JAC Advance Access originally published online on December 2, 2008
Journal of Antimicrobial Chemotherapy 2009 63(2):340-342; doi:10.1093/jac/dkn494
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Original research |
In vitro interactions of micafungin with amphotericin B, itraconazole or fluconazole against the pathogenic phase of Penicillium marneffei
1 Department of Dermatology, Peking University First Hospital, Research Center for Medical Mycology, Peking University, Beijing, P.R. China 2 Department of Dermatology, The First Affiliated Hospital of Guangxi Medical University, Nanning, P.R. China
* Corresponding author. Tel: +86-10-66551122, ext. 3056; Fax: +86-10-66551216; E-mail: lrymm{at}medmail.com.cn
Received 19 July 2008; returned 2 September 2008; revised 9 November 2008; accepted 11 November 2008
 |
Abstract |
|---|
 Top Abstract IntroductionMaterials and methodsResults and discussionFundingTransparency declarationsReferences |
|---|
Objectives: Penicillium marneffei infection is an important disease among human immunodeficiency virus patients in south-east Asia, including southern China. However, therapeutic strategies are limited. Combination regimens with synergistic drugs could provide additional options for treating penicilliosis. We evaluated the in vitro efficacy of combining micafungin with amphotericin B, itraconazole or fluconazole against the pathogenic yeast form of P. marneffei.
Methods: Twenty isolates of P. marneffei were assayed. Drug interactions were assessed with the chequerboard technique using the CLSI (formerly the NCCLS) microdilution method (M27-A2) with minor modifications. The fractional inhibitory concentration index (FICI) was used to classify drug interactions. Results were interpreted as follows: synergy, FICI 
0.5; no interaction, FICI >0.5 and 4.0; or antagonism, FICI >4.0.
Results: The in vitro interactions of micafungin combined with itraconazole showed the highest percentage of synergic interaction (65%); for the micafungin/amphotericin B combination, 50% of the isolates had synergic interaction. Micafungin significantly enhanced the antifungal activity of amphotericin B and itraconazole against P. marneffei. Micafungin, however, did not enhance the activity of fluconazole and no synergism was observed with this combination. Antagonism was not detected for any of the antifungal combinations assayed.
Conclusions: The results of this study suggest that micafungin enhances the efficacy of itraconazole or amphotericin B in vitro and indicate that an echinocandin, such as micafungin, might have a potential role in combination therapy among patients infected with P. marneffei.
Keywords: dimorphic fungi , antifungal susceptibility , combined therapy
|
Introduction |
|---|
|
TopAbstractIntroductionMaterials and methodsResults and discussionFundingTransparency declarationsReferences |
|---|
Penicillium marneffei is an emerging opportunistic dimorphic fungal pathogen that is endemic in south-east Asia and the southern part of China. The fungus can cause a life-threatening widespread disseminated mycosis in humans. In recent decades, P. marneffei infections have increased sharply in patients with human immunodeficiency virus (HIV) and is becoming an indicator disease for AIDS.1
The mortality rate of patients with P. marneffei infection has been very high. Patients who do not receive the appropriate antifungal treatment have a poor prognosis. Amphotericin B is still the drug of choice for primary treatment of this mycosis,2 regardless of the immunological condition of the patient. However, its strong toxicity in many patients makes the effective management of severe infections difficult to impossible. AIDS patients with P. marneffei infection who survive the initial disease phase are treated with a long duration of suppressive therapy to reduce the likelihood of recurrent infection. For suppression therapy, itraconazole and fluconazole are presently the agents of choice. The toxicity associated with amphotericin B necessitates the testing of new therapeutic strategies to reduce the antifungal doses and to obtain better clinical results. A promising approach might be to combine antifungal drugs with different active mechanisms. However, little is known about the action of different antifungal drugs against P. marneffei. The advent of new echinocandins that can be combined with amphotericin B or azoles has revived interest in finding antifungal combinations that may be synergistic, resulting in improved treatment of P. marneffei infections. In this study, we evaluated the combined activity of micafungin, an echinocandin antifungal drug, with amphotericin B and two conventional antifungal azoles (itraconazole and fluconazole) against the pathogenic yeast phase of 20 isolates of P. marneffei using the chequerboard microdilution method.
|
Materials and methods |
|---|
|
TopAbstractIntroductionMaterials and methodsResults and discussionFundingTransparency declarationsReferences |
|---|
Organisms
A total of 20 P. marneffei isolates were evaluated, which were maintained by the Research Center for Medical Mycology, Peking University, People's Republic of China. Fourteen isolates were obtained from patients while five isolates were collected from bamboo rats. P. marneffei type strain FRR2161 was also evaluated in the study, which was generously provided by Professor Alex Andrianopoulos, Department of Genetics, University of Melbourne, Australia.
Antifungal agents were obtained as pure powders. Amphotericin B (Sigma, USA) and itraconazole (Janssen Pharmaceutica, Xian, China) were diluted in 100% dimethyl sulphoxide. Micafungin (Fujisawa Pharmaceutical Co. Ltd, Osaka, Japan) and fluconazole (Pfizer Inc., Madrid, Spain) were diluted in sterile distilled water. Stock solutions were diluted in RPMI 1640 medium (Sigma Chemical Co., St Louis, MO, USA) and then further serially diluted 2-fold, yielding four times the final strength required for the test.
To obtain the yeast phase of P. marneffei, isolates were cultured on brain heart infusion agar containing 1% glucose at 37°C and maintained by continuous weekly passages. The cells were harvested 4–6 days after the last passage and dispersed with a sterile cotton swab in a small volume of sterile saline. The cells were then suspended in saline and thoroughly vortexed. If large aggregates existed, they were allowed to settle for several minutes and the supernatants were collected. The number of yeast cells was estimated by counting with a haemocytometer in lactophenol cotton blue. The suspensions were then diluted in RPMI medium to 1 x 106 to 5 x 106 cfu/mL. The final inocula concentrations in the drug dilutions were 
1 x 104 to 5 x 104 cfu/mL.
Interactions of drugs in vitro
Drug interactions were assessed by a chequerboard microdilution method that also included the determination of the MIC of each drug alone in the same plate using the guidelines presented in the CLSI (formerly the NCCLS) document M27-A2 with minor modifications.3 Antifungal agents were placed in the rows or in the columns of the trays to perform all possible combinations, with concentrations from 8 to 0.03 mg/L for amphotericin B, 4 to 0.0125 mg/L for itraconazole, 64 to 0.25 mg/L for fluconazole and 16 to 0.125 mg/L for micafungin. The plates were incubated at 37°C for 72 h. Candida parapsilosis ATCC 22019 was included as a control. For all the drugs and their combinations, an MIC-0 endpoint criterion was used, which was defined as the lowest concentration resulting in 100% inhibition of visible fungal growth after 72 h of incubation. Duplicate testing was performed on three different days.
The fractional inhibitory concentration index (FICI) was used to classify drug interaction. The FICI is the sum of the FIC of each of the drugs, which in turn was defined as the MIC of each drug when used in combination divided by the MIC of the drug when used alone. The FICI was calculated and defined as: synergic if the FICI was 0.5; neither synergistic nor antagonistic if the FICI was >0.5 and 4; and antagonistic if the FICI was >4.
|
Results and discussion |
|---|
|
TopAbstractIntroductionMaterials and methodsResults and discussionFundingTransparency declarationsReferences |
|---|
Table 1 shows four antifungal drugs combined or alone against the yeast form of 20 isolates of P. marneffei. In micafungin/itraconazole and micafungin/amphotericin B combinations, 65% and 50% of tested isolates showed synergism, respectively. Potentially, one of the most significant results of this study is the reduction of MICs shown by the combination of micafungin with amphotericin B or itraconazole. The geometric mean of the MICs of the three drugs dropped dramatically when they were used in combination. The MICs of both amphotericin B and itraconazole were reduced 2-fold in more than half of the tested isolates, which demonstrated that micafungin could enhance the activity of the two drugs against some isolates of the pathogenic form of P. marneffei. The MICs of micafungin in the two combinations had two to six dilution reductions compared with micafungin tested alone, and were below peak plasma levels achieved by this drug in humans4 and in experimental infections.5 It was previously reported that micafungin had very weak activity against the yeast form compared with the mycelial form of P. marneffei.6 In this study, micafungin tested alone was also inactive against the yeast form of the 20 isolates, but when combined with amphotericin B or itraconazole, micafungin was obviously active against the test isolates.
|
However, the MIC of fluconazole in the micafungin/fluconazole combination had no significant difference when compared with fluconazole tested alone: the MIC ranged from 2 to 8 mg/L, which showed that fluconazole was the least effective against P. marneffei when compared with amphotericin B and itraconazole. Micafungin did not enhance the activity of fluconazole against the yeast form of P. marneffei.
In vitro combinations of echinocandins such as caspofungin or micafungin with amphotericin B and triazoles showed additive or synergic antifungal activity in several fungi.7,8 The mechanisms by which micafungin enhances other antifungal activities are by inhibiting cell wall synthesis and increasing the access of these drugs to the cell membrane.9 In this study, the results were encouraging. Our in vitro data suggest that therapies combined with micafungin would allow for the use of lower doses of amphotericin B and itraconazole without the loss of a clinical response. The results obtained in this study encourage us to conduct further studies with animal models to confirm the potential of these combinations for the treatment of P. marneffei infections in humans.
|
Funding |
|---|
|
TopAbstractIntroductionMaterials and methodsResults and discussionFundingTransparency declarationsReferences |
|---|
This work was supported by the Specialized Research Fund for the Doctoral Program of Higher Education of China, and the Beijing Municipal Natural Science Foundation (no. 7072081).
|
Transparency declarations |
|---|
|
TopAbstractIntroductionMaterials and methodsResults and discussionFundingTransparency declarationsReferences |
|---|
None to declare.
|
Acknowledgements |
|---|
We thank Professor Glenn S. Bulmer for help in preparing this manuscript.
|
References |
|---|
|
TopAbstractIntroductionMaterials and methodsResults and discussionFundingTransparency declarationsReferences |
|---|
1 Vanittanakom N, Cooper CR Jr, Fisher MC, et al. Penicillium marneffei infection and recent advances in the epidemiology and molecular biology aspects. Clin Microbiol Rev (2006) 19:95–110.
2
Supparatpinyo K, Perriens J, Nelson KE, et al. A controlled trial of itraconazole to prevent relapse of Penicillium marneffei infection in patients infected with the human immunodeficiency virus. N Engl J Med (1998) 339:1739–43.
3 National Committee for Clinical Laboratory Standards. Reference Method for Broth Dilution Antifungal Susceptibility Testing of Yeasts—Second Edition: Approved Standard M27-A2 (2002) Wayne, PA, USA: NCCLS.
4 Denning DW. Echinocandin antifungal drugs. Lancet (2003) 362:1142–51.[CrossRef][Web of Science][Medline]
5
Groll AH, Mickiene D, Petraitis V, et al. Compartmental pharmacokinetics and tissue distribution of the antifungal echinocandin lipopeptide micafungin (FK463) in rabbits. Antimicrob Agents Chemother (2001) 45:3322–7.
6
Nakai T, Uno J, Ikeda F, et al. In vitro antifungal activity of micafungin (FK463) against dimorphic fungi: comparison of yeast-like and mycelial forms. Antimicrob Agents Chemother (2003) 47:1376–81.
7
Letscher-Bru V, Herbrecht R. Caspofungin: the first representative of a new antifungal class. J Antimicrob Chemother (2003) 51:513–21.
8
Serena C, Marine M, Pastor FJ, et al. In vitro interaction of micafungin with conventional and new antifungals against clinical isolates of Trichosporon, Sporobolomyces and Rhodotorula. J Antimicrob Chemother (2005) 55:1020–3.
9
Franzot SP, Casadevall A. Pneumocandin L-743,872 enhances the activities of amphotericin B and fluconazole against Cryptococcus neoformans in vitro. Antimicrob Agents Chemother (1997) 41:331–6.
CiteULike 
Connotea 
Del.icio.us What's this?
This article has been cited by other articles:
|
|
 |
|
  H. L VandenBussche and D. A Van Loo A Clinical Review of Echinocandins in Pediatric Patients Ann. Pharmacother., January 1, 2010; 44(1): 166 - 177. [Abstract] [Full Text] [PDF]
|
|
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||