1

A bioassay for determining voriconazole serum levels in patients receiving combination 1

therapy with echinocandins 2

3

Maria Siopi1, Efthymios Neroutsos2, Kalliopi Zisaki3, Maria Gamaletsou4, Maria Piroynaki5, 4

Panagiotis Tsirigotis6, Nikolaos Sipsas4, Aristides Dokoumetzidis2, Evgenios Goussetis3, 5

Loukia Zerva1, Georgia Valsami2, Joseph Meletiadis1 6

7

1 Clinical Microbiology Laboratory, Attikon University Hospital, Medical School, National 8

and Kapodistrian University of Athens, Athens, Greece 9

2 Laboratory of Biopharmaceutics and Pharmacokinetics, Faculty of Pharmacy, National and 10

Kapodistrian University of Athens, Athens, Greece 11

3 Bone Marrow Transplantation Unit, Aghia Sophia Children Hospital, Athens, Greece 12

4 Pathophysiology Department, Laikon General Hospital, Medical School, National and 13

Kapodistrian University of Athens, Athens, Greece 14

5 2nd Department of Internal Medicine, Hematology Unit, Ippokration Hospital, Medical 15

School, National and Kapodistrian University of Athens, Athens, Greece 16

6 2nd Department of Internal Medicine, Hematology Unit, Attikon University Hospital, 17

Medical School, National and Kapodistrian University of Athens, Athens, Greece 18

19

Correspondence: Joseph Meletiadis, Ph. D. 20

Clinical Microbiology Laboratory 21

Attikon University Hospital 22

Rimini 1, Haidari, 124 62 Athens 23

Tel: 210-583-1909, Fax: 210-532-6421 24

Email: jmeletiadis@med.uoa.gr 25

AAC Accepted Manuscript Posted Online 26 October 2015Antimicrob. Agents Chemother. doi:10.1128/AAC.01688-15Copyright © 2015, American Society for Microbiology. All Rights Reserved.

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SUMMARY 26

Voriconazole levels were determined with HPLC and a microbiological agar diffusion 27

assay using a C. parapsilosis isolate in 103 serum samples from HPLC-tested external quality 28

control program (N=39), 21 patients receiving voriconazole monotherapy (N=39) and 7 29

patients receiving combination therapy (N=25). The results of the bioassay were correlated 30

with the results obtained from the external quality control program samples and with the 31

HPLC results in sera from patients on voriconazole monotherapy and on combination therapy 32

with an echinocandin (rs>0.93, mean±SEM % difference <12±3.8%). 33

34

Keywords: TDM, bioassay, combination therapy, voriconazole, echinocandins 35

36

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TEXT 37

Voriconazole is characterized by nonlinear pharmacokinetics due to saturation of its 38

metabolism resulting in unpredictable exposure of standard dosing regimens. Furthermore, it 39

exhibits substantial inter- and intra-patient variability (88-100%) with many physiological, 40

pathological and pharmacological variables affecting serum concentrations (1). A correlation 41

between serum concentrations with toxicity or response has been reported (2) whereas the 42

benefit of TDM of voriconazole in the clinical setting has been demonstrated in many clinical 43

studies including a randomized clinical trial (3–7). Therefore, the TDM of voriconazole is an 44

important tool of individualized therapy leading to dosage optimization in order to maximize 45

the therapeutic effect and minimize toxicity. 46

The clinical use and value of TDM is mainly related to accurate, rapid and cost-47

effective assays. Specifically, voriconazole levels in body fluids are often determined by 48

using chromatographic or microbiological methods. Although high performance liquid 49

chromatography (HPLC) is still considered the gold standard, bioassays are frequently 50

adopted and routinely performed because of their relative technical simplicity and low 51

consumable and equipment costs, while there are several data indicating concordance of 52

results between the two methods (8–13). Nevertheless, current microbiological assays are 53

lacking specificity in cases of antifungal combination therapy as they do not allow the 54

separation and simultaneous quantification of each individual compound. In light of the recent 55

encouraging data from a large prospective randomized clinical trial on antifungal combination 56

therapy (14), voriconazole may be combined with echinocandins in order to increase efficacy 57

and overcome limitations of voriconazole monotherapy such as the long time to reach steady-58

state, the subtherapeutic levels and difficult-to-treat infections (e.g. CNS infections, azole 59

resistant pathogens) (15, 16). We therefore, developed and validated an agar diffusion 60

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bioassay for determination of voriconazole concentration in serum of patients on combination 61

therapy with echinocandins. 62

Isolate. A Candida parapsilosis clinical isolate from the collection of the 63

microbiology laboratory of our hospital (internal identifier number 221) served as the test 64

organism. The in vitro susceptibility of the strain to voriconazole (0.015 mg/L) and the three 65

echinocandins (anidulafungin, caspofungin, micafungin; 0.25, 0.25 and 0.5 mg/L, 66

respectively) was tested using two broth microdilution techniques: the reference method of 67

the Clinical and Laboratory Standards Institute (CLSI) (17, 18) and the colorimetric Sensititre 68

YeastOne® antifungal panel (Trek Diagnostic Systems, Cleveland, OH, USA) (19, 20). The 69

isolate was stored in normal sterile saline with 10% glycerol at -70°C until the study was 70

performed and prior to testing it was revived by subculturing twice onto Sabouraud dextrose 71

agar plates with gentamicin and chloramphenicol (SGC2; bioMerieux) at 30°C for 24 hours to 72

ensure purity and viability. Distinctive colony-forming units (CFU) of the subcultured yeast 73

were tipped and suspended in normal sterile saline. After counting viable cells in a Neubauer 74

chamber, Candida suspension was adjusted to give a final inoculum concentration of 3x105 75

CFU/mL. CFU counts were affirmed each time by spread plate counts on SGC2 plates. 76

Antifungal drugs and medium. Laboratory grade standard powders of voriconazole 77

and anidulafungin (Pfizer Inc., Groton, CT, USA), caspofungin (Merck & Co. Inc., 78

Whitehouse, NJ, USA) and micafungin (Astellas Pharma Inc., Osaka, Japan) were dissolved 79

in sterile dimethyl sulfoxide (DMSO;Carlo Erba Reactifs-SDS, Val de Reuil, France) and 80

stock solutions of 10 mg/mL were stored in small portions at -70oC until use. The medium 81

used throughout was RPMI 1640 medium (with L-glutamine, without bicarbonate) 82

(AppliChem, Darmstadt, Germany) buffered to pH 7.0 with 0.165M MOPS 83

(morpholinepropanesulfonic acid) (AppliChem, Darmstadt, Germany). 84

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Bioassay. The agar diffusion assay is a slight modification of a previously described 85

assay (21). Briefly, the yeast suspension was inoculated in standard medium with 15 g/L agar 86

(60 mL, 50oC) (Oxoid Ltd, Basingstoke, England), which was then dispensed into square 87

sterile plastic plates (10x10 cm) and was left to solidify at room temperature. Thereafter, 88

round wells were cut aseptically using a sterile cork borer in a well-spaced pattern. Sixty μL 89

of each standard, control or clinical sample were pipetted into individual wells of the plate. 90

After overnight incubation (37oC), growth inhibition was quantified by measuring the 91

diameter of zones of inhibited growth. Each run included one blank (drug-free serum control 92

to exclude the possibility that the inhibitory activity was due to serum components), 93

calibration standards and two external quality controls (HPLC-tested with known 94

concentrations of voriconazole provided by the external quality control program UKNEQAS). 95

Calibration standard samples containing 8-0.125 mg/L voriconazole were prepared by serial 96

two-fold dilutions of the stock solutions in different pooled sera from healthy human donors. 97

HPLC assay. A previously validated high performance liquid chromatography 98

(HPLC) method was used for cross-validation (22). Briefly, 40 μL of internal standard (12 99

μg/mL naproxen) were added into 200 μL of serum standard, quality control or serum sample. 100

Voriconazole extraction was performed with 30 μL of phosphate Buffer (pH= 3.1, 0.05 M) 101

and 400 μL MeOH. After vortex mixing (30 s) and centrifugation (1000g, 5 min), the 102

supernatant was evaporated under nitrogen stream, reconstituted in 100 μL of methanol and 103

30 μL were injected into the HPLC system. The chromatographic separation was performed 104

using a LiChrosorb®column (250×4.6 mm, 5 μm i.d.) with a compatible LiChrosorb® RP-105

C18 guard column. The temperature was maintained at 30oC throughout the measurement. 106

The mobile phase consisted of a filtered and degassed mixture of acetonitrile:sodium 107

dihydrogen phosphate (0.05M, pH=3.1) (55:45, v/v) and was delivered at a flow rate of 1.2 108

mL/min in the isocratic mode. Detection was achieved by monitoring the absorbance at 255 109

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nm. Measurements by each methodology (microbiological and chromatographic) were 110

performed blindly by two different investigators in triplicate. 111

Serum samples. A total of 103 serum samples from patients who received 112

voriconazole for different indications were analyzed: 39 from 21 patients receiving 113

voriconazole monotherapy, 39 external quality control program HPLC-tested samples 114

received from an interlaboratory proficiency testing program (NEQAS, North Bristol, UK) for 115

assessment of voriconazole levels and 25 originating from 7 patients receiving concurrent 116

therapy with echinocandins (5 with caspofungin and 2 with anidulafungin). Blood samples 117

were collected in evacuated blood collection tubes containing potassium EDTA (lavender top) 118

just before and 0.5h after drug administration in order obtain trough and peak concentrations, 119

respectively. Samples were then centrifuged at 4000g for 10 minutes and stored at -70°C. 120

Evaluation of bioassay. The diffusion assay was tested for linearity, analytical 121

sensitivity, reproducibility and specificity. The diameters of inhibition zones vs. standard drug 122

concentrations were analyzed with linear regression analysis. Intra- and inter-day 123

reproducibility were assessed by running 16 external quality control program samples with 124

voriconazole concentrations ranging from 0.3-7.5 mg/l in triplicate on non-consecutive days 125

and estimating the coefficient of variation (% CV). The effect of the presence of 126

echinocandins in determining voriconazole levels was evaluated by measuring voriconazole 127

levels with the bioassay in sera spiked with 0.5, 2 and 6 mg/L of voriconazole alone and 128

together with 1, 6 και 12 mg/L of each echinocandin. The latter concentrations were chosen 129

based on the clinically achievable concentrations in serum of patients (23–26). Data were 130

analyzed by conducting repeated measures ANOVA followed by Dunnett's multiple 131

comparison test. 132

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Correlation between bioassay and HPLC. The two methods were compared in a 133

quantitative and qualitative manner considering the HPLC as the reference method. For 134

quantitative analysis, the results of the two methods were analyzed with Spearman's rank 135

correlation coefficient (rs) and linear regression analysis in order to test whether the slope was 136

significantly different than 1. For the qualitative analysis, the categorical agreement between 137

the two methods was estimated as the % of serum samples lying lower than, within or higher 138

than the therapeutic window 2-5 mg/L with both methods (11,,27). 139

All analyses were performed with the statistics software package GraphPad Prism, 140

version 5.0, for Windows (GraphPad Software, San Diego, CA). 141

The standard curve of the diameter of inhibition zone-voriconazole concentration is 142

depicted in Figure 1. The lower limit of quantification (LLOQ) was determined to be 0.25 143

mg/L and the bioassay was internally validated over the range of 0.25 to 8 mg/L which 144

includes the therapeutic window as previously found (28, 29). Drug concentrations correlated 145

linearly with the diameter of inhibition zones (r2=0.98, p<0.0001) with mean (range) intra- 146

and inter-experimental variation 6% (0 to 12%) among all drug concentrations tested, which 147

is within the limits of acceptability of data established by international guidelines (30, 31). 148

None of the echinocandins’ concentrations produced a discernible inhibition zone in the 149

bioassay when tested alone except caspofungin at 12 mg/l. Regarding the effect of 150

echinocandins on voriconazole inhibition zones when the three echinocandins (1, 6 and 12 151

mg/L) were combined with voriconazole (0.5, 2 and 6 mg/L) in spiked human sera, there was 152

no difference between the inhibition zones of voriconazole alone and in the presence of each 153

echinocandin (ANOVA p>0.18). No interaction between voriconazole and echinocandins 154

against C. parapsilosis isolates has been previously reported (32–34). 155

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Voriconazole levels measured by the bioassay were significantly correlated with the 156

external quality control samples [n=39, rs=0.97 (95% CI 0.94-0.99; p<0.0001), slope of the 157

regression line: 1.000 ± 0.044; p=0.995] with mean±SEM % difference of 9±3.5% which 158

corresponded in mean±SEM difference in concentrations 0.17±0.10 mg/L (Figure 2). Drug 159

bioassay levels were also correlated with the HPLC results in sera from patients treated with 160

voriconazole monotherapy [n=39, rs=0.93 (95% CI 0.87-0.96; p<0.0001), slope of the 161

regression line: 1.013 ± 0.037; p=0.728] with mean±SEM % difference of 9±3.1% (0.34±0.12 162

mg/L). High correlation was found between the bioassay and the HPLC results in clinical 163

samples from patients treated with voriconazole-echinocandin combination therapy [n=25, 164

rs=0.98 (95% CI 0.95-0.99; p<0.0001), slope of the regression line: 0.912 ± 0.047; p=0.074] 165

with mean±SEM % difference of 12±3.8% (0.50±0.19 mg/L) (Figure 2). The aforementioned 166

deviations from HPLC values fulfill the criteria established by international recommendations 167

for accepting the accuracy of a method (30, 31). The overall categorical agreement between 168

the bioassay and the HPLC was 94%. For the remaining 6% (6/103) of the samples, the 169

bioassay resulted in mean±SEM 5±11% (0.46±0.60 mg/L) of HPLC concentrations (in 3 170

samples were lower and 3 higher than bioassay levels) at the upper limit of the therapeutic 171

concentration range in 5 patients receiving voriconazole monotherapy and in 1 patient 172

receiving combination therapy. 173

The present study reports for the first time in literature the validation of a simple 174

microbiological bioassay that can be used for TDM of voriconazole in patients on 175

combination therapy with echinocandins. The in house developed technique exhibits good 176

sensitivity (LLOQ 0.25 mg/L) and reproducibility (CV 6%) across the entire concentration 177

range tested, while its accuracy and reliability were ensured by validation with the reference 178

method. The bioassay is well correlated quantitatively and qualitatively with HPLC assay in 179

sera from patients treated with voriconazole alone and in combination with echinocandins 180

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(rs>0.93, 7-12% differences, slope 0.912-1.013). The overall categorical agreement between 181

the two methods was 94% with the remaining 6% of the samples representing borderline 182

deviations from the upper limit of the therapeutic concentration range. 183

The ideal method for performing TDM should be highly accurate (both sensitive and 184

specific), reproducible, rapid, inexpensive and require a relatively small volume of sample for 185

analysis. Several assays have been developed for quantification of voriconazole in human 186

blood. HPLC is still considered the gold standard, but the protocols used are characterized by 187

moderately laborious pre-analytical processes and are costly as specialized equipment and 188

trained personnel are needed, hindering their implementation in daily clinical laboratory 189

practice. In recent years there is a growing trend in developing protocols of liquid 190

chromatography in conjunction with mass spectrometry, which lead to rapid high-resolution 191

separation analysis but are extremely expensive and are not widely available for routine 192

clinical work-ups (35). As a result, microbiological assays represent an attractive alternative 193

testing methodology characterized by greater technical simplicity and lower cost. 194

When voriconazole is co-administered with other antifungal compounds 195

microbiological assays are not suitable to determine blood concentrations since they lack 196

specificity as inhibition zones may be influenced by any metabolite or drug that possesses 197

antifungal activity. However, for the first time in literature we developed and validated an 198

agar diffusion bioassay to quantify voriconazole levels in serum of patients on combination 199

therapy with echinocandins. For this purpose, a C. parapsilosis clinical isolate with high 200

levels of susceptibility to voriconazole (0.015 mg/L) and relatively low to all three 201

echinocandins (0.5-0.25 mg/L) was used. Afterwards, the full range of serum drug 202

concentrations that can be achieved in patients receiving the standard dosages, in accordance 203

with previous pharmacokinetic studies (23–26), was tested in vitro in order to exclude 204

potential interactions between them. Finally, the results were compared to reference method 205

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target values. The small number of serum specimens and patients on combination therapy 206

(particularly with subtherapeutic levels) may be considered a limitation of this study. 207

Nevertheless, the collection of such samples is difficult because antifungal combination 208

therapy has not yet been established and fungal infections are rarely treated empirically 209

following this strategy. 210

In the reported agar diffusion methods for measurement of voriconazole blood levels 211

from patients receiving monotherapy several strains have been used as test organism, i.e. a C. 212

kefyr (8, 12, 13), a S. cerevisiae (11), an azole-hypersusceptible C. albicans mutant (10) and 213

recently a Candida parapsilosis isolate (36). A standard microorganism for the performance 214

of this methodology has not yet been defined. Apparently, any voriconazole-susceptible 215

isolate providing well-defined and symmetrical zones of growth inhibition not affected by the 216

presence of echinocandins, as our own clinical C. parapsilosis strain, may be suitable after 217

performing validation studies. In the present study a simple and widely available medium was 218

used similar to the one recommended by CLSI and EUCAST for antifungal susceptibility 219

testing. A small volume of serum was utilized which may be particularly important for 220

neonates. The LLOQ and the linearity range obtained by our in house technique are slightly 221

better in comparison with those of previously reported methods (9, 11, 12), covering what is 222

currently believed to be the therapeutic range for voriconazole concentrations in human blood 223

(28, 29), while the concordance with the reference method is improved (9, 11, 12) or 224

comparable (8, 10, 13) to data from other studies. Like in a previous published assay (9), 225

voriconazole concentrations measured by HPLC were marginally higher than bioassay levels 226

although non-significant higher drug concentrations were also found with bioassays compared 227

to HPLC (8). 228

Since, in all previous studies the developed bioassays were suitable for use for TDM 229

only in patients who receive monotherapy with voriconazole, our study is unique because the 230

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present bioassay has been extensively validated for both patients on voriconazole 231

monotherapy and on combination therapy with an echinocandin. Studies with bioassays for 232

patients on antifungal combination therapy are limited and restricted to patients treated with 233

5-fluorocytosine plus amphotericin B without extensive validation (37, 38). 234

In conclusion, since voriconazole is often co-administered with echinocandins for 235

difficult-to-treat infections and its blood concentrations are characterized by considerable 236

variability and have been associated with adverse effects and unfavourable clinical response, 237

periodic monitoring of voriconazole levels has been recommended in order to avoid sub-238

therapeutic or toxic levels. In the present study, an easy and reliable microbiological assay 239

was developed for determination of voriconazole levels in serum of patients on combination 240

therapy with echinocandins, with good reproducibility and sensitivity. This method may be a 241

valid alternative tool to HPLC in clinical laboratories without specialized facilities. 242

243

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10

15

20

25

30

35

0.25 0.5 1 2 4 8

r2= 0.9788

Cvoriconazole (mg/L)

Dia

met

er o

f inh

ibiti

on (m

m)

386

387

Figure 1. The new bioassay and the standard curve of the diameter of inhibition zone (y axis) 388

-voriconazole concentration (x axis). Numbers above the holes represent voriconazole 389

concentrations whereas the numbers below the holes are the mean diameter of inhibition 390

zones. The bioassay showed linearity in the range of 0.25-8 mg/L with a correlation 391

coefficient of r2=0.98 in all runs. Error bars represent standard deviations. 392

8 mg/L

(31.5 mm)

2 mg/L

(22.5 mm

0.5 mg/L

(14 mm)

4 mg/L

(27.5 mm)

1 mg/L

(17.5 mm)

serum

(0 mm)

0.25 mg/L

(11.5 mm)

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A. External QC samples (n=39)

0 2 4 6 8 100

2

4

6

8

10

rs=0.9717, p<0.0001slope: 1.000 ± 0.044

HPLC (Cvoriconazole mg/L)

Bio

assa

y (C

voric

onaz

ole

mg/

L)

B. Voriconazole monotherapyclinical samples (n=39)

0 2 4 6 8 10 12 14 160

2

4

6

8

10

12

14

16

rs=0.9332, p<0.0001slope: 1.013 ± 0.037

HPLC (Cvoriconazole mg/L)

Bio

assa

y (C

voric

onaz

ole

mg/

L)

C. Voriconazole-echinocandin combinationtherapy clinical samples (n=25)

0 2 4 6 8 100

2

4

6

8

10

rs=0.9784, p<0.0001slope: 0.912 ± 0.047

HPLC (Cvoriconazole mg/L)

Bio

assa

y (C

voric

onaz

ole

mg/

L)

393 394

Figure 2. Scatter plots of voriconazole serum concentrations measured by HPLC and 395

bioassay in serum samples obtained from external quality program (A), patients on 396

voriconazole monotherapy (B) and patients on combination therapy with an echinocandin (C). 397

rs: Spearman's rank correlation coefficient with the p value, slope of the regression line ± 95% 398

confidence interval. Dotted lines represent the therapeutic window 2 to 5 mg/l. 399

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