Abstract
We evaluated the activities of amphotericin B, itraconazole, voriconazole, caspofungin, and posaconazole against zygomycetes by CLSI M38-A, Etest and Sensititre. The most active drug was posaconazole, followed by amphotericin B and itraconazole. The correlation of the Etest and Sensititre with CLSI M38-A was moderate for posaconazole but poor for the others.
The incidence of zygomycosis is increasing in some institutions, purportedly due to voriconazole prophylaxis (16, 19, 20, 22, 23, 25, 28, 31, 32). The introduction of new antifungal agents and the availability of alternative antifungal susceptibility procedures necessitate studies comparing the in vitro activity of these new antifungal agents against zygomycetes.
In this study, the in vitro activities of amphotericin B (AMB), itraconazole (ITC), voriconazole (VC), caspofungin (CAS), and posaconazole (POS) were assessed against 45 clinical strains of zygomycetes (Table 1). In addition, we compared the results obtained by the CLSI M38-A method with the Etest and Sensititre YeastOne.
TABLE 1.
In vitro activity of the five antifungal agents studied against the 45 zygomycetes isolates obtained by the CLSI M38-A procedure at 24 and 48 h of incubation
| Species (no. of strains) | Antifungal drug | Result (μg/ml) at incubation time (h): |
|||||||
|---|---|---|---|---|---|---|---|---|---|
| 24 |
48 |
||||||||
| MIC50 | MIC90 | MECa | MIC range | MIC50 | MIC90 | MECa | MIC range | ||
| Overall (45) | AMB | 1 | 2 | 0.125-4 | 1 | 4 | 0.125->16 | ||
| ITC | 1 | >16 | 0.25->16 | 4 | >16 | 0.5->16 | |||
| VC | 16 | >16 | 4->16 | >16 | >16 | 8->16 | |||
| POS | 0.5 | 1 | 0.125-2 | 0.5 | 2 | 0.25->16 | |||
| CAS | 256 | >256 | 128 | 64->256 | >256 | >256 | 256 | 128->256 | |
| Absidia spp. (8) | AMB | 1 | 4 | 0.5-4 | 0.5 | 4 | 0.5-4 | ||
| ITC | 1 | >16 | 1->16 | 1 | >16 | 1->16 | |||
| VC | >16 | >16 | 16->16 | >16 | >16 | >16 | |||
| POS | 0.5 | 1 | 0.25-1 | 0.5 | >16 | 0.5-16 | |||
| CAS | 256 | >256 | 64 | 128->256 | >256 | >256 | 256 | 128->256 | |
| Cunninghamella spp. (4) | AMB | 2 | 4 | 0.25-4 | 2 | >16 | 0.25->16 | ||
| ITC | 1 | 4 | 1-4 | 1 | 4 | 1-4 | |||
| VC | >16 | >16 | 16->16 | >16 | >16 | >16 | |||
| POS | 0.5 | 0.5 | 0.25-0.5 | 0.5 | 0.5 | 0.5 | |||
| CAS | 256 | >256 | 128 | 128->256 | 256 | >256 | 128 | 128->256 | |
| Mucor spp. (19) | AMB | 1 | 1 | 1 | 1 | 2 | 1-2 | ||
| ITC | 1 | >16 | 1->16 | 4 | >16 | 1->16 | |||
| VC | >16 | >16 | 8->16 | >16 | >16 | 8->16 | |||
| POS | 0.5 | 2 | 0.25-2 | 0.5 | 2 | 0.25->16 | |||
| CAS | 256 | >256 | 128 | 64->256 | >256 | >256 | 128->256 | ||
| Rhizomucor spp. (1) | AMB | 1 | 1 | 1 | 1 | 1 | 1 | ||
| ITC | 2 | 2 | 2 | >16 | >16 | >16 | |||
| VC | 16 | 16 | 16 | >16 | >16 | >16 | |||
| POS | 0.5 | 0.5 | 0.5 | 2 | 2 | 2 | |||
| CAS | 256 | 256 | 32 | 256 | >256 | >256 | 64 | >256 | |
| Rhizopus spp. (11) | AMB | 1 | 2 | 0.125-2 | 1 | 2 | 0.125-4 | ||
| ITC | 1 | >16 | 0.25->16 | 4 | >16 | 0.05->16 | |||
| VC | 16 | 16 | 8-16 | 16 | >16 | 8->16 | |||
| POS | 0.5 | 0.5 | 0.125-1 | 0.5 | 1 | 0.25->16 | |||
| CAS | >256 | >256 | 128 | 128->256 | >256 | >256 | 256 | 128->256 | |
| Syncephalastrum spp. (2) | AMB | 0.125 | 1 | 0.125-1 | 0.125 | 2 | 0-0.125 | ||
| ITC | 1 | 1 | 1 | 1 | 8 | 1-8 | |||
| VC | 4 | >16 | 4->16 | >16 | >16 | >16 | |||
| POS | 0.25 | 0.5 | 0.25-0.5 | 0.5 | 0.5 | 0.5 | |||
| CAS | 256 | >256 | 8 | 256->256 | 256 | >256 | >256 | 256->256 | |
The MEC was calculated only for caspofungin.
CLSI M38-A method.
The antifungal drugs used were AMB (Sigma Chemical Co., St. Louis, MO), ITC (Janssen Pharmaceutical Research and Development, Madrid, Spain), VC (Pfizer Pharmaceutical Group, New York, NY), POS (Schering-Plough, Kenilworth, NJ), and CAS (Merck Research Laboratories, Rahway, NJ). The broth microdilution method was performed according to CLSI guidelines (21). The final concentration of the VC, ITC, POS, and AMB in the wells ranged from 0.03 to 16 μg/ml. The final concentration of CAS in the wells ranged from 0.250 to 256 μg/ml. The MIC endpoint was defined as the lowest concentration producing complete inhibition of growth (MIC-0) for all the antifungal drugs studied. The minimum effective concentration (MEC) endpoint for CAS was defined as the lowest concentration at which an abnormal growth was observed.
Quality control was ensured by testing Aspergillus flavus ATCC 204304 and Aspergillus fumigatus ATCC 204305. All results were within the recommended limits of CLSI M38-A.
Etest and Sensititre YeastOne procedures.
Etest strips (AB Biodisk, Solna, Sweden) of ITC, VC, CAS, and AMB were supplied by Tec Laim (Madrid, Spain), and those of POS were supplied by Schering-Plough (Kenilworth, NJ). Inoculum suspensions were prepared in the same way as for the CLSI method. The MIC was defined as the lowest drug concentration at which the border of the elliptical inhibition zone intercepted the scale on the antifungal strip.
The activities of AMB, VC, ITC, and CAS were studied by means of Sensititre YeastOne (Trek Diagnostic Systems, Ltd., East Grinstead, United Kingdom). The trays containing serial twofold dilutions of the drug (0.008 to 16 μg/ml) were used. Inoculum suspensions were prepared in the same way as for the CLSI method, and the adjusted suspensions were diluted at 1:100 in RPMI.
The CLSI microtiter panels, Sensititre panels, and Etest plates were incubated at 35°C and read after 16, 24, 36, 48, and 72 h of incubation.
We compared CLSI M38-A results (incubation at 24 h) with the alternative methods (incubations at 16, 24, 36, 48, and 72 h). MIC endpoint discrepancies of no more than ±2 dilutions were used to calculate the percentage of agreement between the two methods (14, 28).
POS (MIC90, 1 μg/ml) was the most active drug, followed by AMB, ITC and VC, and CAS. The activity of CAS was determined using the MIC and MEC and showed values of >128 μg/ml, irrespective of the parameter used. We did not find significant variations between the MIC90 obtained at any time of incubation, with the exception of the MICs of POS against Absidia (1 μg/ml and >16 μg/ml, respectively). Absidia and Mucor showed the highest MICs for POS (ranging from 0.25 μg/ml to >16 μg/ml). In addition, Absidia and Cunninghamella were less susceptible to AMB than the other species (ranging from 0.25 μg/ml to >16 μg/ml). VC and CAS were inactive against all the isolates tested. VC and CAS showed very high MIC90s, 8 to 16 μg/ml and 128 to >256 μg/ml, respectively.
AMB has traditionally been the treatment of choice for zygomycosis (1, 2, 4, 12, 18, 24). The limited clinical use of ITC in the treatment of zygomycosis does not allow us to draw any conclusions from the in vitro data (8, 15, 17). CAS and VC proved to be quite resistant in vitro, and this correlates well with previous reports on the clinical inefficacy of both drugs in the treatment of zygomycosis (6, 7, 10, 27, 29). While VC has been considered responsible for increased incidence in some institutions (16, 19, 20, 22, 23, 25, 28, 31, 32), POS seems to be very promising for the treatment of zygomycosis (3, 11, 13, 26, 30).
This is the first study to evaluate the correlation between the CLSI M38-A procedure and the Etest and Sensititre YeastOne methods for testing the antifungal activity of POS, ITC, AMB, CAS, and VC against zygomycetes at different incubation times. The percentages of strains whose Sensititre YeastOne and Etest MICs differed by ±1, ±2, and ≥±3 dilutions compared with the CLSI procedure are shown in Table 2.
TABLE 2.
Percent agreement between the CLSI (24 h) method and Sensititre YeastOne and Etest procedures at different incubation times
| Drug | Incubation time (h) | % Agreement of CLSI result with that of E/Ya within no. of dilutions: |
||||||||
|---|---|---|---|---|---|---|---|---|---|---|
| ≥−3 | −2 | −1 | 0 | +1 | +2 | ≥+3 | ±1 | ±2 | ||
| AMB | 16 | 86.4/90.9 | 4.55/9.09 | 9.09/ | 13.6/9.09 | |||||
| 24 | 43.2/70.5 | 25/25 | 18.2/4.55 | 11.4 | 2.27 | 29.5/4.55 | 54.5/29.5 | |||
| 36 | /59.1 | 11.4/9.09 | 20.5/27.3 | 13.6/4.55 | 15.9 | 9.09 | 29.5 | 50/31.8 | 70.5/40.9 | |
| 48 | /65.9 | 4.55/27.3 | 25/6.82 | 9.09 | 18.2 | 13.6 | 29.5 | 52.3/6.82 | 70.5/34.1 | |
| 72 | /59.1 | /22.7 | 6.82/18.2 | 6.82 | 9.09 | 4.55 | 72.7 | 22.7/18.2 | 27.3/40.9 | |
| CAS | 16 | /9.09 | /90.9 | 100 | 100/90.9 | 100/90.9 | ||||
| 24 | /6.67 | /93.3 | 100 | 100/93.3 | 100/93.3 | |||||
| 36 | /100 | 100 | 100/100 | 100/100 | ||||||
| 48 | /100 | 100 | 100 | 100/100 | ||||||
| 72 | /100 | 100 | 100 | 100/100 | ||||||
| ITC | 16 | 6.82/27.3 | 4.55/30.3 | 11.4/21.2 | 25/18.2 | 20.5/3.03 | 9.09 | 22.7 | 56.8/42.4 | 70.5/72.7 |
| 24 | /20.5 | /22.7 | 11.4/29.5 | 22.7/20.5 | 13.6/6.82 | 18.2 | 34.1 | 47.7/56.8 | 65.9/79.5 | |
| 36 | /6.82 | /9.09 | 6.82/29.5 | 13.6/25 | 4.55/4.55 | 75/25 | 20.5/54.5 | 25/68.2 | ||
| 48 | /13.6 | /27.3 | 2.27/20.5 | 15.9/2.27 | /6.82 | 4.55/6.82 | 77.3/22.7 | 18.2/29.5 | 22.7/63.6 | |
| 72 | /4.55 | /22.7 | /22.7 | 15.9/2.27 | 2.27 | /4.55 | 81.8/43.2 | 18.2/25 | 18.2/52.3 | |
| POS | 16 | 4.55 | 22.7 | 22.7 | 31.8 | 6.82 | 11.4 | 77.3 | 88.6 | |
| 24 | 6.82 | 27.3 | 29.5 | 18.2 | 18.2 | 63.6 | 81.8 | |||
| 36 | 4.55 | 11.4 | 11.4 | 72.7 | 15.9 | 27.3 | ||||
| 48 | 2.27 | 4.55 | 13.6 | 79.5 | 6.82 | 20.5 | ||||
| 72 | 2.27 | 2.27 | 4.55 | 90.9 | 4.55 | 9.09 | ||||
| VC | 16 | 13.6/40.6 | 13.6/28.1 | 18.2/15.6 | 43.2/12.5 | 11.4/3.13 | 72.7/31.3 | 86.4 | ||
| 24 | /25.6 | /23.3 | 9.09/30.2 | 47.7/16.3 | 29.5/4.65 | 13.6 | 86.4/51.2 | 100/74.4 | ||
| 36 | /6.82 | /13.6 | 22.7 | 45.5/45.5 | 29.5/11.4 | 22.7 | 2.27 | 75/79.5 | 97.7/93.2 | |
| 48 | /18.2 | /18.2 | 50 | 45.5/11.4 | 29.5/2.27 | 22.7 | 2.27 | 75/63.6 | 97.7/81.8 | |
| 72 | /9.09 | /11.4 | 47.7 | 45.5/20.5 | 29.5/11.4 | 22.7 | 2.27 | 75/79.5 | 97.7/90.9 | |
E, Etest; Y, Sensititre YeastOne.
The highest agreements between AMB, ITC, and POS Etest and the reference MICs (70.5%, 70.5%, 88.6%) were at 48, 16, and 16 h of incubation, respectively. Our results show a moderate correlation between the Etest and CLSI M38-A for POS at 16 h of incubation (88.6%). The highest agreements between the AMB and ITC Sensititre YeastOne and the reference MICs (40.9%, 79.5%) were at 36 and 24 h of incubation, respectively. Although the Etest and Sensititre YeastOne presented good correlations with CLSI M38-A for VC and CAS, both agents presented very poor activity.
Sensititre YeastOne and the Etest have been successfully compared with the CLSI procedure in other filamentous fungi, such as Aspergillus fumigatus (14). The authors found that the correlation was very good for azoles at 48 h of incubation, although that for zygomycetes seems to be different. Other authors have found a good correlation between Etest/Sensititre and CLSI, but the agreement used by them (±3 dilutions) may explain the differences with our results (9).
In conclusion, POS and AMB have good in vitro activity against zygomycetes. The correlation of the Etest and Sensititre YeastOne (±2 dilutions) with CLSI M38-A was moderate (below 80%), with the exception of POS. The Etest can be used to determine the activity of POS at 16 h of incubation. CLSI M38-A should remain the reference procedure for antifungal susceptibility testing against zygomycetes.
Acknowledgments
We thank Thomas O'Boyle for his help in the translation of the article.
This study does not present any conflict of interest.
We thank Schering-Plough for the posaconazole Etest strips.
This study was partially financed by grants from Red Española de Investigación en Patología Infecciosa C/03/14 (REIPI). J.G. is contracted by Fondo de Investigación Sanitaria (FIS) CM05/00171.
Footnotes
Published ahead of print on 28 December 2006.
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