Antifungal Susceptibilities of Aspergillus fumigatus Clinical Isolates Obtained in Nagasaki, Japan

Abstract

We investigated the triazole, amphotericin B, and micafungin susceptibilities of 196 A. fumigatus clinical isolates in Nagasaki, Japan. The percentages of non-wild-type (non-WT) isolates for which MICs of itraconazole, posaconazole, and voriconazole were above the ECV were 7.1%, 2.6%, and 4.1%, respectively. A G54 mutation in cyp51A was detected in 64.2% (9/14 isolates) and 100% (5/5 isolates) of non-WT isolates for itraconazole and posaconazole, respectively. Amphotericin B MICs of ≥2 μg/ml and micafungin minimum effective concentrations (MECs) of ≥16 μg/ml were recorded for two and one isolates, respectively.

TEXT

The clinical importance of Aspergillus infection has increased as the number of immunocompromised patients has risen (16). Antifungals recommended for treatment of patients with invasive pulmonary aspergillosis (IPA) or chronic pulmonary aspergillosis (CPA) are triazoles, amphotericin B, and echinocandins (13, 15, 37). Patients with CPA often need years of treatment (13, 37). Although oral therapy is important for carrying out long courses of treatment, azoles (with the exception of fluconazole) are the only class of oral drugs licensed for the treatment of aspergillosis (14, 37).

Aspergillus fumigatus is the most common and pathogenic species of Aspergillus (34, 37). Antifungal resistance of A. fumigatus, especially to azoles, is one of the concerns in treatment of aspergillosis. During the last decade, many cases of treatment failure due to azole-resistant Aspergillus infection have been reported, and in the past few years a growing body of papers about antifungal susceptibilities of A. fumigatus has been accumulating (1, 3–6, 9, 10, 12, 18, 23–27, 31–33, 35, 36). Even though an increased rate of azole resistance has been reported recently in the Netherlands and the United Kingdom, the prevalence of azole resistance reportedly remains low in other countries (1, 3, 6, 9, 12, 23, 25, 33).

The azole target protein lanosterol 14α-demethylase of Aspergillus is encoded by the cyp51A gene, and mutations of cyp51A are a major mechanism of azole resistance (8, 17, 19, 20, 22, 32). Some mutational hotspots, such as G54, M220, and TR/L98H, have been identified as causes of azole resistance (2, 21, 22). Of these mutations, TR/L98H is especially prevalent in the Netherlands. An environmental origin (resulting from agricultural antifungal drug usage) is suspected, in spite of the fact that the mechanism(s) of mutation induction has not been shown definitively (24, 31, 32).

We studied the antifungal susceptibility of 196 A. fumigatus clinical isolates obtained in the Pneumology Department of Nagasaki University Hospital, Nagasaki, Japan. The isolates were collected between February 1994 and April 2010. All of the isolates were subjected to susceptibility testing and cyp51A sequence analysis. All isolates were identified as A. fumigatus by macroscopic colony morphology, micromorphological characteristics, and the ability to grow at 48°C. Non-wild-type (non-WT) isolates were subjected to additional molecular identification by amplification of ribosomal internal transcribed spacers (ITSs) and ribosomal large-subunit D1-D2 sequencing as described previously (11). MICs of itraconazole, posaconazole, voriconazole, and amphotericin B and minimum effective concentrations (MECs) of micafungin were determined using the Clinical and Laboratory Standards Institute (CLSI) M38-A2 broth microdilution method. Assays were performed using RPMI 1640 broth (0.2% dextrose), final inoculum concentrations ranging from 0.4 × 10⁴ to 5 × 10⁴ CFU/ml, and 48 h of incubation at 35°C (7). The MIC was defined as the lowest drug concentration that produced complete growth inhibition; the MEC was read as the lowest concentration of drug that led to the growth of small, rounded, compact hyphal forms compared to the hyphal growth seen in the control well. Susceptibility tests of non-WT isolates were performed at least three times for each isolate; each test was performed on different days. Because clinical breakpoints have not been established yet, we used epidemiological cutoff values (ECVs) to evaluate azole susceptibility (9, 25, 29). Wild-type (WT) isolates of A. fumigatus (MIC ≤ ECV) were distinguished from non-WT isolates (MIC > ECV), which may exhibit acquired low-susceptibility mechanisms. ECVs used in this study were as follows: itraconazole, 1 μg/ml; posaconazole, 0.5 μg/ml; voriconazole, 1 μg/ml, all as previously suggested (9, 25).

For sequence analyses, genomic DNA was extracted from non-WT isolates using a MasterPure yeast DNA purification kit (Epicentre Biotechnologies, Madison, WI). The full coding region of the cyp51A gene was amplified as previously described (32). DNA sequences were determined using a BigDye Terminator version 1.1 cycle sequencing kit (ABI) and an ABI 3100xl DNA analyzer. Sequence alignments were performed against the sequence from an azole-susceptible strain (GenBank accession no. AF338659) using MacVector10.0 software (MacVector, Inc., Cary, NC) (20). Mutations were confirmed three times by repeating the PCR and sequencing the relevant region using the closest primer.

In this study, using the ECVs, the percentages of non-WT isolates for which MICs of itraconazole, posaconazole, and voriconazole were above the ECV were 7.1%, 2.6%, and 4.1%, respectively (Table 1). To exclude the possibility of increased proportions of non-WT isolates due to clonal spread (notably, the potential repeated isolation of a drug-resistant strain originating from one patient), we confirmed those proportions on a per-case basis, which (for non-WT isolates) were 7.5%, 4.3%, and 6.5% for itraconazole, posaconazole, and voriconazole, respectively. These proportions of non-WT isolates were not much different from previous data from other regions, with the exception of data for the Netherlands and the United Kingdom (3, 9, 12, 23, 25, 33). All the itraconazole-resistant isolates (MIC ≥ 4 μg/ml) were obtained from 1998 to 2001. No consistent trend of increased proportion of non-WT isolates was observed. Amphotericin B MICs of ≥2 μg/ml were recorded for 1.0% of the isolates (2/196); micafungin MECs of ≥16 μg/ml were recorded for 1.0% of the isolates (2/196) (Table 1). For these antifungals, the proportions of resistant isolates were low and similar to those in previous reports (3, 10, 23, 26).

Table 1.

MIC and MEC distributions of five antifungals

Antifungal agent	No. of isolatesa											% of non-WT isolates
	Total	With a MIC or MECb (μg/ml) of:
		≤0.03	0.06	0.125	0.25	0.5	1.0	2.0	4.0	8.0	≥16
Triazoles
Itraconazole	182 (14)			1	28	107	46	(8)	(4)	(1)	(1)	7.1
Posaconazole	182 (14)	14	108	35	24 (2)	(8)	(1)	1 (2)			(1)	2.6
Voriconazole	182 (14)			1 (3)	20 (7)	101 (2)	53 (1)	7 (1)				4.1
Polyene
Amphotericin B	182 (14)		6	8 (1)	19 (1)	90 (7)	57 (5)	2
Echinocandin
Micafungin	182 (14)	177 (14)	2	1							2

^aMIC distributions for each agent were obtained by subtracting from the total isolates tested the 14 non-WT isolates resistant to itraconazole. The MIC distribution for the 14 non-WT isolates is in parentheses.

^bMICs are shown for amphotericin B, itraconazole, posaconazole, and voriconazole; MECs are shown for micafungin.

In Japan, posaconazole has not been approved for clinical use; nonetheless, non-WT isolates for posaconazole already existed (Table 1). Resistance in these isolates might reflect native biological variability. Alternatively, this phenomenon could be associated with cross-resistance between itraconazole and posaconazole, because 80% (4/5) of non-WT isolates for posaconazole were also non-WT isolates for itraconazole (Table 2). In addition, non-WT isolates for itraconazole tended to be more resistant to posaconazole, though not to voriconazole (Table 1). Cross-resistance between itraconazole and posaconazole, but not with voriconazole, may result from the G54 mutation of cyp51A, which was present in 64.2% (9/14) of the non-WT isolates for itraconazole and also present in 100% (5/5) of the non-WT isolates for posaconazole (Table 2). There is a known structural basis for the association of the G54 mutation with this pattern of cross-resistance among the azoles: unlike voriconazole, itraconazole and posaconazole have long side chains that clash with the amino acid side chain of the residue replacing G54 in the mutated CYP51A protein (8, 27, 32, 38).

Table 2.

MICs and Cyp51A substitutions in 22 non-WT Aspergillus fumigatus isolates

Isolate	MIC (μg/ml)			Cyp51A substitution
	Itraconazole	Posaconazole	Voriconazole
MF-452	>8	0.5	0.5	I266N
MF-469	8	1	0.25	G54E, I266N
MF-460	4	2	0.25	G54E, I266N
MF-357	4	0.5	0.5	I266N
MF-468	4	0.5	0.25	G54E, I266N
MF-329	4	0.5	0.25	None
MF-331	2	>16	0.25	G54W
MF-327	2	2	0.12	G54R
MF-439	2	0.5	0.25	G54E, I266N
MF-473	2	0.5	0.25	G54E, I266N
MF-454	2	0.5	0.12	G54E, I266N
MF-472	2	0.5	0.12	G54E, I266N
MF-843	2	0.25	2	None
MF-748	2	0.25	1	NDa
MF-1011	1	2	0.12	G54W
MF-855	1	0.25	2	None
MF-336	1	0.25	2	None
MF-486	1	0.25	2	None
MF-520	1	0.25	2	None
MF-1091	0.5	0.25	2	None
MF-474	0.5	0.25	2	None
MF-303	0.5	0.12	2	None

^aND, not determined.

Among mutations of the cyp51A gene, TR/L98H has received the most attention, notably because this mutation was seen in a specific country and found in A. fumigatus isolated from the environment (17, 22, 24, 31–33). Similarly, TR/L98H was recently detected in a multi-azole resistant isolate in China (17), suggesting that the TR/L98H mutation could be selected in Asia as well as in Europe. Of all 22 non-WT isolates in our study of Japanese isolates, CYP51A mutations were detected as follows: G54W, two isolates; G54R, one isolate; I266N, two isolates; G54E plus I266N, seven isolates (Table 2). No TR/L98H-bearing isolates were detected. The I266N mutation, which has (to our knowledge) not been reported previously, was also seen in other azole-susceptible isolates; therefore, it might not be directly related to azole resistance. Of 21 non-WT isolates, 9 had no CYP51A substitution (Table 2). Interestingly, most non-WT isolates for voriconazole did not possess a cyp51A mutation. Although Bueid et al. reported an increase of frequency of azole-resistant isolates without cyp51A mutations, other possible resistant mechanisms (e.g., upregulation of efflux pump) have not yet been fully identified (6, 28, 30), and further analysis is warranted.

Only a few previous analyses have examined changes in susceptibility over time; therefore, it is not clear that the frequency of azole-resistant A. fumigatus is increasing worldwide (12, 25, 33). Nevertheless, mechanisms of resistance induction in clinical settings or the environment (e.g., selection following agricultural antifungal exposure) remain poorly understood. Given that azole usage varies from one country to another, the mechanism of azole resistance may differ between regions.

In this study, we found a low prevalence of resistance to triazoles in Japanese isolates of A. fumigatus, a clinically important fungus of increasing concern in respiratory medicine. The proportions of non-WT isolates were similar to those previously reported for other countries. In the future, Japanese A. fumigatus isolates may develop azole resistance by different mechanisms (such as TR/L98H); therefore, we urge the continued monitoring of azole susceptibility in this species.