 1.5- 2 lag/ml) the fluorescing particles tended to decrease after 4 h compared with the 2-h values. The cause for this finding has not been delineated; possibly, it derived from autolytic processes and DNA degradation in the damaged cells occurring over time. Another possibility is that severely damaged yeast cells fragmented to yield particles that were no longer detected under the given assay conditions. Indirect support for this derived from the observation that the average rate of particle counts also decreased at high Amphotericin B concentrations. Figure 3

Fig. 2. Changes of flow cytometric parameters of C. albicans induced by Amphotericin B. Yeasts were incubated in serum containing 0.5 lag/ml or 2.0 lag/ml Amphotericin B for 2 h, 37~ and analyzed after treatment with deoxycholate and propidium iodide. Two thousand particles were acquired for each analysis. Dot plots are shown for forward scatter (FSC) and sideward scatter (SSC) (size and granularity), and for fluorescence 3 (red =propidium iodide fluorescence). Fluorescence 1 (green) served only as the second axis for the dot plots. The percentage of red fluorescent particles was calculated as shown in the histograms. Compared to the negative control (0.5 lag/ml Amphotericin B, left panels), yeasts incubated with 2.0 btg/ml Amphotericin B (right panels) exhibited an overall decrease in size, and a large population was observed to become permeable to propidium iodide

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summarizes the results of 18 experiments conducted with sera from 15 donors. Depicted are the FSC/SSC ratios calculated at 2 h (Fig. 3 A) and the mean values of PI positivity (red fluorescence in %) observed after 2h and 4h (Fig. 3B). The fluorescence assays displayed fairly wide variations, but the overall tendency was clear and reproducible. Consideration of both parameters (FSC/SSC ratio at 2 h and percentage of fluorescing particles after 2 and 4h) enabled us to broadly differentiate three categories of fungicidal activity: (1) subfungicidal ( < 0.75 gg/ml AmphotericinB; FSC/SSC ratio >0.9 ; < 10% red fluorescence after 4 h); (2) fungicidal (0.75-1.5gg/ml AmphotericinB; FSC/SSC ratio 0.8-0.4; 2-h red fluorescence < 4 - h red fluorescence value); (3) strongly fungicidal (> 2gg/ml Amphotericin B; FSC/SSC ratio <0.4; 2-h red fluorescence ->4-h red fluor- escence).

Applicability of the bioassays for monitoring Amphotericin B levels in patients

Pilot experiments were performed in four leukemia patients undergoing Amphote- ricinB therapy. All patients were neutropenic and presented with antibiotic- resistant fever for over 5 days. Serum samples were obtained immediately prior to application of the next infusion of Amphotericin B. Fungicidal activities were assessed by flow cytometry and by colony countings, and the results were always concordant with each other. In all cases, the three major types of responses could

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Fig. 4A, B. Flow cytometric assessment of serum fungici- dal activities observed in two patients undergoing Am- photericin B therapy. Serum samples were obtained 18- 21 h post infusion. Graphs shown in the upper parts de- pict FSC/SSC ratios and red fluorescence; the lower parts display the dose of Amphote- ricin B administered on the corresponding days. A Patient who received a pro- tracted regimen; therapy dur- ation of 8 days, maintenance dose 1 mg/kg per day. Note that significant serum fungic- idal activity did not become detectable until day 4. B Patient who received a maintenance dose of 1 mg/kg per day from day 3 onward. First fungicidal activity was detected on day 3 and strong fungicidal activity from day 12 onward

be distinguished. Two examples are shown in Fig. 4. Patient A received a protracted therapy regimen. No serum fungicidal activity was detected in this patient on days 1-3. On days 4 and 5, fungicidal activities were observed that correspond to moderate levels of Amphotericin B. Here, the 4-h values for PI positivity exceeded the 2-h values, and the average size ratio ranged from 0.8-0.7. On days 6 and 8, higher fungicidal levels of Amphotericin B were present. The 4-h PI values were similar to the 2-h values, and the FSC/SSC ratios dropped to approximately 0.6. The maintenance dosage of 60mg (1 mg/kg) per day was considered to generate acceptable drug levels in this patient.

Patient B received the maintenance dose (40 mg, 1 mg/kg) on day 3 onwards. Serum fungicidal activity was first detected on day 3 in this patient, and high levels were found on day 5 onwards. FSC/SSC ratios decreased to values of --~ 0.4 at day 3-5, and the 2-h PI fluorescence values were sometimes similar to or exceeded the

124

4-h values. Retrospectively, it was considered that the maintenance dosage in this patient possibly could have been reduced, since the measured fungicidal activity representing the trough concentration was continuously at the upper limit of the therapeutic window.

Discussion

The described flow cytometric method for detecting serum fungicidal activity of Amphotericin B is very simple and yields useful information within 5 h. It is based on two observations. First, membranes of C. albicans treated with fungicidal concentrations of Amphotericin B become susceptible to damage by deoxycholate, so that PI diffuses into the cells to generate a red fluorescence. The finding is conceptually related to the early observation of Lehrer [27] that phagocytic killing of C. albicans and other yeasts followed by deoxycholate treatment allows these cells to be selectively stained and identified. Bjerknes [5] has previously used PI to stain dead yeast cells for flow cytometry, and Pore [35] has recently developed a flow cytometric method for antibiotic susceptibility testing of C. albicans based on PI staining. However, since the latter investigator did not treat yeast cells with deoxycholate, his results cannot be directly compared to ours. Second, damaged yeast cells exhibit a relative decrease in size and granularity that is dose dependent. Based on the flow cytometric data, serum fungicidal activity can be grouped into three broad categories: (1) no fungicidal activity; (2) detectable fungicidal activity; (3) strong fungicidal activity. In normal human serum, these three categories correspond to Amphotericin B levels of 0-0.5 gg/ml, 0.75-1.5 lag/ml, and concen- trations exceeding 2 lag/ml, respectively.

Concern has been voiced that measurements of serum Amphotericin B may not permit conclusions to be drawn regarding the concentrations and therapeutic efficacy of this agent in tissues, and that therapeutic outcomes of deep-seated fungal infections may not correlate well with serum concentrations [1, 3, 12]. However, previous HPLC measurements have shown that conventional mainten- ance doses that are therapeutically successful generate Amphotericin B serum peak levels of 0.5-3.5 gg/ml [1, 16, 20, 22, 39]. Since levels below 0.5 gg/ml are without fungicidal activity in vitro, and because approximately 50% of peak serum levels are found 18-20h post infusion [22], we tentatively estimate the therapeutic window of pre-infusion Amphotericin B concentrations to be 0.75-1.5 lag/ml.

Applying this criterion, we found that the protracted regimen used in one of the patients studied failed to generate sufficient fungicidal plasma levels in the first 3 days. The same conclusion was reached earlier by Koren et al. [24] and Atkinson and Bennet [1] and suggests that the current trend towards increasing initial doses of Amphotericin B in order to attain serum fungicidal activity more rapidly is justified [3, 22]. Saral [36] has reported improved therapeutic effects in patients with invasive aspergillosis who received a high-dose Amphotericin B therapeutic regimen.

Assaying effective fungicidal action in whole serum ex vivo could prove more useful than measuring absolute Amphotericin B concentrations by HPLC, since the latter method does not take into account the individual variations in Amphotericin B binding capacity of serum components. This consideration may not be trivial, especially amongst patients with malignancies. Resistance of C. albicans towards Amphotericin B is uncommon. Therefore, the use of one test

125

strain should generally be acceptable for routine testing. Optimal testing can be per formed whenever a causative yeast strain has been isolated f rom the patient. The routine flow cytometr ic me thod renders possible the analysis o f large numbers o f serum samples and, thus, will enhance the prospects o f objectifying the true value of therapeutic drug moni tor ing. The method may also become useful for assessing the effects o f combina t ion therapy with other fungicidal agents.

Acknowledgements. This investigation was supported by the Deutsche Forschungsgemeinschaft (SFB 311, D9/10) and the Verband der Chemischen Industrie. We thank Drs. E. Riichel and G. H~inel for helpful and stimulating discussions, and Drs. C. Huber, W. Aulitzky and C. Peschel (Department of Hematology, University of Mainz) for their collegial help in supplying patients' data.

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