In Vitro Interactions between Antifungals and Immunosuppressants against Aspergillus fumigatus Isolates from Transplant and Nontransplant Patients

Abstract

We performed in vitro antifungal checkerboard testing on 12 Aspergillus fumigatus clinical isolates (6 transplant recipients and 6 nontransplant patients) with three antifungal agents (amphotericin B, voriconazole, and caspofungin) and three immunosuppressants (FK506, cyclosporine, and rapamycin). We were not able to detect a difference in calcineurin inhibitor antifungal activity against isolates from transplant recipients and nontransplant patients.

The incidence of invasive aspergillosis (IA) (13, 23) has increased significantly in the last several decades, paralleling the increase in the number of immunocompromised patients. While there has been a recent expansion of the antifungal armamentarium against IA (32), optimal treatment remains elusive, and newer agents with novel mechanisms are desperately needed. The immunosuppressants cyclosporine (CsA), tacrolimus (FK506), and rapamycin all suppress T-cell proliferative responses. CsA and FK506 inhibit calcineurin, a Ca²⁺/calmodulin-dependent protein phosphatase important in cell signaling (4, 17), while rapamycin functions through a separate TOR signaling pathway (28). These immunosuppressants have revolutionized modern transplantation, but their role as potential antifungal agents is only beginning to be understood (3, 12). The calcineurin pathway has been shown to be critical in fungal survival and stress response in several fungi (26, 29), including in vitro antifungal activity against Saccharomyces cerevisiae, Candida albicans, and Cryptococcus neoformans (5-9, 25) and fungicidal synergism between calcineurin inhibitors and the normally fungistatic azole antifungals (19-22, 27).

We previously reported the inherent in vitro antifungal activities of CsA and FK506 against Aspergillus fumigatus, as well as a positive interaction between CsA or FK506 and caspofungin against A. fumigatus (31). These results highlight the potential role that inhibition of the calcineurin stress response pathway could play in the treatment of IA. Here we investigated the in vitro antifungal activity of calcineurin inhibition against A. fumigatus clinical isolates from transplant patients already receiving calcineurin inhibitors as part of their immunosuppressive regimen versus the antifungal activity against clinical isolates from patients who did not receive a transplant and have never received a calcineurin inhibitor. Since it is known that patients on calcineurin inhibitors can develop IA, it is important to examine the development of direct drug resistance as a failure to prevent IA.

Six strains of A. fumigatus from nontransplant patients and six strains of A. fumigatus from transplant recipients (Table 1) were used. The nontransplant A. fumigatus strains were all clinical isolates from patients with IA from Duke University Medical Center, and the transplant recipient A. fumigatus strains were all clinical isolates from patients with IA at the University of Pittsburgh.

TABLE 1.

A. fumigatus clinical isolates from Transplant recipients and nontransplant patients

Patient group and isolate	Transplant or underlying disease	Immunosuppressant(s) patient receiving (no. of days received when culture obtained)	Antifungal prophylaxis	Site of IA	Treatment of IAa	Patient outcome
Transplant recipients
1	Heart	CsA (75), mycophenolate	AmB	Pulmonary	ABLC, posaconazole	Alive
2	Double lung	FK506 (90), azathioprine	Itraconazole	Pulmonary	ABLC	Alive
3	Liver	FK506 (21)	None	Pulmonary	ABLC	Died
4	Liver (2nd)	FK506 (36)	None	Disseminated	L-AmB	Died
5	Liver or kidney	FK506 (25)	None	Pulmonary	ABLC	Alive
6	Single lung	FK506 (80)	None	Pulmonary	ABLC, caspofungin	Died
Nontransplant patients
1	AMLb	None	None	Pulmonary	AmB	Alive
2	Competent	None	None	Osteomyelitis	AmB	Alive
3	Competent	None	None	Endocarditis	N/A	Died
4	Sarcoidosis	None	None	Disseminated	AmB	Died
5	Lung cancer	None	None	Paravertebral	AmB, itraconazole	Died
6	Competent	None	None	Osteomyelitis	AmB	N/A

^aABLC, amphotericin B lipid complex; L-AmB, liposomal amphotericin B; AmB, amphotericin B; NA, data unknown.

^bAML, acute myelogenous leukemia.

Amphotericin B (Fungizone; Bristol-Myers Squibb Co., New York, N.Y.), caspofungin (Cancidas; Merck & Co., Rahway, N.J.), and voriconazole (Vfend; Pfizer, Inc., New York, N.Y.) were obtained as powders and prepared as outlined in the National Committee for Clinical Laboratory Standards (NCCLS) M38-A document (24). Rapamycin was obtained from the National Cancer Institute, FK506 was supplied by Fujisawa Healthcare, Inc. (Deerfield, Ill.), and CsA was purchased from Alexis Corporation.

In vitro MIC testing with amphotericin B, voriconazole, caspofungin, and the immunosuppressants was performed by the NCCLS M38-A microdilution method (24), modified for diluting the five immunosuppressants, with Candida parapsilosis ATCC 22019 as an antifungal control as previously described (31). We used an MIC-0 endpoint (optically clear well) for voriconazole and amphotericin B and an MIC-2 endpoint (prominent growth reduction or a 50% reduction in optical density) for caspofungin and the immunosuppressants. Additionally, for caspofungin-treated wells, we reported the minimum effective concentration, which we defined as the formation of aberrant hyphal tips seen by microscopy (1).

We also performed a total of 108 checkerboard tests, one for each immunosuppressant with each antifungal for each of the 12 isolates. The fractional inhibitory concentration (FIC) of a drug was defined as the MIC of the drug given in a combination divided by the MIC of the drug alone. An FIC index (FICI) (ΣFIC) is the sum of the two FICs of the individual drugs (11). An FICI value of <0.5 indicated synergy, values of 0.5 to 4 indicated indifference, and a value of >4 indicated antagonism. We used nonparametric methods (Wilcoxon rank sum) to generate P values. Analyses were completed using Stata 8.1 statistical software (Stata Corp., College Station, Tex.).

CsA and FK506 both showed consistent in vitro antifungal activity against all 12 A. fumigatus isolates (Table 2). Rapamycin showed excellent activity at 24 h (data not shown) but lost all antifungal activity by 48 h. This work confirmed that FK506 and CsA possess inherent in vitro antifungal activity against A. fumigatus as previously described (31). We were not able to detect a difference in the MICs for the transplant and nontransplant clinical isolates.

TABLE 2.

In vitro 48-h MIC for antifungals and Nontransplant immunosuppressants against A. fumigatus isolates^a

Antifungal	MICb (μg/ml) (range) of drugs against isolates from:		P valuec
	Transplant recipients (n = 6)	Nontransplant patients (n = 6)
Amphotericin B	2.24 (1-4)	1.85 (1-2)	0.14
Voriconazole	0.38 (0.25-2)	0.24 (0.125-0.5)	0.09
Caspofungind	0.82 (0.5-2)	0.65 (0.5-1)	0.79
FK506	0.36 (0.097-1.56)	0.21 (0.097-3.125)	0.39
Cyclosporine	4.96 (1.56-50)	3.37 (1.56-25)	0.55
Rapamycin	50 (N/Ae)	50 (N/A)	0.99

^aMIC-0 for voriconazole and amphotericin B; MIC-2 for the other drugs.

^bGeometric mean.

^cP values for the values for the isolates from transplant recipients and nontransplant patients.

^dMinimum effective concentration given for caspofungin.

^eN/A, data unknown.

Checkerboard microdilution assays (Table 3) with isolates from transplant recipients versus isolates from nontransplant patients detected no difference between the two groups of isolates. The antifungal plus immunosuppressant combination with the lowest FICI was caspofungin plus FK506, while the combinations of voriconazole and an immunosuppressant yielded higher FICIs. These results are similar to our previous studies with other strains of A. fumigatus (31). In the isolates tested, there was a general interaction indifference between the antifungals and immunosuppressants on the basis of FICI calculation.

TABLE 3.

FICIs for antifungal plus immunosuppressant combinations against A. fumigatus isolates

Antifungal combination	FICIa (range) of drugs against isolates from:		P valueb
	Transplant recipients (n = 6)	Nontransplant patients (n = 6)
Amphotericin B + FK506	1.68 (1-2.5)	1.64 (1-3)	0.80
Amphotericin B + CsA	2.12 (1.5-3)	2.09 (1-3)	0.80
Amphotericin B + rapamycin	1.60 (1.25-2)	1.65 (1.5-2)	0.85
Voriconazole + FK506	3.97 (2.5-6.1)	3.39 (2.25-6.1)	0.46
Voriconazole + CsA	3.34 (2-4)	3.21 (2.25-4.5)	0.75
Voriconazole + rapamycin	2.69 (2-5)	3.06 (2-5)	0.49
Caspofungin + FK506	0.85 (0.57-1.5)	1.05 (0.57-1.24)	0.23
Caspofungin + CsA	1.50 (1.06-3)	1.16 (0.56-2)	0.57
Caspofungin + rapamycin	2 (N/Ac)	2 (N/A)	0.99

^aGeometric mean.

^bP values for the values for the isolates from transplant recipients and nontransplant patients.

^cN/A, data unknown.

Host immunosuppression is a well-documented risk factor for IA (23), and optimal management of immunosuppressant therapy is crucial in the treatment of IA in transplant recipients. There is a critical balance between maintaining immunosuppression to sustain the transplanted graft and increased immunosuppression predisposing the patient to opportunistic fungal infection.

This in vitro antifungal activity of calcineurin inhibitors may have clinical benefits. For instance, the use of CsA yielded a 54% decrease in the incidence of IA in 126 heart transplant patients (15). A study of liver transplant recipients with IA showed a significantly lower rate of disseminated (30 versus 62%) and central nervous system infection (0 versus 46%) in a cohort of patients who received FK506 (30).

There has been limited previous in vitro evaluation of calcineurin inhibitors against A. fumigatus (2, 14, 16, 18, 31). Transplant recipients are one of the highest risk patient groups to develop IA (10), and it is this group of patients who are generally already receiving calcineurin inhibitors as part of their immunosuppressive regimen. Since the calcineurin inhibitors are being used in patients who developed IA, it is possible to hypothesize several reasons for its failure as an antifungal. First, the immunosuppressive activity is greater than the direct antifungal activity, and the increased host immunosuppression outweighs the antifungal effect. Second, there is the potential inability to achieve sustained levels of calcineurin inhibitors in serum or tissue that are above the inhibitory concentration for Aspergillus. Third, there could be a rapid development of drug resistance to the anti-Aspergillus activity of the calcineurin inhibitors in vivo. In this study, we were not able to detect any evidence for the third possibility of rapid development of drug resistance, since the in vitro anti-Aspergillus activity of the calcineurin inhibitors was sustained in groups with or without exposure to FK506 or cyclosporine.

This study again demonstrates the inherent in vitro antifungal activity of calcineurin inhibitors against A. fumigatus. Importantly, this is the first study evaluating calcineurin inhibitors utilizing clinical isolates from transplant recipients who were receiving calcineurin inhibitors versus isolates from nontransplant patients who were not receiving calcineurin inhibitors. We were unable to detect a difference in antifungal activity in the isolates from the different groups of patients. One plausible explanation relates to small sample size; specifically, despite testing 108 checkerboard assays, we tested only a total of 12 isolates. Alternatively, these results could imply that preexposure to calcineurin inhibitors did not alter their in vitro antifungal activity against the clinical isolates.

The patients at highest risk for IA are often already on immunosuppressants, so determining the optimal combination and balance of immunosuppressive and antifungal activity to combat IA will be crucial. The calcineurin inhibitors in use at this time have similar in vitro activities in isolates from transplant recipients and nontransplant patients, suggesting that long-term exposure to these drugs did not decrease their antifungal activity. Despite this in vitro antifungal activity of the calcineurin inhibitors, patients receiving these agents developed and succumbed to this infection. Therefore, in our opinion, the focus for this area of antifungal drug development with calcineurin inhibitors is the development of nonimmunosuppressive inhibitors with more potent anti-Aspergillus activity.