Abstract
The aim of the present study was to identify retrospectively trends in the species distributions and the susceptibility patterns of Aspergillus species causing fungal infections in Spanish medical centers from 2000 to 2002. The susceptibilities of 338 isolates to amphotericin B, itraconazole, and voriconazole were tested. Aspergillus fumigatus was the most common species (54.7%), followed by Aspergillus terreus (14.8%) and Aspergillus flavus (13.9%). Non-A. fumigatus species were encountered in 45.3% of the samples studied. The majority of Aspergillus isolates were obtained from respiratory tract specimens, followed by ear and skin samples. The geometric mean (GM) MIC of amphotericin B was 0.56 μg/ml, and the amphotericin B MIC was >2 μg/ml for 16 isolates (4.7%). Nine of them were A. terreus. The GM MIC of itraconazole was 0.37, and the itraconazole MIC was >4 μg/ml for 12 (3.5%) isolates. The voriconazole MICs were also high for 8 of the 12 strains for which itraconazole MICs were high (voriconazole MIC range, 2 to 8 μg/ml).
The rates of opportunistic fungal infections have increased in the past decade, particularly those caused by Aspergillus species (10, 23). This invasive mycosis is a major complication in highly immunosuppressed patients, such as bone marrow or solid-organ transplant recipients, and in patients with prolonged neutropenia caused by hematological malignancies or steroid therapy (15). Aspergillus is a very large genus containing more than 185 species, to which humans are constantly exposed. Among these species, Aspergillus fumigatus is responsible for the majority (85 to 90%) of cases of infection, although other Aspergillus spp. have been associated with severe infections in the immunosuppressed host (2, 5, 7, 11, 12, 22).
Amphotericin B (AMB), itraconazole (ITR), voriconazole (VOR), and caspofungin have been approved for the treatment of aspergillosis, although their efficacies are limited for several reasons, such as problems associated with the diagnosis of invasive aspergillosis, the seriousness of the underlying diseases, and the limited number of therapeutic options. Resistance to antifungal drugs is not as great a concern as resistance to antibacterial agents, but there has been an increase in the number of reported cases of both primary and secondary resistance among strains causing human mycoses (8).
The National Committee for Clinical Laboratory Standards (NCCLS) Subcommittee on Antifungal Susceptibility Tests has approved recently a standard (document M38-A) for testing in vitro the susceptibilities of filamentous fungi to several antifungal agents (19). The experience with this procedure to date indicates that the AMB MICs above 2 μg/ml and ITR MICs above 8 μg/ml are associated with treatment failure and clinical resistance to these agents (6, 13). Some investigators have reported that Aspergillus flavus and Aspergillus terreus are resistant to AMB in vitro (14, 21). In addition, it is known that A. fumigatus can develop resistance to ITR (6).
The purpose of this study was to determine the susceptibility patterns of clinically important Aspergillus species to three antifungal agents. The data were analyzed to determine the most active antifungal agents, establish any differences based on the infection site, and compare the susceptibility patterns of individual species.
MATERIALS AND METHODS
Fungi.
A total of 338 clinical isolates of Aspergillus spp. were included in the study. All strains were recovered from 81 Spanish hospitals over a period of 3 years (2000 to 2002). The 338 isolates were obtained from a variety of sources, including blood (n = 2; 0.6%), the respiratory tract (n = 247; 73.1%), skin (n = 19; 5.6%), eyes (n = 5; 1.5%), ears (n = 25; 7.4%), tissue biopsy specimens (n = 8; 2.4%), catheters (n = 3; 0.9%), environmental samples (n = 13; 3.8%), and other locations (n = 16; 4.7%). Each isolate was obtained from a different patient. A. fumigatus ATCC 9197 and Paecilomyces variotii ATCC 22319 were included as control isolates in each set of experiments. The isolates were maintained as a suspension in sterile distilled water at 4°C until testing was performed.
Antifungal agents.
The antifungal agents used were AMB (Sigma-Aldrich Química, Madrid, Spain), ITZ (Janssen Pharmaceutica, Madrid, Spain), and VOR (Pfizer Ltd., Sandwich, United Kingdom). They were obtained as standard powders, and stock solutions were prepared in 100% dimethyl sulfoxide (Sigma-Aldrich Química). Solutions of each drug were kept at −70°C.
Antifungal susceptibility testing.
A broth microdilution test was performed by following the NCCLS reference method (19), with minor modifications. The modifications included the use of RPMI 1640 with l-glutamine buffered to pH 7 with 0.165 M morpholinepropanesulfonic acid and 10 M NaOH and supplemented with 18 g of glucose per liter (RPMI-2% glucose; Oxoid, Madrid, Spain) and an inoculum size of 1 × 105 to 5 × 105 CFU/ml (3). This inoculum size is 10-fold higher than that recommended by the NCCLS M38-A protocol. However, some reports have demonstrated that inoculum sizes of 1 × 105 to 5 × 105 CFU/ml generate reproducible in vitro susceptibility data for Aspergillus spp. that can predict clinical outcomes. In addition, the higher inoculum size does not have a significant influence on the MICs (6). Table 1 displays the susceptibility results for the reference strains and each antifungal agent tested. Table 1 also includes the MICs obtained after 30 repetitions on different days as well as the MICs obtained by the NCCLS M38-A reference procedure.
TABLE 1.
MIC ranges for control strainsa
| Antifungal agent | MIC range (μg/ml)
|
|||
|---|---|---|---|---|
|
P. variotii ATCC 22319
|
A. fumigatus ATCC 9197
|
|||
| Modified method | M38-A procedure | Modified method | M38-A procedure | |
| AMB | 0.25-1.0 | 0.12-0.50 | 0.12-0.50 | 0.12-0.50 |
| ITR | 0.06-0.25 | 0.12-0.25 | 0.25-0.50 | 0.12-0.50 |
| VOR | 0.06-0.25 | 0.03-0.12 | 0.25-1.0 | 0.25-1.0 |
Inoculum suspensions were prepared from fresh, mature (3- to 5-day-old) cultures by a previously reported method (1). Briefly, the colonies were covered with 5 ml of distilled sterile water containing 0.1% Tween 20 (Sigma-Aldrich Quimica). The conidia were then carefully rubbed with a sterile cotton swab (Collection swab; EUROTUBO, Madrid, Spain) and transferred to a sterile tube; the resulting suspensions were homogenized for 15 s with a gyratory vortex mixer at 2,000 rpm (MS 1 Minishaker, IFA; Cultek, Madrid, Spain). The inoculum size was adjusted to a range of 1.0 × 106 to 5.0 × 106 spores/ml by microscopic enumeration with a cell-counting hemocytometer (Neubauer chamber; Merck, S.A., Madrid, Spain). All adjusted suspensions were quantified by plating on Sabouraud agar plates.
Initial solutions of the antifungal agents were diluted in dimethyl sulfoxide. The final concentrations ranged from 16 to 0.03 μg/ml for AMB, 8 to 0.015 μg/ml for ITZ, and 64 to 0.12 μg/ml for VOR. Sterile plastic microtitration plates each with 96 flat-bottom wells were used. These plates contained serial twofold dilutions of the antifungal drugs and two drug-free wells with medium only as sterility and growth controls. The inoculum suspension was diluted 1:10 with sterile water to obtain a final working inoculum of 1 × 105 to 5 × 105 CFU/ml. Each well of the plates was inoculated with 0.100 ml. The plates were incubated at 35°C for 48 h in a humid atmosphere. Visual readings were performed with the help of a mirror.
Endpoint determination.
MICs were defined as the lowest concentration of the antifungal agent that completely inhibited fungal growth.
Data analysis.
Differences in proportions were determined by Fisher's exact test or χ2 analysis. The significance of the differences in MICs was determined by Student's t test (unpaired, unequal variance). A P value <0.01 was considered significant. In order to approximate a normal distribution, the MICs were transformed to log2 values to established susceptibility differences between species. Both on-scale and off-scale results were included in the analysis. The off-scale MICs were converted to the next concentration up or down.
Statistical analysis was done with the Statistical Package for the Social Sciences (version 10.0; SPSS S.L., Madrid, Spain).
RESULTS
The MICs of the three agents for the control organisms were consistent within 2 or 3 twofold dilutions. These values are displayed in Table 1. No differences in the MICs obtained by NCCLS reference procedure and those obtained by the modified method were observed.
Regarding the species distribution, A. fumigatus was the species recovered the most frequently, with a total of 185 of 338 (54.7%) isolates recovered being A. fumigatus. The second and third most common species recovered were A. terreus (50 of 338 isolates; 14.8%) and A. flavus (47 of 338 isolates; 13.9%), respectively. Table 2 displays the distribution of strains for each isolation site. No differences in species distributions were observed over the 3-year study period.
TABLE 2.
Distribution of species by isolation sitea
| Site of isolation | No. (%) of specimens | Species | No. of isolates (% of isolates by site) |
|---|---|---|---|
| Respiratory | 247 (73.1) | A. fumigatus | 150 (60.7) |
| A. terreus | 38 (15.4) | ||
| A. flavus | 19 (7.7) | ||
| A. nidulans | 12 (4.9) | ||
| A. sydowii | 10 (4.0) | ||
| A. niger | 6 (2.4) | ||
| Others | 12 (4.8) | ||
| Skin | 19 (5.6) | A. fumigatus | 7 (36.8) |
| A. terreus | 5 (26.3) | ||
| A. flavus | 1 (5.2) | ||
| A. niger | 1 (5.2) | ||
| A. nidulans | 1 (5.2) | ||
| Others | 4 (21.1) | ||
| Environment | 13 (3.8) | A. fumigatus | 5 (38.5) |
| A. flavus | 7 (53.8) | ||
| A. nidulans | 1 (7.7) | ||
| Ear | 25 (7.4) | A. fumigatus | 3 (12.0) |
| A. flavus | 13 (52.0) | ||
| A. terreus | 4 (16.0) | ||
| A. niger | 3 (12.0) | ||
| Others | 2 (8.0) | ||
| Others | 34 (10.1) | A. fumigatus | 20 (58.8) |
| A. flavus | 7 (20.6) | ||
| A. terreus | 3 (8.8) | ||
| A. niger | 1 (2.9) | ||
| Others | 3 (8.8) | ||
| Total | 338 (100) |
The distributions for the most frequent isolation sites (total number, 338) are presented here.
The majority of Aspergillus isolates were obtained from respiratory tract specimens, followed by ear and skin samples. In contrast, only two strains were isolated from blood. The species the most frequently recovered from respiratory tract specimens was A. fumigatus (150 of 247 isolates; 60.7%). In fact, this species was significantly associated with respiratory tract locations (P < 0.01 by χ2 analysis), with 150 strains of the 185 isolates tested (81.1%) being from the respiratory tract. A. flavus was associated with ear samples (13 of 47 isolates; 27.6% [P < 0.01]). In contrast, A. terreus and Aspergillus niger were not significantly associated with any particular location. Other associations were not analyzed because of the small number of isolates.
The in vitro activities of AMB, ITZ, and VOR are summarized in Table 3. Wide ranges of MICs were observed by species tested. The geometric mean (GM) MIC of AMB was 0.56 μg/ml, and the MIC ranged between 0.06 and 16 μg/ml. Differences in susceptibilities were seen between species. Overall, 322 of the 338 Aspergillus isolates tested (98.22%) were inhibited by ≤2 μg of AMB per ml. The data in Table 3 indicate that AMB is highly active against A. fumigatus in vitro, with a GM MIC of 0.36 μg/ml. It can be stressed that this species was significantly more susceptible to AMB than the other Aspergillus spp. tested (P < 0.01 by Student's t test). The AMB MIC was >2 μg/ml for 16 isolates. These strains resistant in vitro belonged to different species: A. terreus (9 of 50 isolates), A. flavus (2 of 47 isolates), Aspergillus versicolor (1 of 8 isolates), Aspergillus ustus (1 of 2 isolates), Aspergillus candidus (1 of 3 isolates), Aspergillus ochraceus (1 of 3 isolates), and Aspergillus spp. (1 of 2 isolates). The AMB MICs for A. terreus and A. flavus isolates were significantly higher than those for the other species tested (P < 0.01), ranging from 0.12 to 16 μg/ml (GM MIC, 1.56 μg/ml) and 0.25 to 4 μg/ml (GM MIC, 0.85 μg/ml), respectively. Other species for which AMB MICs were high were not included in the comparative statistical analysis because of the small numbers of isolates. AMB MICs were not greater than 2 μg/ml for any of the A. fumigatus isolates.
TABLE 3.
In vitro susceptibilities of the Aspergillus spp. included in the studya
| Species | AMB
|
ITZ
|
VOR
|
||||||
|---|---|---|---|---|---|---|---|---|---|
| No. of isolates | MIC (μg/ml)
|
No. of isolates | MIC (μg/ml)
|
No. of isolates | MIC (μg/ml)
|
||||
| GM | Range | GM | Range | GM | Range | ||||
| A. fumigatus | 185 | 0.36 | 0.06-2 | 185 | 0.39 | 0.06-8 | 179 | 0.53 | 0.12-4 |
| A. flavus | 47 | 0.85 | 0.25-4 | 47 | 0.33 | 0.06-2 | 47 | 0.96 | 0.5-2 |
| A. terreus | 50 | 1.56 | 0.12-16 | 50 | 0.21 | 0.03-8 | 50 | 0.83 | 0.25-4 |
| A. niger | 11 | 0.28 | 0.12-0.5 | 11 | 1.37 | 0.25-8 | 11 | 0.73 | 0.12-2 |
| A. sydowii | 10 | 0.71 | 0.06-1 | 10 | 0.61 | 0.12-8 | 10 | 0.93 | 0.25-2 |
| A. nidulans | 14 | 0.71 | 0.5-1 | 14 | 0.37 | 0.6-8 | 14 | 0.22 | 0.06-1 |
| A. versicolor | 8 | 0.92 | 0.25-4 | 8 | 0.92 | 0.25-8 | 8 | 0.77 | 0.25-8 |
| A. ustus | 2 | 1.41 | 0.25-8 | 2 | 11.31 | 8->8 | 2 | 8 | 8 |
| A. candidus | 3 | 1 | 0.25-16 | 3 | 0.06 | 0.015-0.12 | 3 | 0.2 | 0.06-0.5 |
| A. ochraceus | 3 | 1.59 | 1-4 | 3 | 0.16 | 0.06-0.5 | 3 | 0.5 | 0.25-1 |
| A. flavipes | 1 | 0.5 | 1 | 0.25 | 1 | 1 | |||
| A. glaucus | 1 | 0.25 | 1 | 0.03 | 1 | 0.5 | |||
| A. sclerotiorum | 1 | 0.5 | 1 | 2 | 1 | 0.5 | |||
| Aspergillus spp. | 2 | 2 | 1-4 | 2 | 0.06 | 0.06 | 2 | 1 | 1 |
| Total | 338 | 0.56 | 0.06-16 | 338 | 0.37 | 0.015-16 | 332 | 0.62 | 0.06-8 |
A total of 338 isolates were evaluated.
The GM MIC of ITZ was 0.37 μg/ml, and the MIC range was 0.015 to > 8 μg/ml. A total of 96.4% of the organisms (326 of 338) analyzed were inhibited by less than 4 μg of ITZ per ml. However, ITZ MICs were >4 μg/ml for three A. fumigatus isolates, two A. terreus isolates, two A. niger isolates, one Aspergillus sydowii isolate, one Aspergillus nidulans isolate, one A. versicolor isolate, and two Aspergillus ustus isolates. Six of them were recovered from the respiratory tract, and six were of other origins. These data represent a resistance rate of 3.5% (12 of 338 isolates). A. terreus appeared to be the species most susceptible to ITZ, being significantly more susceptible than A. fumigatus (P < 0.01). In contrast, the ITZ MICs for A. niger were markedly greater (GM MIC, 1.37 μg/ml) and ranged between 0.25 and 8 μg/ml. The highest ITZ MICs (≥8 μg/ml) detected in the study were for two A. ustus isolates.
For most of the strains, the in vitro activity of VOR was comparable to that of ITZ, although VOR MICs were slightly higher than those of ITZ for the most common species, such as A. fumigatus, A. flavus, and A. terreus, for which the GM MICs of VOR and ITZ were 0.53 and 0.39 μg/ml, 0.96 and 0.33 μg/ml, and 0.83 and 0.21 μg/ml, respectively. Although breakpoints have not been established for interpretation of the susceptibility testing results for VOR, 91.6% (304 of 332) of the Aspergillus isolates tested were inhibited by less than 2 μg of VOR per ml. Interestingly, for 8 of the 12 strains for which ITZ MICs were high (>4 μg/ml), the MIC of VOR also increased, ranging from 2 to 8 μg/ml.
DISCUSSION
A. fumigatus has been reported to be the cause of approximately 85% of all forms of aspergillosis, followed by A. flavus (5 to 10%), A. niger (2 to 3%), and A. terreus (2 to 3%). In addition, some Aspergillus spp. are associated with certain clinical forms of aspergillosis, such as A. niger with external otitis, A. flavus with sinusitis, and A. glaucus with joint infection (20). The results of our study agree with findings presented in previous reports showing that A. fumigatus is the most common species isolated from clinical specimens, but non-A. fumigatus species were encountered in 45.4% of the samples studied. A. terreus was isolated from 14.8% of the samples, and A. flavus was isolated from 13.9%. These findings emphasize the increasing role of the non-A. fumigatus species in mold infections. At the beginning of the past decade, one-fifth of the cases of aspergillosis were caused by these species, but their prevalence is growing. Some investigators state that the massive use of AMB against fungal infections has led to the emergence of less susceptible species, such as A. terreus and A. flavus (16).
The results of susceptibility testing showed that AMB appears to be the antifungal agent that is the most active against A. fumigatus. On the contrary, ITZ and VOR were the most active compounds against non-A. fumigatus species in vitro. Some reports have shown that A. terreus is consistently resistant to AMB in vitro. In addition, disseminated infections caused by this organism resulted in death, despite treatment (13). Our results show that in vitro AMB exhibits less potent activity against this species than against other Aspergillus spp. However, A. terreus isolates were not invariably resistant to the polyene. The AMB MIC was >2 μg/ml for 9 of the 50 (18%) A. terreus isolates tested.
Another point to consider is the azole resistance of Aspergillus spp. Recently reported data have shown rates of ITZ resistance close to 4% among Aspergillus spp. and 2% among A. fumigatus isolates. ITZ resistance was defined as an MIC >4 μg/ml (17, 18). The same rate of resistance was found among the strains included in our study.
Breakpoints for VOR have not been established, and so discussion of the rate of resistance is not possible. However, some Aspergillus isolates seem to show cross-resistance to ITZ and VOR. Espinel-Ingroff et al. (9) noted that this phenomenon was not universal and may vary according to the strain of Aspergillus (4). Other investigators have reported on ITZ-resistant strains with high levels of susceptibility to VOR. In our study, for 8 of the 12 strains for which the ITZ MIC was >4 μg/ml, the VOR MIC was >2 μg/ml. These values may indicate cross-resistance. However, it was interesting that for the two most prevalent non-A. fumigatus species, A. flavus and A. terreus, the GM MICs of VOR were higher than that for A. fumigatus. The same was not observed for ITZ, the GM MICs of which were lower for A. flavus and A. terreus than for A. fumigatus. This finding could be due to the fact that different patterns of cross-resistance can exist, suggesting marked differences in the activities of azole agents and in the mechanisms of resistance to azole agents (18).
These findings reinforce the need for continued surveillance programs that analyze the factors that may have an influence on trends in species distributions and the antifungal susceptibility profiles of the isolates responsible for aspergillosis. This report also points to the need to maintain a consistent method of in vitro susceptibility testing so that MICs are comparable between relevant studies.
Acknowledgments
This work was partially supported by a grant from the Fondo de Investigaciones Sanitarias, Instituto de Salud Carlos III (grant 99/0198), and by research project 99/1199 from the Instituto de Salud Carlos III. A. Gomez-Lopez is a fellow of the Instituto de Salud Carlos III (grant 99/198).
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