Posaconazole Enhances the Activity of Amphotericin B against Hyphae of Zygomycetes In Vitro

Abstract

The in vitro activity of posaconazole plus amphotericin B against conidia and hyphae of 30 clinical zygomycetes was investigated. The combination of posaconazole with amphotericin B was found to be significantly more synergistic (40%) against hyphae (P < 0.05) than against conidia (10%). Antagonism was not observed.

Infections with zygomycetes are increasingly recognized in immunocompromised patients (7, 8, 18). The species within zygomycetes that are identified most commonly are those of the genera Rhizopus, Rhizomucor, Mucor, and Absidia (7). Presently, the standard therapy for treating these life-threatening infections consists of the removal of the predisposing factors, widespread surgical debridement, and high doses of intravenous amphotericin B (AMB) (2, 7). Nevertheless, even for patients who receive therapy, the rate of mortality is often above 50% (1, 7).

Posaconazole is a novel member of the triazole class of antifungals (12) which exhibits activity against zygomycetes in vitro and in vivo (1, 2, 5, 8, 19, 20). At present few data on combination treatments against zygomycetes are available (3, 4, 6, 10). The combination of posaconazole with caspofungin (10), as well as terbinafine with either AMB or voriconazole, showed synergistic effects against zygomycetes in vitro (6).

The aim of this study was to analyze the antifungal activity of posaconazole in combination with AMB against hyphae of zygomycete species, since they represent invasive disease. The results obtained with conidia served as the control.

A total of 30 clinical isolates of zygomycetes (Absidia corymbifera [n = 8], Rhizopus spp. [n = 6], Rhizomucor spp. [n = 5], Mucor spp. [n = 4], Syncephalastrum racemosum [n = 2], and Cunninghamella bertholletiae [n = 5]) were tested. MICs of posaconazole (kindly provided by Schering-Plough Research Institute, Kenilworth, NJ) and AMB (Sigma Aldrich, Vienna, Austria) for conidia were tested according to the Antifungal Susceptibility Testing Subcommittee of the European Committee on Antimicrobial Susceptibility Testing (AFST-EUCAST) (14). RPMI 1640 medium supplemented with 2% glucose as the assay medium and an inoculum size ranging from 2 × 10⁵ to 5 × 10⁵ CFU/ml were used (14). MICs for hyphae were evaluated by the method of Lass-Flörl et al. (15). In short, the conidial stock solutions were prepared as described above. Then, 100-μl samples of these solutions were added onto 96-well plates (Costar, Vienna, Austria) and incubated at 30°C for 16 to 22 h and allowed to form hyphae. This allowed the outgrowth of more than 95% of conidia, with a hyphal length that varied from 50 to 70 μm, as determined by examination with an inverted microscope. Wells were washed and refilled with 100 μl of RPMI 1640 medium-2% glucose, and the antifungal agents were added. All tests were performed twice and in duplicate. All drug end points were read visually as total growth inhibition for conidia and hyphae after 24 h of incubation at 35°C.

Drug combinations were assessed using a checkerboard method. Synergy tests were evaluated by using MIC end points of each drug. The fractional inhibitory concentrations (FIC) of each drug for an individual isolate were calculated (11). FIC index (FICI) values were interpreted as follows: a FICI value of ≤0.5 was synergistic, >0.5 to ≤4 was indifferent, and >4 was antagonistic. The results of the in vitro tests were compared by using the log-rank test. Significance was defined as a P value of <0.05. All tests were performed twice and in duplicate.

For the control, the metabolic activity was determined by the isolates' abilities to reduce the tetrazolium compound 2,3-bis(2-methoxy-4-nitro-5-sulfophenyl)-2 H-tetrazolium-5-carboxanilide (XTT) as described previously (9).

In our study, the MICs of posaconazole and AMB for conidia ranged from 0.25 to 8 μg/ml and from 0.5 to 4 μg/ml, respectively. Similar data were found by others (5, 19). In our study, the in vitro activity of posaconazole in combination with AMB against conidia of zygomycetes ranged from indifferent (90%) to synergistic (10%), as shown in Table 1. For this drug combination, no antagonism (FICI value of >4) was observed (Table 2). The comparison of the visually determined end points with the results of the XTT assay method revealed that 92% of the visually determined MICs corresponded to a 95% or greater reduction in metabolic activity, as measured by optical density.

TABLE 1.

FICI values and synergy results for conidia and hyphae of the zygomycetes tested

Species	Conidia		Hyphae
	FICI valuea	Synergy resultb	FICI value	Synergy result
Rhizomucor spp.	0.5	S	0.75	I
Rhizomucor spp.	0.8	I	0.75	I
Rhizomucor spp.	0.6	I	1	I
Rhizomucor spp.	0.5	S	1	I
Rhizomucor spp.	1	I	1	I
Cunninghamella bertholletiae	1.1	I	1	I
Cunninghamella bertholletiae	2.5	I	0.5	S
Cunninghamella bertholletiae	2.5	I	0.5	S
Cunninghamella bertholletiae	2.5	I	1	I
Cunninghamella bertholletiae	2	I	0.5	S
Mucor spp.	1.25	I	0.5	S
Mucor spp.	1.5	I	0.5	S
Mucor spp.	2	I	0.5	S
Mucor spp.	2	I	2	I
Rhizopus oryzae	0.75	I	1	I
Rhizopus oryzae	1.5	I	1.5	I
Rhizopus oryzae	1.25	I	1.25	I
Rhizopus microsporus	0.37	S	0.37	S
Rhizopus microsporus	1.5	I	0.37	S
Rhizopus microsporus	1.25	I	0.37	S
Absidia corymbifera	2	I	1	I
Absidia corymbifera	2	I	0.75	I
Absidia corymbifera	1.25	I	0.75	I
Absidia corymbifera	2	I	2	I
Absidia corymbifera	2	I	0.5	S
Absidia corymbifera	1	I	0.37	S
Absidia corymbifera	0.75	I	1.12	I
Absidia corymbifera	1.5	I	0.37	S
Syncephalastrum racemosum	1	I	0.75	I
Syncephalastrum racemosum	1.25	I	0.6	I

^aValues at 24 h. The FICI is the sum of FICs of each of the drugs and is defined as the MIC of each drug when used in combination divided by the MIC of the drug when used alone.

^bI, indifferent; S, synergistic.

TABLE 2.

FICI value results for the zygomycetes tested

Synergy result	Drug	% of the FICI values for conidia	% of the FICI values for hyphaea
Synergistic	POS-AMB	10	40*
Indifferent	POS-AMB	90	60

^a*, P < 0.05.

Few in vitro studies of combination therapies against zygomycete infection have been performed. Interestingly, combination therapies have all been shown to be effective against zygomycete infection in vitro and did not exert antagonism (3, 6, 10).

Like aspergilli, zygomycetes are respiratory pathogens, and infections are usually acquired through inhalation of conidia. In immunocompromised hosts, conidia are able to germinate, and the invasive form is associated with the appearance of wide ribbonlike, aseptate hyphae (2, 7). Consequently, an agent must be active against the hyphal form in order to be clinically effective. For hyphae, the MICs of posaconazole and AMB ranged from 1 to 8 μg/ml. The posaconazole-AMB combination exerted strong activity against hyphae of zygomycetes (Table 1) as FICI values were synergistic for 40% of isolates. Therefore, the synergism for hyphae was significantly more frequent (P < 0.05) than that observed for conidia (10%) (Table 2). The comparison of visually determined end points with the XTT assay method showed an agreement level of 89%. Similar results were obtained for Aspergillus species (17).

Our results suggest that a combination of AMB and posaconazole might be effective in some cases of zygomycosis. However, the precise mechanism of this finding is unknown. One possible explanation for the synergy could be that the binding of a polyene (AMB) to the fungus destabilizes the membrane and facilitates the entry of the azole (posaconazole). It is reasonable to suppose that the mechanisms involved in the inhibition of conidial germination are different from those that prevent hyphal elongation. Differences in the sterol compositions, the fungal cell membrane transporters, and the cell wall compositions within conidia and hyphae could account for our findings.

Due to the high mortality rate from infections with zygomycetes and the relatively limited efficacy of the current agents, the use of a combination therapy presents an interesting treatment option (11, 13). The potential advantages of combination therapy include the possibility of increased antifungal effects, as well as a lower dosing of toxic drugs and hence fewer side effects (13). Successful treatment with combination therapy has been reported for patients with zygomycosis (4, 16).

The lack of antagonism and the synergistic effects against hyphae found in our study suggest the use of posaconazole-AMB as an antifungal combination therapy against zygomycete infection. It will, however, be necessary to carry out further synergy tests in vivo to ascertain the clinical relevance of this drug combination.