Antifungal Drug Resistance in Aspergillus

Antifungal Drug Resistance in Aspergillus

Journal of Infection (2000) 41, 203–220 doi:10.1053/jinf.2000.0747, available online at http://www.idealibrary.com on REVIEW ARTICLE Antifungal Drug...

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Journal of Infection (2000) 41, 203–220 doi:10.1053/jinf.2000.0747, available online at http://www.idealibrary.com on

REVIEW ARTICLE

Antifungal Drug Resistance in Aspergillus C. B. Moore1, N. Sayers2, J. Mosquera2, J. Slaven2 and D. W. Denning*2,3 1

Department of Microbiology, Hope Hospital, Salford, 2University of Manchester and 3Department of Infectious Diseases and Tropical Medicine, North Manchester General Hospital, Manchester, U.K.

Introduction This article overviews the emerging problem of antifungal drug resistance in Aspergillus. Fluconazole and ketoconazole (KCZ) are inactive against Aspergillus. Validation of testing procedures for the detection of itraconazole (ITZ) resistance in Aspergillus fumigatus in 19971,2 were further validated with posaconazole (SCH-56592)3,4 and voriconazole (VCZ)5 (Hitchcock, personal communication). Work is ongoing to validate testing procedures for azoles in non-fumigatus Aspergilli. In contrast to the successful validation of azole susceptibility testing of Aspergillus, successful validation of amphotericin B (AmB) testing has been problematic, with the probable exception of Aspergillus terreus.6,7 This article summarizes data from our laboratory, contrasts it with that in the literature, and makes recommendations for subsequent work to further clarify the situation. The new group of antifungal drugs, the candins, is not discussed and information on terbinafine is tabulated only for comparison. Resistance in flucytosine is also not discussed.

Resistance to Amphotericin B AmB, a polyene antifungal agent, is the drug of choice against a wide range of fungal pathogens. Certain fungal species are intrinsically resistant to AmB, such as Pseudallescheria boydii and Trichosporon beigelii.8 Failure of AmB treatment against invasive aspergillosis is common. Correlating that failure to AmB resistance of Aspergillus spp. in vivo is difficult to prove. Therefore, the true rate of AmB resistance is unknown. Moreover, few attempts have been made to correlate clinical outcome and AmB resistance in Aspergillus spp. either in vitro or in animal * Please address all correspondence to: Dr D. W. Denning, Department of Infectious Diseases and Tropical Medicine, North Manchester General Hospital, Delaunays Road, Manchester M8 6RB, U.K. Accepted for publication 7 September 2000.

0163-4453/00/060203;18 $35.00/0

models. This is further complicated by the fact that individuals who develop Aspergillus infections are often severely immunocompromised. Host-related factors might therefore be more significant than the AmB susceptibility of Aspergillus spp. Other confounding factors in the ability to correlate clinical outcome with in vitro and in vivo susceptibility to AmB are the test systems and animal models used.9,10 MICs vary depending upon test format and the species of Aspergillus tested, highlighting the need for standardization.10–12 Verweij et al.13 demonstrated different efficacy of AmB in a transient neutropenic mouse model infected with two A. fumigatus isolates with similar MIC values (2 ␮g/ml and 1 ␮g/ml). Johnson et al.7 used 54 in vitro test formats to determine MIC values for three A. fumigatus isolates and one A. terreus isolate. After comparing MICs determined in vitro with efficacy in a neutropenic mouse model of invasive aspergillosis, the authors found no correlation between clinical outcome and A. fumigatus MIC values. They did find a correlation between a high MIC value and poor clinical outcome with A. terreus; however, this was dependent upon the in vitro test format used. The survival rates for patients with invasive pulmonary or disseminated aspergillosis caused by A. terreus are very poor. In vitro resistance to AmB in this species was also shown by Sutton et al.10 in 101 clinical isolates that had a mean MIC value of 3.37␮g/ml. Lass-Flörl et al.6 found a positive correlation between an AmB MIC of 2␮g/ml and survival in neutropenic patients infected with A. fumigatus or A. flavus. However, MICs of 2␮g/ml were highly associated with a fatal outcome, as was infection with A. terreus, all of which had high MIC values. Testing of Aspergillus against lipid-associated AmB usually yields higher MIC values.14 AmB colloidal dispersion (ABCD) has the lowest geometric mean MICs of all three commercially available lipid-associated AmBs (Table I). In all cases, however, minimum fungicidal concentrations (MFCs) were substantially higher than those with conventional AmB. Comparison of AmBisome and AmB lipid complex (Abelcet) in a murine model showed differential activity (AmBisome being superior) that was not reflected in vitro.16 © 2000 The British Infection Society

C. B. Moore et al.

204

Table I. Comparative activities of antifungal drugs against Aspergillus (polyenes). Geometric mean MICs* (number tested). Species

d-AB15

ABCD14

ABLC14

L-AB14

Nystatin14

Liposomal nystatin14

A. fumigatus, ITZ-S A. fumigatus, ITZ-R A. flavus A. terreus A. niger

0.40 (51) 0.40 (9) 2.35 (13) 3.56 (12) 0.34 (13)

0.24 (25) 1.37 (11) 5.2 (8) 20.7 (8) 0.20 (8)

0.97 (25) 5.84 (11) 29.3 (8) 32 (8) 0.10 (8)

0.59 (25) 4 (11) 16 (8) 32 (8) 0.30 (8)

5.58 (25) 16 (11) 12.3 (8) 32(8) 5.7 (8)

1.84 (25) 2 (11) 2.4 (8) 8.7 (8) 1.4 (8)

Total

0.65 (98)

0.90 (60)

2.5 (60)

2.1 (60)

9.5 (60)

2.3 (60)

*For the purpose of analysis, all values 16 ␮g/ml were classed as 32 ␮g/ml. AB:amphotericin B; d-AB:fungizone (deoxycholate AB); ABCD:Amphocil/Amphotec (AB colloidal dispersion); ABLC:Abelcet (AB lipid complex); L-AB:AmBisome (liposomal amphotericin B); ITZ:itraconazole. A microtitre method using RPMI-1640 with 2% glucose and a final inoculum of 5105 conidia/ml was used for all drugs.

Although AmB is fungicidal for some isolates in vitro, it is not generally so in vivo. Tissues can be sterilized in some models, but usually not. This is consistent with data from patients.17 It is possible that MFCs would be a better measure of AmB activity, and predictive of clinical response, but methodology varies.11 Substantial work would be necessary to validate this approach. A better understanding of the mechanisms of resistance to AmB in fungi may be obtained by the generation of AmB resistant mutants in vitro. Manavathu et al.18 generated genetically stable AmB resistant mutants of A. fumigatus by UV irradiation. Alternatively, AmB resistant isolates of a variety of fungi, including Candida albicans, Cryptococcus neoformans, A. nidulans and A. fennelliae have all been generated by repeated sub-culturing on agar containing increasing amounts of polyene antifungals.19 However, meaningful and reproducible in vitro test systems for measuring the susceptibility of Aspergillus spp., and in particular A. fumigatus, must be developed in order to accurately assess polyene antifungal resistance in the outcome of clinical infections.

Mechanism of Action and Resistance in Aspergillus AmB binds to sterols in membranes of eukaryotic cells, with a 10-fold higher affinity for ergosterol (predominant in fungi) than the human equivalent cholesterol, possibly due to the presence of two additional double bonds in ergosterol as compared to cholesterol.20 This affinity to sterols is conferred by the structure of AmB, a large macrolide ring with two distinct regions. The first, hydrophobic, region consists of a series of seven conjugated double bonds. The second, hydrophilic, region consists of

a carbon chain with eight hydroxyl groups and a mycosamine sugar moiety. Incorporation of the AmB molecule into cell membranes, with a stoichiometry of 1 : 1 sterol to AmB, leads to the formation of pores with the hydrophilic region forming the inner wall, through which small ions such as potassium can pass.21,22 This cellular leakage was originally believed to lead ultimately to cell death.22 However, despite the fact that AmB-mediated potassium leakage has been demonstrated in A. fumigatus, the association between potassium leakage and cell death has yet to be proven.23 Additional mechanisms of cell death may be operative. This suggestion is supported by a number of experimental observations. Kotler-Brajtburg et al.24 described two ways in which AmB could incorporate into the membranes of Saccharomyces cerevisiae: either reversibly (most likely by pore formation), or irreversibly in an energy-dependent interaction.

Sterol Alterations 8

Sterling and Merz reviewed 44 cases of clinical resistance to AmB in nine fungal species, including six Candida spp. (n:39), T. beigelii (n:2), C. neoformans (n:2) and A. fumigatus (n:1). Resistance in all 44 cases was attributed to either the absence or reduction in ergosterol, which in the one case of A. fumigatus infection was probably due to prior treatment with ITZ. If AmB resistance was solely the consequence of decreased ergosterol content in phospholipid bilayers, then defects in the enzymes which constitute the sterol biosynthetic pathway, resulting in the formation of intermediates other than ergosterol (such as ⌬7 sterols and ⌬8 sterols), would result in resistance to AmB.25 In addition, Pierce et al.26 suggested that an increase in esterification

205 Antifungal Aspergillus Drug Resistance of sterols increased sensitivity to AmB due to alterations in sterol conformation or membrane fluidity, which facilitate the binding of AmB. However, resistance to AmB in fungal isolates cannot be fully explained by variations in quantity or type of sterols in their membranes. This has been shown with certain isolates of C. albicans,26 Kluyveromyces species27 and our own unpublished data in A. fumigatus. Joseph-Horne et al.25 described two Ustilago maydis isolates that were resistant to AmB and nystatin but sensitive to the azole triadimenol in vitro. Ergosterol content of the isolates was within normal limits of the parental wild type, suggesting a non-sterol related mechanism of AmB resistance. Broughton et al.,28 who also isolated AmB resistant isolates of C. albicans that differed neither in sterol nor fatty acid contents compared to wild type, suggested that an increase in membrane fluidity might confer resistance to AmB. In Candida species, the concentration of AmB required to cause leakage from the cell membrane is lower than that required to kill the cell, indicating that leakage does not always result in death.29–31 This observation has led to the theory that two separate types of AmB resistance exist: the first is resistance to leakage and killing, and the second is resistance to killing alone.29 Resistance to leakage and killing may arise simply from the inability of AmB to interact with the fungal membrane arising

Figure 1.

either because of alterations in the sterol content, increased rigidity of the membrane,28 or because of the unavailability or obstruction of the membrane surface. Membrane surface obstruction may occur by sequestration within phagocytes or by the presence of surface adherent leukocytes during the inflammatory response.

Amphotericin B as an Oxidizing Agent Resistance to killing alone is postulated to be independent of the interactions between membrane sterols and AmB with blockage of an undefined second step, which is crucial for the ultimate demise of the cell. Evidence exists to suggest that killing of the cell may be the result of AmB acting as an oxidizing agent,19,32 leading to the formation of reactive oxygen species (ROS),33 which result in oxidative damage through membrane lipid peroxidation (summarized in Figure 1). The cytotoxic action of AmB on host tissue has been partially attributed to oxidative damage, as seen with erythrocytes.35 Sandhu23 showed that AmB inhibited respiration in A. fumigatus. Sandhu23 and Manavathu et al.18 found that AmB exerted its effects on actively metabolizing A. fumigatus only. Sokol-Anderson et al.30 have shown that AmB-mediated lysis of C. albicans protoplasts and whole cells was greatly reduced in the absence of oxygen and in the presence of exogenous

Oxidation and propagation of reactive oxygen species in a biological membrane (adapted from Kagan34).

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C. B. Moore et al.

catalase and superoxide dismutase; in contrast, leakage of potassium was not affected. Using AmB resistant strains of C. albicans, Sokol-Anderson et al.31 also showed that these strains were significantly less sensitive to hydrogen peroxide and produced less hydrogen peroxide upon exposure to menadione than controls. They also showed that these resistant strains in the presence of AmB produced significantly more intracellular and extracellular catalase than controls. Therefore, resistance to AmB may arise from the ability of strains to cope more efficiently with oxidative stress initiated by AmB. During normal aerobic metabolism, molecular oxygen undergoes reduction via the addition of four electrons to give water; this is achieved without the production of intermediate species. However, at physiological pH and in the presence of a catalysing transition metal, e.g. manganese or iron, molecular oxygen can undergo a number of sequential reductions to produce a range of ROS. The ROS involved include hydrogen peroxide, hypochlorous acid, hypohalous acid, and peroxynitrite in addition to free radical species including superoxide, hydroxyl and nitric oxide.36 Possibly the most reactive of these species is the hydroxyl radical. The hydroxyl radical is capable of oxidizing a wide range of proteins, DNA and lipids and is important in initiating the lipid peroxidation cascade (see Figure 1). One ROS which acts as major source of the hydroxyl radical is hydrogen peroxide. Therefore, removing hydrogen peroxide from the system, achieved by the action of catalases, will ameliorate oxidative damage.

Amphotericin B as an Antioxidant The presence of seven conjugated double bonds in AmB means that it is prone to auto-oxidation.30,37 This tendency to auto-oxidation means that AmB could also act like an anti-oxidant, but perhaps only at low oxygen tensions. In the presence of molecular oxygen, AmB may interact with peroxyl free radicals, producing a chain-propagating species akin to the ␤-carotene peroxyl radical. This peroxyl radical can trigger further oxidation reactions by the abstraction of hydrogen ions from neighbouring polyunsaturated fatty acids.38 This would lead to dysfunction of the plasma membrane (and possibly intracellular membranes such as the endoplasmic reticulum), resulting in cell death. Whether this is also the case for AmB and Aspergillus, in the presence of molecular oxygen, is not known. The impetus for the (presumptive) auto-oxidation of AmB is not fully elucidated. ROS could initiate auto-oxidation of AmB, which under certain conditions (e.g. the presence of an optimum oxygen tension) may result in the propagation of lipid peroxidation of membrane bound fatty acids. As AmB must span the fungal membrane for

leakage to occur,39 either intracellular or extracellular ROS may initiate auto-oxidation of AmB. In vivo, ROS are released from phagocytes as part of the respiratory burst. Given that Aspergillus hyphae are large, killing proceeds extracellularly with neutrophils, monocytes and macrophages attached to the hyphal surface, all discharging phagolysosomes onto the hyphal surface. Thus, host-generated ROS will come into contact with the surface of the hyphae in a discontinuous way, both spatially and temporally. Under aerobic conditions, intracellular ROS will be produced. Bursts of ROS are produced in Neurospora crassa, associated with certain stages of differentiation.40 Aspergillus fumigatus has been shown to produce intracellular hydrogen peroxide independent of external stimulus.41 This evidence is supported by the fact that Aspergillus produces at least three superoxide dismutases42 and at least three catalases.43 In addition, A. fumigatus produces gliotoxin, which is believed to play an important role in aspergillosis through its immunosuppressive and apoptotic actions. Gliotoxin can also stimulate the production of hydrogen peroxide via redox cycling.44 In the absence of oxygen, and in common with carotenoids and retinoids, AmB may act as an antioxidant and therefore a chain terminator of this putative propagative peroxidation process. Indeed, it may partially protect the Aspergillus cell from attack by phagocytes. In these circumstances AmB binding to ergosterol may simply result in leakage but not cell death. In addition, the ability of Aspergillus to quickly counteract the deleterious effects of ROS, by rapid production of large amounts of exogenous and endogenous catalase and superoxide dismutase, may also result in leakage but not cell death. Aspergillus fumigatus has also been shown to secrete mannitol, a potential ROS scavenger, that may also aid protection.45 Neutropenic patients, who are also monocytopenic, will be at a major disadvantage compared with non-neutropenic patients, because in the absence of neutrophil attack the fungus is likely to grow faster, invade more tissue and cause more tissue hypoxia. As a result, AmB switches to being an anti-oxidant and thus the initiation of a peroxidation cascade is prevented.

New Polyenes Nystatin has recently been lipid incorporated and the product Nyotran tested in vitro, in vivo and in patients with invasive aspergillosis.14,16,46 It has activity in vitro and, in direct contrast to AmB, MICs are lowered by lipid incorporation (Table I). In studies that used extensive dosing ranges in vivo, Nyotran was active against an AmB ‘resistant’ strain and substantially more active than AmBisome and Abelcet in this model.16 Clinical data are awaited.

Antifungal Aspergillus Drug Resistance

Azole Resistance Detection of Azole Resistance in Aspergillus In order to detect azole resistance in Aspergillus many techniques have been described. Validated resistant strains are necessary to confirm that a method can detect resistance. Many studies have not tested such strains, and in some cases they have yet to be identified. With each different method MICs vary, and it is difficult to compare results. In a review by Denning et al.11 ITZ MIC results of 18 methods were reviewed, showing a MIC range from 0.01–100 ␮g/ml. However, resistance had not been convincingly demonstrated at that time. Resistance to ITZ in two strains of A. fumigatus was demonstrated in vivo and in vitro in 1997.1,2 In recent years, a trend towards standardization has been observed to reduce inter- and intra-laboratory variation, especially since the publication of the M-27A standardized method for yeast antifungal susceptibility testing.47 Recently, the M-38P reference method for conidium-forming filamentous fungi (proposed standard) has been published.48 The M38-P method has been validated with one strain in vivo.49 The methodology of the NCCLS method is similar to that used to describe ITZ resistance1,2 (summarized in Table II). In Tables III and IV it can be seen that most of the recently published studies on susceptibility testing on Aspergillus (which includes a substantial number of isolates) are variations of both methods and show a narrow MIC range for ITZ and VCZ. These relatively uniform MIC results are due to a degree of standardization of Table II. Comparison of validated methods for susceptibility testing Aspergillus.

Format Media Drug dilutions Itraconazole diluent Inoculum counts Final inoculum (CFU/ml) Incubation temperature MIC reading

Validated method1,2

NCCLS48

Microtitre RPMI-1640 (2% glucose) Doubling 1 : 1 acetone/ 0.2M HCl

Microtitre RPMI-1640 (0.2% glucose) Batch Dimethyl sulphoxide

Haemocytometer

Spectrophotometer

5105

0.4–5104

37 °C 48 h visual 100% inhibition

35 °C 46–50 h AmB visual 100% inhibition ITZ visual 50–75% inhibition

207

methodology and allow comparison between studies to a certain extent. Most isolates have an ITZ MIC lower than 2 ␮g/ml. For VCZ, slightly higher MICs have been reported, but isolates with significantly elevated MICs are rare. Nevertheless one must be careful in interpreting in vitro data since the change of some of the variables involved in in vitro susceptibility testing methodologies can lead to significant variations in the MIC for a single strain. Here follows a discussion of key factors in susceptibility testing in Aspergillus.

Macro versus Microdilution Espinel-Ingroff et al.63 showed a moderately poor correlation for A. fumigatus and A. flavus between macro and microdilution methods for ITZ. Oakley et al.5 showed relatively good agreement for ITZ and VCZ, although some discrepancies were observed. Manavathu et al.64 reported that, using the microdilution method, ITZ MIC values were consistently four-fold higher than those obtained by the macrodilution method. Denning et al.2 compared macro and microdilution methods in order to distinguish two in vivo ITZ-resistant isolates. The results for the microdilution method showed the better correlation. No other published study has used confirmed resistant and susceptible strains to validate either test format.

Media Different media have been tested. In a study by Manavathu et al.,64 the use of peptone yeast extract glucose (PYG) and RPMI-1640 media yielded essentially identical MIC values for ITZ. The same was shown by Rath,54 when comparing yeast nitrogen broth and RPMI-1640. The NCCLS recommend RPMI-1640 when testing any filamentous fungus against any antifungal.48 Espinel-Ingroff et al.65 tested 15 selected isolates of three species of Aspergillus comparing different media. Normal RPMI-1640 (0.2% glucose) was better than RPMI-1640 with 2% glucose, antibiotic medium 3 (AM3; 0.1% glucose) and AM3 with 2% glucose, in distinguishing clinically resistant isolates with MICs 8 ␮g/ml and a VCZ-resistant laboratory mutant. Denning et al.2 showed that RPMI-1640 with 2% glucose was superior to normal RPMI-1640, Sabouraud broth and AM3 with 2% glucose in distinguishing in vivo ITZresistant A. fumigatus isolates, in vitro.

Drug Dilutions It is vitally important to get the desired drug concentration in the wells of the microtitre plate. In the validated method2

C. B. Moore et al.

208 Table III.

Recent studies of ITZ susceptibility in Aspergillus giving MICs and methodology.

Species

A. fumigatus (n:913)a

n

12 10 12 156 8 21 62 50 35 107 35 142b 6 150

A. flavus (n:135)a

A. terreus (n:39)a

MT

Media

Drug dilutions

IC

Final inoculum (CFU/ml)

IT

MIC Reading

Reference

0.12–16 (GM 0.24) 0.25–0.5 0.5–1

m

RPMI

Batch

S

0.9–4.7104

35

50%

50

m m

RPMI 2%G RPMI

Batch Batch

? S

0.4–5104 0.4–5104

35 35

51 52

m

RPMI

Batch

H

1104

35

80% 75–100% 72 h 50–75%

0.12–16 (MIC90:2) 0.06–0.25 0.03–1 0.5–2 0.13–16 (GM:0.77) 0.06–32 (MIC90:0.5) 0.125–32

4

53

m m m m

RPMI RPMI RPMI 2%G RPMI 2%G

Doubling Doubling Doubling ?

? H S H

~510 1104 ~1105 5105

35 35 35 37

~75% 100% 100% 100%

54 55 56 57

m

RPMI 2%G

Doubling

H

5105

37

100%

5

M

RPMI

Batch

S

35

100%

58

0.25–2

M

RPMI 2%G

Doubling

H

0.9–4.5104 (in 1 ml) 1103(in 2 ml)

37

0.25–4 (MIC90:1) 0.03–0.06

M

PYG

Doubling

?

1104(in 1 ml)

35

100% 40–42 h 100%

M

YNG 2%G

Doubling

S

~1104(in 1 ml)

28

0.06–1

A

RPMI 2%G

Doubling

S

1107

35

50 51 52

58

m m m

RPMI RPMI 2%G RPMI

Batch Batch Batch

S ? S

0.9–4.710 0.4–5104 0.4–5104

35 35 35

22 7

0.12–1 0.125–0.25

m m

RPMI RPMI

Batch Doubling

H ?

1104 2.5104

35 37

8 10 8

m m m

RPMI RPMI RPMI 2%G

Doubling Doubling Doubling

? H H

~5104 1104 5105

35 35 37

10

0.03–0.06 0.125–0.25 0.125–16 (MIC90:0.5) 0.125–1

50% 80% 75–100% 72 h 50–75% 50% 96 h ~75% 100% 100%

M

RPMI

Batch

S

35

100%

8

0.06–0.125

M

RPMI 2%G

Doubling

H

0.9–4.5104 (in 1 ml) 1103 (in 2 ml)

37

15

0.25–1

M

PYG

Doubling

?

100% 40–42 h 100%

24

0.015–0.12

M

YNG 2%G

Doubling

20

0.25–1

m

RPMI

0.25

m

RPMI

S

1104 (in 1 ml) ~1104 (in 1 ml)

28

Batch

H

1104

35

Doubling

?

2.5104

37

4

35 37 35

2 8 3

0.06–0.125 0.06–0.125 0.125

m m M

RPMI RPMI 2%G RPMI

Doubling Doubling Batch

? H S

8

0.06–0.125

M

RPMI 2%G

Doubling

4

0.03–0.06

M

YNG 2%G

15 4 10 7 7

0.12–1 0.5 0.5–2 0.06–1 0.06–1

m m m m M

36

0.25–8 (MIC90:4)

M

35

H

~510 5105 0.9–4.5104 (in 1 ml) 1103 (in 2 ml)

37

Doubling

S

~1104 (in 1 ml)

28

RPMI RPMI RPMI RPMI 2%G RPMI 2%G

Batch Doubling Doubling Doubling Doubling

H ? H H H

1104 ~5104 1104 5105 1103 (in 2 ml)

35 35 35 37 37

PYG

Doubling

?

~1104 (in 1 ml)

35

59 60

0.06–0.12 0.12–0.25 0.25–1

4

5

~100% 72 h 100%

11 10 10

2

A. niger (n:78)a

MIC range

51

53 62 54 55 5

5 59

~100% 72 h 50–75%

60

50% 96 h ~75% 100% 100%

62

100% 40–42 h ~100% 72 h 50–75% ~75% 100% 100% 100% 40–42 h ~100%

53

54 5 58 5 60 53 54 55 5 5 59 (continued)

Antifungal Aspergillus Drug Resistance

209

Table III. continued Species

A. nidulans (n:24)a

n

MIC range

MT

Media

Drug dilutions

IC

06

0.015–0.06

M

YNG 2%G

Doubling

17

0.12–16 (MIC90:4; MIC50:0.25) 0.06–0.125 0.25–0.5 0.06–0.125

m

RPMI

m m M

RPMI RPMI 2%G RPMI 2%G

05 02 02

Final inoculum (CFU/ml)

IT

MIC Reading

Reference

S

~1104 (in 1 ml)

28

~100% 72 h

60

Batch

H

1104

35

50–75%

53

Doubling Doubling Doubling

? H H

~5104 5105 1103 (in 2 ml)

35 37 37

~75% 100% 100% 40–42 h

54 05 05

a

Strains from reference 5 tested twice. Includes 28 laboratory-induced resistant strains from a parental strain with MIC:0.25 ␮g/ml. MT:method; IC:inoculum counts; IT:incubation temperature in °C; GM:Geometric mean; MIC reading as visual reduction in growth (after 48 h incubation if not given); m:broth microdilution; M:broth macrodilution; A:agar dilution; H:haemocytometer; S:spectrophotometer; 2%G:2% glucose. b

the easier doubling drug dilution system is employed instead of the potentially more accurate but cumbersome batch dilution NCCLS method.48 Different diluents for ITZ have been used (1 : 1 acetone: 0.2 M HCl and dimethyl sulphoxide), but we have not found important differences in the MIC results against ITZ between the use of both solvents. There is a problem with the solubility of ITZ at concentrations higher than 2 ␮g/ml. Crystals can be observed in RPMI-1640 in the wells of a microtitration plate with an ITZ concentration 2 ␮g/ml using an inverted microscope after 48 h incubation at 37 °C. In the presence of a high inoculum of conidia of A. flavus, crystals may even be seen at an ITZ concentration of 2 ␮g/ml. Crystals are more abundant and larger in the wells with higher ITZ concentrations. For this reason it may be that any reported MIC value higher than 2 ␮g/ml using published microtitre methods should be taken as an MIC 2 ␮g/ml. The use of a low inoculum may help to overcome the reduced MIC range due to this limitation.

Inoculum The importance of inoculum size when testing Aspergillus species against ITZ was studied by Gehrt et al.66 They tested eight A. fumigatus and A. flavus strains, each one with four different final inocula within a 1000-fold range (1–5102 to 1–5105 CFU/ml). The mean MIC elevations of A. fumigatus and A. flavus MICs at 48 h were only 0.18–0.45 ␮g/ml (2.5-fold) and 0.19–0.88 ␮g/ml (4.6-fold), respectively. Nevertheless, studies in our laboratory show that inoculum size is a vital factor when testing strains with relative MICs higher than the mean against ITZ (data not published). Recent work in our laboratory has shown that the final inoculum used in the validated method with A. fumigatus (5105 CFU/ml) is not appropriate for some isolates of

A. flavus.67 The appearance of trailing end points at higher inocula prevented a clear-cut endpoint determination. Other work in our laboratory shows that some clinical isolates of A. fumigatus show a 64-fold increase in the MIC (from 0.5 to 16 ␮g/ml), with a 100-fold increase in inoculum from 4103 CFU/ml. Trailing end points have been reported in the literature as a problem when testing Aspergillus.64,67 We also found an inoculum of 2.5104 to be better than 5105 CFU/ml in avoiding the appearance of trailing end points and minimizing the effect of the inoculum in raising the MICs when A. flavus is tested. The range recommended in the NCCLS M38-P document48 (0.4–5104 CFU/ml) gives variable MICs with some strains. Two authors have examined the importance of inoculum of germinated vs. ungerminated conidia on MICs.68,69 No differences in MICs were found with AmB, ITZ, VCZ and posaconazole (SCH-56592). Since inoculum size is such an important factor in in vitro susceptibility testing against ITZ and probably other azoles, a method with the best possible accuracy should be employed for preparation of inocula. We recommend that conidia are harvested by rolling a cotton swab over the surface of the colony instead of scraping the colonies with the tip of a Pasteur pipette, in order to get less conidial chains. At the same time, heavy particles from the conidial suspension should be allowed to settle for 5 min before collection of the upper homogeneous suspension. Thus, hyphal fragments and conidial clumps that could give variable and false results are less likely to be present in the final inoculum suspension. The spectrophotometric method, used as recommended in the NCCLS M38-P document, gives a variation of 4 to 10-fold48,70,71 for A. fumigatus and A. flavus. A counting chamber, while not perfect, has a lower error rate of about 20%. Studies will be

C. B. Moore et al.

210

Table IV. Recent studies of voriconazole susceptibility data showing MICs and methodology. Species

A. fumigatus (n:444)a

n

MIC range

MT

Media

Drug dilutions

IC

Final inoculum (CFU/ml)

IT

MIC Reading

Reference

12

0.06–0.5

m

RPMI

Batch

S

0.9–4.7104

35

50%

50

10 12

0.25–0.5 0.25–0.5

m m

RPMI 2%G RPMI

Batch Batch

? S

0.4–5104 0.4–5104

35 35

51 52

21 62

0.03–0.5 0.25–1 ? (MIC50:0.5; MIC90:1) 0.25–2 0.25–1

m M

RPMI RPMI 2%G

Doubling Doubling

H S

1104 ⬃1105

35 35

80% 75–100% 72 h 100% 100%

m M

RPMI 2%G RPMI 2%G

Doubling Doubling

H H

37 37

M

PYG

Doubling

?

A

RPMI 2%G

Doubling

S

11

0.25–4 (MIC90:2) 0.25–8 (MIC90:0.25) 0.5–1

5105 1103 (in 2 ml) 1104 (in 1 ml) 1107

m

RPMI

Batch

S

10 10

0.25–1 0.12–0.5

m m

RPMI 2%G RPMI

Batch Batch

10 8 8

0.25–0.5 0.5–2 0.25–4

m m M

RPMI RPMI 2%G RPMI 2%G

15

0.25–2

M

8

0.25–1

m

35 35 142 150 A. flavus (n:64)a

A. terreus (n:109)a

101

A. niger (n:53)a

A. nidulans (n:2)a

55 56

35

100% 100% 40–42 h 100%

59

35

100%

61

0.9–4.7104

35

50%

50

? S

0.4–5104 0.4–5104

35 35

51 52

Doubling Doubling Doubling

H H H

35 37 37

PYG

Doubling

?

35

RPMI 2%G

Doubling

H

1104 5105 1103 (in 2 ml) 1104 (in 1 ml) 5105

80% 75–100% 72 h 100% 100% 100% 40–42 h 100%

37

100%

35

80% 24 h 100% 40–42 h 100%

4

0.06–0.5

M

RPMI

Batch

H

110

8

0.25–1

M

RPMI 2%G

Doubling

H

37

10

0.25–1

m

RPMI

Doubling

H

1103 (in 2 ml) 1104

7 7

0.5–1 0.125–0.5

m M

RPMI 2%G RPMI 2%G

Doubling Doubling

H H

37 37

36

M

PYG

Doubling

?

2

0.5–4 (MIC90:4) 0.125–0.25

m

RPMI 2%G

Doubling

H

5105 1103 (in 2 ml) 1104 (in 1 ml) 5105

2

0.125–0.25

M

RPMI 2%G

Doubling

H

1103 (in 2 ml)

35

5 5

55 5 5 59 5 10 5 55

35

100% 100% 40–42 h 100%

5 5

37

100%

5

37

100% 40–42 h

5

59

a

Strains from reference 5 tested twice. Includes 28 laboratory-induced resistant strains from a parental strain with MIC:0.25 ␮g/ml. MT:method; IC:inoculum counts; IT:incubation temperature in °C; MIC reading as visual reduction in growth (after 48 hours incubation if not given); m:broth microdilution; M:broth macrodilution; A:agar dilution; H:haemocytometer; S:spectrophotometer; 2%G:2% glucose. b

needed to find the most accurate method to reliably produce the optimum inoculum to detect resistance in vitro.

Incubation Regarding temperature and duration of incubation, few important differences have been found in ITZ MIC results.2,64 Either 35 °C or 37 °C is adequate, and 48 h is preferred. An exception, where 24 h was preferred, has

been reported when a low inoculum was used.66 Espinel-Ingroff et al.65 tested 15 selected isolates of three species of Aspergillus and showed that 48 h was better than 24 or 72 h in distinguishing resistant isolates.

Reading In a study of 30 filamentous fungi where seven Aspergillus strains were included, Llop et al.72 reported that the MICs

Antifungal Aspergillus Drug Resistance of ITZ did not vary significantly when read at 48 or 72 h visually, with or without agitation, or spectrophotometrically. Espinel-Ingroff et al.65 showed that a no growth end point was better than prominent growth inhibition in distinguishing resistant isolates, in agreement with prior work.2 A variable affecting MICs are floating spores, since they can grow faster than those at the bottom of the wells or tubes (Mosquera, personal observations). Thus, they can alter a MIC interpretation by about 1–2 wells. The probability of floating spores increases with the use of higher inocula. Other means of reading end points, such as the use of Alamar Blue, are being explored.73

Mechanism of Action and Resistance in Azoles Azole antifungals are classified as triazoles or imidazoles depending on whether they have two or three nitrogens in the five-membered azole ring. Totally synthetic, they act on the ergosterol biosynthetic pathway at the C-14 demethylation stage by binding to the ERG11 gene product lanosterol 14␣-demethylase (14-DM). The 14-DM enzyme is a cytochrome P450 enzyme that has an active site containing a heme moiety. The unhindered nitrogen of the imidazole or triazole ring binds to the heme iron as a sixth ligand and inhibits demethylation of lanosterol.74 This leads to ergosterol depletion and the accumulation of lanosterol and other 14␣-methylated sterols (Fig. 2). A second nitrogen is thought to interact directly with the 14-DM apoprotein. Azoles bind weakly to the mammalian P450 enzyme and so drug toxicity is reduced in humans.

Target Mutation The decreased affinity of azole derivatives for the ERG11 gene product 14-DM is an important mechanism of resistance in yeasts and fungi. A defective 14-DM will result in the accumulation of the 14␣ methyl sterols. These insert into the plasma membrane and alter plasma membrane fluidity. This causes growth arrest in S. cerevisiae76 and C. albicans77 as well as making the cell more sensitive to reactive oxygen.78 Amino acid mutations responsible for resistance have been predicted in C. albicans 14-DM by comparing ERG11 gene sequences from resistant and sensitive isolates. The amino acid mutation R467K (where arginine (R) is replaced with a lysine (K) at amino acid position 467) is thought to be responsible for changing the position of the iron atom in the heme co-factor.79 This causes structural or functional alteration of the enzyme that is associated with the positioning of the heme. Synthetic mutations have also been introduced into the ERG11 gene to determine their effect on enzyme activity.

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For example, the T315A mutation in C. albicans 14-DM was predicted to be situated in the active site pocket of the enzyme.80 Substrates or inhibitors enter the active site of the enzyme through a channel that is only accessible by movement in an alpha helix of the protein. As expected, the mutation resulted in reduced enzymatic activity and a reduction in azole binding to the active site. A recent study has described five other mutations in the ERG11 gene that lead to azole resistance in C. albicans.81 They were discovered by functional expression of PCR-amplified ERG11 genes from resistant C. albicans in S. cerevisiae. The effect of each mutation on azole affinity was measured and was dependent on the azole used in the assay. These mutations were found to occur simultaneously in some resistant isolates. A mutation in the 14-DM was associated with resistance to triadimenol in some Uncinula necator and U. maydis strains82,83 and possibly related with azole resistance in Penicillium italicum.84 Alteration of the ERG11 gene as a cause of resistance can also be inferred in the absence of gene sequence data. In A. fumigatus, there was a high percentage of ergosterol in two ITZ-resistant isolates (AF90 and AF91) and a concomitant absence of the intermediate sterols ergostatetraenol and 14␣-methyl fecosterol.1 These isolates contained wild-type levels of cytochrome P450 enzyme, indicating that the inability of ITZ to bind effectively to 14-DM was the primary mode of resistance. Sequencing of the ERG11 gene in these isolates and comparison with wild type isolate sequence may highlight amino acid residues involved in the resistance phenotype. Analysis of sterol patterns in azole-resistant isolates indicates that the alteration of other enzymes in the ergosterol biosynthetic pathway can lead to resistance. These include mutation of the ERG3 gene that encodes the C-5 sterol desaturase enzyme. Mutation of this enzyme can lead to its inactivation and accumulation of 14␣ methyl fecosterol that replaces the function of the absent ergosterol in the cell membrane. The accumulation of 14␣ methyl fecosterol in two azole-resistant C. albicans clinical isolates was consistent with the alteration or absence of the C-5 sterol desaturase enzyme.85,86 However, as null mutants of ERG3 are unavailable in C. albicans, it is still unclear whether this enzyme can be solely responsible for azole resistance in this organism. Studies in Candida glabrata have shown that deletion of both the ERG3 and ERG11 genes was required for azole resistance and that deletion of ERG3 alone was insufficient.87 Minor changes in the sterol profile of three ITZ-resistant A. fumigatus isolates argued against ERG3 being a mechanism of resistance.1 Changes in sterol composition was proposed as a possible mechanism of resistance to flusilazole in Cercospora beticola strains.88

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Squalene ERG1

2,3-Oxidosqualene ERG7

Lanosterol

ERG11

4,4-Dimethyl-cholesta-8,24-triienol

24-Methylenedihydrolanosterol

ERG24

4,4-Dimethyl-cholesta-8,24-dienol

4,4-Dimethylergosta-8,14,24(28)-trienol

ERG25, X, Y

4-Methylcholesta-8,24-dienol

4,4-Dimethylergosta-8,24(28)-dienol

ERG25, X, Y Zymosterol

4-Methylergosta-8,24(28)-dienol

ERG6

14α Methyl fecosterol ERG2 Episterol ERG3

Ergosta-5,7,24(28)-trienol ERG5

Ergosta-5,7,22,24(28)-tetraenol ERG4 Ergosterol

Figure 2. Ergosterol biosynthetic pathway from squalene to ergosterol. The pathway shows the ERG gene designations and sterol intermediates. All of the genes listed have been cloned except for X and Y. These enzymes, together with ERG25, catalyse the removal of two methyl groups from C-4.75

Antifungal Aspergillus Drug Resistance

Target Overexpression Overexpression of ERG11 mRNA has been described as a mechanism of azole resistance in C. albicans and C. glabrata. It is always accompanied by mutations in other enzymes of the ergosterol biosynthetic pathway or overexpression of drug efflux pumps. It has not been shown to be a cause of resistance in Aspergillus species or other moulds. Increased gene copy number is a common mechanism of resistance in eukaryotic cells, but has not been found to play a major role in antifungal resistance.75 Amplification of the ERG11 gene, and a corresponding increase in ERG11 mRNA levels, was responsible for azole resistance in a single C. glabrata isolate.89 Increased drug efflux and changes in ERG7 activity had also been noted in this isolate.19 ERG11 levels, enzyme activity and drug efflux all reverted to normal after 159 subcultures despite the isolate retaining partial resistance to fluconazole. Recent studies link amplification of the ERG11 gene to chromosome duplication in this isolate. This also resulted in the altered expression of several other proteins.89 This mechanism of resistance has not so far been described in Aspergillus species.

Increased Drug Efflux Decreased intracellular accumulation of drugs or cytotoxic compounds has long been known as a mechanism of resistance in mammalian and bacterial systems.90 The first described case of drug efflux in Aspergillus species was fenarimol resistance in A. nidulans.91,92 Accumulation levels of this fungicide followed a transient pattern with maximum levels found in mycelia 10 min after addition. Similar results have been described for other fungicides in P. italicum93,94 and Penicillium digitatum.95 It was proposed that the transient levels of azole fungicide in wild type cells was due to an inducible energy dependent efflux mechanism, while efflux activity in mutants was thought to be constitutive. Induction of azole efflux from the cell coincided with a rapid increase in expression of two efflux transporters, suggesting they might be responsible for the phenotype.96 Drug efflux has been proposed as a possible mechanism of resistance to tioconazole in A. nidulans and Trichophyton rubrum isolates97 and to fenarimol in Nectria haematococca strains.98 Two different types of efflux pumps are responsible for the active removal of drugs from eukaryotic cells – ATP binding cassette transporters (ABCTs) and major facilitators (MFs). Together they account for almost half (over 50) of the solute transporters encoded in the genome of micro-organisms.75 The ABCT proteins transport toxic

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molecules from the cell, which are often hydrophobic or lipophilic, including azoles. MF transporters play a lesser role in azole resistance. At present, they are thought to only transport one azole substrate in fungi – fluconazole.99 Although 30 ABCTs have been described in S. cerevisiae, only 13 have so far been found in C. albicans which are putative efflux pumps.75 The C. albicans ABCTs can be subdivided into families as in S. cerevisiae. The PDR5 family is known as the CDR family in Candida species, for Candida drug resistance. The most prominent of these are the CDR1 and CDR2 gene products, which both play an important role in azole resistance.99,100 Homologues of these genes have been found in other Candida species such as C. dubliniensis, C. krusei and C. glabrata.101 The first member of the CDR family discovered in C. albicans was CDR1. It was isolated by functional complementation of a PDR5-deficient mutant of S. cerevisiae that was hypersensitive to several drugs and metabolic inhibitors. Complementation with CDR1 restored the resistance of the mutant to several drugs, including azoles.102 In another study, five azole-resistant strains of C. albicans showed an increase in CDR1 expression compared to azole-sensitive controls.103 Sanglard et al.104 showed that three out of five matched sets of sensitive and resistant isolates displayed increased CDR1 expression levels. Increased CDR1 expression has also been correlated to increasing MICs of fluconazole in a series of clinical isolates.79 Studies have shown that deletion of both alleles of CDR1 results in hypersusceptibility to terbinafine, amorolfine, and several metabolic inhibitors and to azole drugs.104 These data provide compelling evidence that overexpression of CDR1 mRNA is correlated with drug resistance. The importance of ABCTs compared with MF transporters in azole resistance has been demonstrated in C. albicans and S. cerevisiae. Presumably the same is true of Aspergillus species. The first documented case of drug efflux in Aspergillus was that of fenarimol resistance in A. nidulans,92 and so far 13 ABCTs have been identified in Aspergillus. These include AfuMDR1 and AfuMDR2 from A. fumigatus, AflMDR1 from A. flavus and atrA, atrB, atrC, atrC2, atrD and AbcA through to AbcD from A. nidulans.105–107 Given that there are seven ABCTs in the S. cerevisiae genome with highly similar sequence to PDR5, then the number of ABCTs with a role in drug resistance in A. fumigatus and A. flavus looks certain to increase. An EST library of A. nidulans indicates that additional putative members of the ABCT protein family also exist in the A. nidulans genome.106 The first Aspergillus ABCT gene described with a putative role in drug efflux was atrB from A. nidulans.96 atrB cDNA was able to increase the MIC of cycloheximide and

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imazilil by three-fold in a drug hypersusceptible PDR5-deficient mutant of S. cerevisiae. Expression levels of atrB in A. nidulans were also strongly enhanced by several unrelated compounds. Another gene isolated from the same library screen, atrA, was not found to confer resistance to any drugs in the PDR5-deficient mutant. However, experiments have shown that atrA mRNA levels are upregulated in A. nidulans germlings, which coincides with reduced accumulation levels of azole. It is interesting to note that both atrA and atrB gene products have similar protein topologies to Pdr5p and Snq2p. These proteins are characterized by having two domains in each half of the protein. One of them is hydrophilic and responsible for the binding and hydrolysis of ATP. The hydrophilic region precedes the hydrophobic region, which contains six transmembrane domains. The physiological significance of this domain structure on protein function has yet to be elucidated. Studies using [3H]-ITZ demonstrated that one clinical isolate, AF72, was resistant to ITZ due to decreased accumulation of drug compared to susceptible isolates.1 This was probably due to drug efflux given that treatment of AF72 cultures with an efflux pump inhibitor restored ITZ levels to those of other susceptible isolates (Microcide Pharmaceuticals, personal communication). Our laboratory has cloned two ABCT genes from A. fumigatus and studies are now underway to determine whether they play a role in drug efflux. One of the genes, adr1, has 36% sequence identity to CDR1 and PDR5. The expression of adr1 was upregulated in isolate AF72, but only in cultures grown in the presence of ITZ. This might indicate that adr1 is part of a stress response in this isolate.108 Studies are currently underway to establish the substrate range of adr1 in a PDR5-deficient mutant of S. cerevisiae.

Reduced Permeability Another study has suggested that two ITZ-resistant isolates generated by exposure to miconazole (MCZ) have a decreased permeability to the drug as opposed to an efflux mechanisim.109 This observation is based on a fall in intracellular [3H]-ITZ concentrations after exposure to the respiratory inhibitor carbonyl cyanide m-chlorophenyl hydrazone. Few other observations of reduced permeability have been made. More studies are therefore required to resolve the mechanisms of decreased drug accumulation in A. fumigatus.

Frequency of Azole Resistance Natural azole resistance has been reported in filamentous fungi, especially in crop pathogens. Resistance to

agriculturally used azoles in the field is an increasing problem in fungal crop management.110 With regard to Aspergillus, clinical strains that are naturally less susceptible in vitro than the majority to some azoles have been reported. In our laboratory we have analysed clinical isolates of Aspergillus received during a recent 3-year period (1 June 1997–31 May 2000). Using a microtitre method (RPMI-1640 with 2% glucose and final inoculum of 5105 conidia/ml), we tested 94 isolates of Aspergillus consisting of 67 (71%) A. fumigatus, seven (7%) A. terreus and 10 (11%) each of A. flavus and A. niger. These were predominantly from respiratory sites such as sputa and bronchial washes (60%) and ear swabs (16%). Overall, four isolates (4.2%) were found to have higher MIC values to ITZ (8 ␮g/ml) – three A. fumigatus (from two patients) and one A. flavus. In addition, we tested 86 of the 94 isolates received against VCZ using the same method, and if a similar breakpoint is presumed, three isolates (3.5%) had elevated MICs – two A. fumigatus and one A. flavus. Cross-resistance may be a possibility since two of the isolates (one of the A. fumigatus isolates and the A. flavus isolate) were less susceptible to both drugs. Additional in vivo studies would be required to clarify this issue. Furthermore, at least one of these patients had been receiving ITZ and had maintained therapeutic levels (5 ␮g/ml) of the drug. However, no pre-therapy isolate was available in order to ascertain whether resistance had developed as a result. In another study using the same microtitre format, we reported nine A. fumigatus isolates (from six patients) with ITZ MICs 16 ␮g/ml.57 We have also previously reported two isolates of A. flavus that had ITZ MICs 16 ␮g/ml.5,57 These strains, however, have shown MICs less than 1 ␮g/ml in other in vitro test formats and one is susceptible in vivo.67 Therefore, there remains uncertainty whether our current method of testing is appropriate for A. flavus. MIC results against ITZ of about 900 A. fumigatus strains are reported in the recent literature. In total, we have found 19 A. fumigatus isolates (from no more than 14 patients) and one of A. nidulans, which are resistant to ITZ. This resistance represents about 2.1% of the reported strains or 1.5% when two or more resistant strains isolated from the same patient are assumed as being the same (hence only 14 resistant isolates). Chryssanthou58 reported four A. fumigatus isolates with MICs higher than 32 ␮g/ml (from three patients) from a total of 107 isolates of that species obtained from various Swedish hospitals. The MIC range (without the resistant ones) was 0.125–1 ␮g/ml. The author suggests that the four isolates gradually developed resistance to ITZ

Antifungal Aspergillus Drug Resistance during treatment. Isolates were obtained at different time points and MICs rose from 0.125–0.25 to 32 ␮g/ml from the first to last isolate from each patient. Unfortunately, the genetic identities of the in vitro susceptible and resistant strains from the same patient were not studied, so it was not proven that they were identical. Dannaoui et al.53 reported four isolates of A. fumigatus (from three patients) with MICs greater than 16 ␮g/ml out of 156 tested (MIC90:2 ␮g/ml), and one A. nidulans with a MIC greater than 16 ␮g/ml, out of 17 tested (MIC90:4 ␮g/ml). Of the three patients with cultures of A. fumigatus, only two had received prior treatment with ITZ. The same authors reported an isolate resistant to ITZ in a murine model.49 It had a MIC of 16 ␮g/ml (employing an NCCLS-based broth microdilution technique) and, interestingly, was isolated from a patient who had not received ITZ. Verweij et al.61 tested 150 A. fumigatus isolates from The Netherlands, of which 130 were clinical and 20 were environmental. They presented a MIC90 equal to 0.25 ␮g/ml, with no evidence of ITZ resistance even in a Table V. Laboratory-induced resistance to azoles and pyrimidines in Aspergillus and other moulds. Species

Agent

Reference

A. nidulans A. nidulans A. nidulans Ustilago maydis

Fenarimol Triadimefon Tioconazole Triadimenol – cross resistance to diclobutrazole, hexaconazole, procholaz, tebuconazole, fenarimol Flusilazole Fenarimol Tioconazole Fenarimol – cross resistance to bitertanol, etaconazole, imazalil Imazalil

111 112 97 83

Cercospora beticola Nectria haematococca Trichophyton rubrum Penicillium italicum Penicillium italicum

88 98 97 113 114

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patient who had received long-term treatment. However, they did note an isolate with a VCZ MIC of 8 ␮g/ml. No other isolates resistant to VCZ in vitro have been reported in the literature. Abraham et al.59 reported higher MIC values for both ITZ and VCZ, but it seemed to be a trend for all the Aspergillus species tested in their study. Mean MIC values were also high. A broth macrodilution method with peptone yeast extract was employed, possibly accounting for the higher values obtained. Resistance rates of Aspergillus to ITZ appear to be low; nevertheless, documented cases do exist, hence supporting the need for clinically relevant and standardized susceptibility testing.

Resistance Selection/Induction Laboratory-induced selected resistance in Aspergillus and other moulds to non-clinically used azoles has been reported (Table V). In addition, resistance is seen to the chemically related pyrimidines, which often show cross-resistance to triazoles and imidazoles. Regarding clinically used azoles, Abraham et al.59 induced resistance to ITZ in A. fumigatus isolates with MICs ranging from 2 to 16 ␮g/ml. They were selected from a parental susceptible strain (MIC:0.25 ␮g/ml) by selecting conidia in plates containing ITZ at a concentration of 8 ␮g/ml. Mikami et al.115 tried unsuccessfully to induce resistance in an A. fumigatus strain by subculturing serially on brain heart infusion agar containing gradiently increasing concentrations of MCZ and ITZ.

Cross-resistance Cross-resistance data has also been reported for agriculturally used azoles.116 Some data has also been reported on cross-resistance to clinically used azoles. Table VI shows the comparative activities of four azole drugs.

Table VI. Comparative activities of antifungal drugs against Aspergillus (azoles and terbinafine). Geometric mean MICs* (number tested). Species

Itraconazole (ITZ)15

Voriconazole5

Posaconazole (SCH-56592)4

Ravuconazole (BMS 207147)57

Terbinafine15

A. fumigatus, ITZ-S A. fumigatus, ITZ-R A. flavus A. terreus A. niger

0.33 (51) 32 (9) 0.43 (13) 0.31 (12) 0.85 (13)

0.63 (32) 1.26 (3) 1 (7) 0.54 (8) 0.89 (6)

0.07 (36) 0.79 (3) 0.23 (7) 0.05 (7) 0.09 (7)

1.25 (41) 1.36 (9) 2.83 (10) 3.03 (10) 2.64 (10)

19.89 (51) 14.81 (9) 0.10 (13) 0.16 (12) 0.19 (13)

Total

0.58 (98)

0.71 (56)

0.086 (60)

1.71 (80)

2.85 (98)

* For the purpose of analysis, all values 16 ␮g/ml were classed as 32 ␮g/ml. A microtitre method using either casitone with 2% glucose (terbinafine) or RPMI-1640 with 2% glucose (all other drugs) and a final inoculum of 5105 conidia/ml was used.

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The geometric mean MIC value for posaconazole is 10 times higher when comparing ITZ-resistant isolates to those that were ITZ-susceptible. However, only three ITZ-resistant isolates were tested in this study. Neither VCZ nor ravuconazole appear to show similar findings. Two A. fumiga-tus isolates resistant in a murine model have been tested against VCZ and posaconazole, presenting only cross-resistance to the latter.4,5 This was confirmed by Espinel-Ingroff, who tested A. fumigatus isolates with high MICs for ITZ, but they did not seem to show cross-resistance to VCZ.50 Abraham et al.59 tested 28 A. fumigatus isolates with induced ITZ resistance and did not show cross-resistance to VCZ. Manavathu et al.109 tested two ITZ-resistant A. fumigatus isolates (laboratory selected) against MCZ and KCZ and showed a 5- to 11.2-fold increase in their MIC values. Perhaps more importantly, cross-resistance between azoles appears to exist in vivo. Oakley et al.3 showed that with an A. fumigatus in vivo ITZ-susceptible strain, posaconazole at 5, 10 and 25 mg/kg and ITZ at 25 mg/kg yielded a 90–100% survival rate. With an in vivo ITZ-resistant strain, for the same posaconazole doses, survival rates were 20, 60 and 100%, respectively, and 0% with ITZ.

Prevention of Azole Resistance in Aspergillus Of immediate medical concern is how the reduced accumulation of azoles in A. fumigatus can be reversed. The development of novel antifungals that cannot be used as substrates for efflux transporters is one option. Screening for these novel agents can be performed once A. fumigatus strains containing deletions in several ABCT genes become available. The direct targeting of ABCTs themselves by compounds that inhibit their activity might also be successful in reducing drug efflux. Currently, there is a lack of fungal efflux pump inhibitors (FEPIs) specifically developed for Aspergillus spp., although one of the Candida FEPIs reduces the MIC of ITZ in an A. fumigatus isolate (AF72) which accumulates reduced levels of ITZ (Microcide Pharmaceuticals, personal communication). Thus, FEPIs are a promising approach for the future treatment of A. fumigatus infection if used in combination with azole antifungals. Combination therapy with antifungal agents and immuno-modulators that increase host defences might help prevent resistance. The objectives would be to increase the number of phagocytic cells and/or to activate the phagocytes to kill the fungal cell more efficiently. Cytokines such as granulocyte colony-stimulating factor may be useful in the treatment of fungal disease by increasing the number of mainly hyphal-killing neutrophils and promoting an increase in the number of phagocytes in patients at risk of aspergillosis.

Newer Azoles with Activity Against Aspergillus Several azoles are also currently under investigation for their efficacy against Aspergillus spp. Voriconazole The novel triazole, VCZ, is currently in phase III clinical trials. Derived from a synthetic modification of fluconazole, it has improved antifungal activity and potency towards 14-DM compared with fluconazole.117 The intrinsic activity of VCZ is approximately equal to ITZ against Aspergillus, but more often fungicidal in vitro.5,56 It is also more selective at targeting ergosterol instead of mammalian cholesterol biosynthesis compared with KTZ or ITZ.117 In immunocompromised guinea pigs with pulmonary aspergillosis, VCZ was significantly (P0.05) more effective than ITZ at similar dosage concentrations.118 In another study, VCZ was more effective than ITZ at preventing A. fumigatus endocarditis in guinea pigs.119 In both these studies however, serum concentrations of VCZ and ITZ were not reported. VCZ is also active in invasive aspergillosis in humans.120,121 Only rarely have VCZ-resistant isolates of Aspergillus been identified. Posaconazole (SCH-56592) The novel triazole, posaconazole, also shows broad-spectrum activity against fungal pathogens, including Aspergillus spp.122 Currently in phase II/III clinical trials, it is at least 10 times more potent than ITZ at inhibiting 14-DM activity in A. fumigatus and A. flavus.123 In a temporary neutropenic mouse model of disseminated aspergillosis (lungs and kidneys) infected with ITZ-susceptible and -resistant A. fumigatus isolates, posaconazole was superior to ITZ and AmB.3 There is a degree of in vitro cross-resistance seen between posaconazole and ITZ (Table VI), the clinical significance of which is unclear. Ravuconazole (BMS 207147) Ravuconazole is another novel azole compound with anti-Aspergillus activity.57 Currently it is in phase II clinical studies. Its intrinsic activity against Aspergillus is either equal to or slightly inferior to ITZ, but there is no cross-resistance with ITZ (Table VI). It has a much longer half-life than any other azole in development (approximately 100 h). In vivo models have demonstrated good efficacy in invasive aspergillosis.124 In vitro/in vivo correlation and clinical significance At the current state of knowledge some scepticism is still appropriate in the interpretation of in vitro results.

Antifungal Aspergillus Drug Resistance However, if an established standardized methodology is used, a reliable MIC range and the frequency of microbiological resistance may be ascertained for azoles, but not polyenes. Due to the small patient population of Aspergillus infections, the use of animal models is the only method employed in order to detect azole resistance in vivo. However, discrepancies have been found with regards to the susceptibility of the same strain when tested in different models.49 It seems that the response of a strain in animal models depends on the treatment dose and the onset timing of the treatment. In most in vivo experiments ITZ has shown a dose-dependent response2,4,125 as has posaconazole against an ITZ in vivo resistant strain.3 Another problem in interpretation when using animal models is the variable ITZ absorption, highlighting the importance of documenting adequate ITZ levels. Furthermore, the Aspergillus patient population is heterogeneous. Factors affecting outcome include drug pharmacokinetics, compliance, drug–drug interactions and severity of disease. Thus, clinical outcome is probably affected not only by the intrinsic susceptibility of a strain against any given antifungal drug, but to an important extent by the state of the patient. For this reason, and the low frequency of resistance, establishing breakpoints for resistance will be difficult based on clinical studies alone. Break points have yet to be formally proposed.

Future Directions Refinements and optimization of the susceptibility testing of azoles against Aspergillus will enable a reproducible and clinically predictive test to be made available in the near future. Breakpoint setting will follow. Substantial work needs to be done to identify a methodology for reliably testing Aspergillus against polyenes, whether lipid incorporated or not.

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