Pakistan Journal of Medical Sciences


ISSN 1681-715X





Volume 23

April - June 2007 (Part-II)

Number 3


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In vitro activities of amphotericin-B in combination
with rifampin against Aspergillus species

Majid Zarrin1, Mohamed Reza Najafi2

Objective: The main goal of study was finding the synergism effect of amphotricin B (AMB) and rifampin (RIF) on 3 species of Aspergillus.
Methodology: Activities of amphotericin B in combination with rifampin were tested in buffered yeast-nitrogen base using checkerboard method. Plates were inoculated with 20µl spores suspensions of each organism and incubated at 30°C for 24h. For this method, the MICs were defined as the lowest antimicrobial concentration inhibiting visible fungal growth on the plates. Minimal fungicidal concentration was defined as the first tube showing no growth on the plate.
Results: The MIC of amphotericin B for 100% of isolates of A. fumigatus and A. flavus were inhibited by 4mg/lit amphotericin B. 100% of isolates of A. niger were inhibited by 8mg/lit amphotericin B. When amphotericin B was combined with rifampin, amphotericin B MICs decreased to 2, 1 and 4mg/lit in A. fumigatus, A. flavus rephrase and A. niger respectively.
Conclusion: The results indicate that combination of amphotreicin B and rifampin was synergistic on A. fumigatus, A. flavus and A. niger.

KEY WORDS: Aspergillus, Amphotreicin B, Rifampin, Synergism.

Pak J Med Sci   May - June 2007   Vol. 23 No. 3    323-325

1. Dr. Majid Zarrin
2. Dr. Mohamed Reza Nagafi
1-2: Department of Medical Parasitology and Mycology,
Jundi shapour University of Medical Sciences,
Ahwaz – Iran.


Dr. Majid Zarrin,

* Received for Publication: September 8, 2007
* Accepted: December 28, 2006


Invasive fungal infections (IFI) are most important causes of death in severely immunocompromised patient.1,2 A move in the epidemiology of IFIs has been observed over the past two decades, associated with an expansion of the antifungal armamentarium and a widening spectrum of immunosuppressive agents. Since the early 1990s, invasive aspergillosis has become the predominant IFI.1,2
Amphotericin B (AMB) is the drug of choice for treatment, but its use is restricted by its narrow therapeutic index.3 A few studies have been performed to test antifungal combinations against fungi. Combination of amphotericin B with rifampin has been confirmed to be synergistic in vitro against yeasts,4,5 dimorphic fungi,6,7 and Rhizopus.8 Interaction between amphotericin B and flucytosine has been revealed to be additive or synergistic against yeasts in vitro and in vivo in animal models as well as in patients.9 Concerning Aspergillus, indifferent-to-synergistic and in some instances antagonistic interactions have been reported for this combination.10
In vitro studies have indicated a possible role for combining rifampin with amphotericin againsts Aspergillus spp., though reports have provided conflicting results.11,12 Therapy with rifampin or flucytosine combined with amphotricin B was shown to increase survival over amphotericin B alone in a mouse model of disseminated A. fumigatus infection.13
In this in vitro study, we combined amphotericin B (AMB) with relevant concentration of rifampin (RIF) to screen for active combinations against A. fumigatus, A. flavus, and A. niger.


Organisms: Strains of A. fumigatus, A. flavus, & A. niger collected by the medical mycology laboratory at the Ahwaz Jondishapor of Medical Sciences. Cultures of A. fumigatus, A. flavus, and A. niger were grown on Sabouraud’s dextrose agar at 37°C for three days. Spores were obtained by swabbing the surface of the colonies with glass beads suspended in distilled water including tween 80. Final suspensions were adjusted to 90% transmittance at 530nm, corresponding to 106 CFU/m.
Drug solutions: Stock solutions of amphotericin B (Squibb) and Rifampin (Hakim) were dissolved in dimethy sulfoxide. These solutions were incubated at room temperature for 30 minutes for self-sterilisation. The drug solutions were then stored at -70°C for further experiences.
Method: The susceptibility testing of amphotericin B and rifampin were performed in yeast nitrogen base glucose (YNBG) agar buffered with phosphate buffer (pH 7). In the first step both amphotericin B and rifampin were tested separately. Final drug concentrations for amphotericin B and rifampin were 64, 32, 16, 8, 4,2,1,0.5 and 0.25mg/l. Combination rows each contained amphotericin B in dilutions from 8 to 0.5mg/ml with rifampin from 64 to 0.5mg/ml. Drug activity was assessed by a checkerboard method. Growth control tube contained media with and without each of drugs and dimethy sulfoxide in plates. Plates were inoculated with 20µl spores suspensions of each organism and incubated at 30°C for 24h. For this method, the MICs were defined as the lowest antimicrobial concentration inhibiting visible fungal growth on the plates. Minimal fungicidal concentration (MFC) was defined as the first tube showing no growth on the plate.
Statistics: P values were calculated by using the paired t test.


The checkerboard approach was used in this study to investigate the interaction between AMB and rifampin against isolates of A. fumigatus, A. flavus and A. niger. The susceptibility results obtained for the isolates are reported in Table-I.
All isolates were uniformly resistant to rifampin. In this study, 14 isolates of A. fumigatus, 30 isolates of A. flavus and 34 isolates of A. niger were tested. All isolates of A. fumigatus and A. flavus were inhibited by 4mg/lit amphotericin B. While all isolates of A. niger were inhibited by 8mg/lit amphotericin B (Table-I). Amphotericin B MICs ranged from 2.5 to 3.5mg/lit for A. fumigatus. The range of MICs for A. flavus and A. niger were 2.5-4 and 5.5-7 respectively.

The addition of rifampin to AMB gave variable results. When AMB was combined with rifampin, there were significant reductions in concentration of AMB. Synergy was demonstrated against A. fumigatus, A. flavus and A. niger. In all isolates, the concentration of AMB in combination with rifampin was reduced.
Table-I shows a list the MIC at which 100% of the strains were inhibited by AMB and amphotericin B + rifampin. When inhibitory effects of AMB were tested with rifampin, amphotricin B MICs decreased to 2,1 and 4mg/lit in A. fumigatus, A. flavus and A. niger respectively (Table-I). While AMB MIC for A. flavus was 2mg/lit with 16mg/lit of rifampin, it was decreased to 1mg/lit using 32mg/lit rifampin. Addition of 16mg/lit rifampin prevented growth of A. niger and A. fumigatus and felled the concentration of AMB to 4 and 2mg/lit respectively. The Table-I shows the activities of AMB with different concentration of rifampin.
Rifampin alone was inactive against all isolates tested. However, when sub inhibitory concentrations of AMB were used, rifampin MICs decreased significantly in isolates. No antagonism was noted with these drug combinations against any of the isolates tested.


The antifungal activity of AMB rifampin combination has been previously proven in vitro against a variety of fungal species including Candida spp,4 Histoplasma capsulatum,6 Coccidioides immitis,7 Cryptococcus neoformans,5 and zygomycetes.14 In each study, rifampin alone had no effect on the Aspergillus strains, while the addition of AMB resulted in obvious synergy at clinically achievable drug concentrations.
Kitahara et al15 previously verified a decrease of MICs of AMB against A. fumigatus with the addition of rifampin or flucytisine in concentrations higher than clinically achievable. Recently, amphotericin B has been tested in combination with many other drugs to determinate whether it possibly has improved activity when used in combinations.16,17 In vitro and in vivo studies have shown wide variations in effect when the polyene is combined with fluconazole or itraconazole.16,18
The lack of efficient antifungal therapy encouraged us to test the Aspergillus isolates against combinations of drugs that have been shown activity against other fungi. With establishing dose-response based on fungal spore suspensions, we were able to confirm our finding by the checkerboard method. Our data suggests that MIC strains were relatively higher to amphotericin B and the concentration of the amphotericin B was decreased significantly when added rifampin was added in the media. The lowest MIC was 1mg/l for A. flavus and the highest was 4mg/l for A. niger. Based on our data the addition of rifampin to amphotericin B might be expected to have enhanced activity against selected isolates. This study indicates that susceptibility tests in vitro may be valuable predictors of the most appropriate drug or drugs to use clinically against a given infecting isolate.


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