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Original article In vitro susceptibility testing of Aspergillus spp. against voriconazole, itraconazole, posaconazole, amphotericin B and caspofungin SHI Jun-yan, XU Ying-chun, SHI Yi, LÜ Huo-xiang, LIU Yong, ZHAO Wang-sheng, CHEN Dong-mei, XI Li-yan, ZHOU Xin, WANG He and GUO Li-na Keywords: aspergillus; antifungal agents; drug resistance; antifungal activity Background During recent years, the incidence of serious infections caused by opportunistic fungi has increased dramatically due to alterations of the immune status of patients with hematological diseases, malignant tumors, transplantations and so forth. Unfortunately, the wide use of triazole antifungal agents to treat these infections has lead to the emergence of Aspergillus spp. resistant to triazoles. The present study was to assess the in vitro activities of five antifungal agents (voriconazole, itraconazole, posaconazole, amphotericin B and caspofungin) against different kinds of Aspergillus spp. that are commonly encountered in the clinical setting. Methods The agar-based Etest MIC method was employed. One hundred and seven strains of Aspergillus spp. (5 species) were collected and prepared according to Etest Technique Manuel. Etest MICs were determined with RPMI agar containing 2% glucose and were read after incubation for 48 hours at 35°C. MIC50, MIC90 and MIC range were acquired by Whonet 5.4 software. Results The MIC90 of caspofungin against A. fumigatus, A. flavus and A. nidulans was 0.094 μg/ml whereas the MIC90 against A. niger was 0.19 μg/ml. For these four species, the MIC90 of caspofungin was the lowest among the five antifungal agents. For A. terrus, the MIC90 of posaconazole was the lowest. For A. fumigatus and A. flavus, the MIC90 in order of increasing was caspofungin, posaconazole, voriconazole, itraconazole, and amphotericin B. The MIC of amphotericin B against A. terrus was higher than 32 μg/ml in all 7 strains tested. Conclusions The in vitro antifungal susceptibility test shows the new drug caspofungin, which is a kind of echinocandins, has good activity against the five species of Aspergillus spp. and all the triazoles tested have better in vitro activity than traditional amphotericin B. Chin Med J 2010;123(19):2706-2709
I
n recent years, the number of immunocompromised people has been increasing. Invasive Aspergillus infection has become the predominant cause of deaths in individuals with bone marrow transplantations, stem cell transplantations, solid organ transplantations and hematologic malignancies.1 Invasive aspergillosis is mainly caused by A. fumigatus whereas infections caused by non-A. fumigatus such as A. flavus, A. niger, A. terrus, and A. nidulans are on the rise. These non-A. fumigatus are usually insensitive to traditional antifungal agents; for example, A. terrus has intrinsic resistance against amphotericin B.2 A. fumigatus resistant against itraconazole has also been reported.3 New antifungal agents, such as posaconazole and caspofungin, offer more options for the antifungal treatments; however, the number of antifungal agents is much less than that of antibacterial drugs. How to optimize antifungal treatments is of great concern for the clinicians. In vitro susceptibility testing is an important step in solving the problem. The methods for in vitro antifungal susceptibility testing include: the broth micro dilution (BMD) procedure recommended by CLSI, the disk diffusion method and agar-based Etest MIC method. The BMD method has been proven to be very reproducible and useful in both clinical testing and antifungal surveillance studies, however, with drawbacks
of being time-consuming and labor-intensive it can not meet all clinical needs. The Etest method is easy to DOI: 10.3760/cma.j.issn.0366-6999.2010.19.016 Clinical Microbiology Laboratory, Peking Union Medical College Hospital, Peking Union Medical College, China Academy of Medical Science, Beijing 100730, China (Shi JY, Xu YC, Wang H, Guo LN) Department of Respiratory, Nanjing General Hospital of Nanjing Military Command, Nanjing, Jiangsu 210002, China (Shi Y) Clinical Laboratory, Zhejiang Provincial People’s Hospital, Hangzhou, Zhejiang 310014, China (Lü HX) Clinical Laboratory, Shengjing Hospital of China Medical University, Shenyang, Liaoning 110001, China (Liu Y) Clinical Microbiology Laboratory, Jiangsu Province Hospital, Nanjing, Jiangsu 210029, China (Zhao WS) Clinical Laboratory, First Affiliated Hospital of Sun Yat-Sen University, Guangzhou, Guangdong 510080, China (Chen DM) Department of Dermatology, Second Affiliated Hospital of Sun Yat-Sen University, Guangzhou, Guangdong 510080, China (Xi LY) Department of Respiratory, Shanghai First People’s Hospital, Shanghai 200080, China (Zhou X) Correspondence to: Dr. XU Ying-chun, Peking Union Medical College Hospital, Peking Union Medical College, China Academy of Medical Science, Beijing 100730, China (Tel: 86-10-65295606. Fax: 86-10-65295406. Email:
[email protected]) This study was supported by a special fund granted by the Ministry of Health for public profession (No. 200802026).
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Chinese Medical Journal 2010;123(19):2706-2709 Table. MICs to voriconazole, itraconazole, posaconazole, amphotericin B and caspofungin determined by Etest method Species (n) A. fumigatus (50)
A. flavus (35)
A. niger (8)
A. terrus (7)
A. nidulans (7)
Antifungal agents Voriconazole Itraconazole Posaconazole Amphotericin B Caspofungin Voriconazole Itraconazole Posaconazole Amphotericin B Caspofungin Voriconazole Itraconazole Posaconazole Amphotericin B Caspofungin Voriconazole Itraconazole Posaconazole Amphotericin B Caspofungin Voriconazole Itraconazole Posaconazole Amphotericin B Caspofungin
MIC range 0.094–0.38 0.064–>32 0.016–1.5 0.006–6 0.032–0.125 0.094–0.5 0.047–4 0.001–0.75 1–>32 0.016–0.19 0.25–0.75 1–>32 0.125–1 0.047–>32 0.023–0.19 0.25–0.5 0.047–0.125 0.032–0.094 >32–>32 0.016–0.19 0.094–0.125 0.19–0.75 0.064–0.125 0.38–2 0.047–0.094
perform so this method is a premium option in the clinical setting. This study was aimed to assess in vitro activities of established and emerging antifungal agents against 5 Aspergillus species and therefore give certain evidence for antifungal therapy. METHODS Organisms A total of 107 isolates of Aspergillus spp. obtained from 8 hospitals in China during 2005–2008 were tested. The common species such as Aspergillus fumigatus, Aspergillus flavus and Aspergillus niger met at least one of the following criteria: (1) isolates were from aseptic locations or specimens such as bronchia alveolus fluid, a protected specimen brush, or a lung biopsy tissue; (2) isolates from specimens had more than one colony which was of the same species; (3) isolates were cultured more than once from reduplicate specimens. Only the first isolated strains were included in this study. The collection of isolates included 50 of A. fumigutus, 35 of A. flavus, 8 of A. niger, 7 of A. terrus and 7 of A. nidulans. The quality control strain was Candida parapsilosis ATCC 22019. Antifungal agents Etest stripes were from AB Biodisk (Solna, Sweden), including voriconazole, itraconazole, posaconazole, amphotericin B and caspofungin. Etest methods The agar-based Etest MIC method (AB Biodisk, Solna, Sweden) was employed to determine the MICs of the five antifungal agents. The strains were cultivated on PDA (OXOID, Hampshire, England) for 7 days at 35°C until
MICs (μg/ml) MIC50 0.19 1 0.094 1 0.094 0.25 0.75 0.094 >32 0.047 0.38 >32 0.38 0.125 0.125 0.38 0.094 0.064 >32 0.064 0.125 0.5 0.094 1 0.064
MIC90 0.25 1.5 0.19 2 0.094 0.38 1.5 0.25 >32 0.094 0.75 >32 1 >32 0.19 0.5 0.125 0.094 >32 0.19 0.125 0.75 0.125 2 0.094
Geom.Mean 0.178 1.031 0.097 0.724 0.068 0.25 0.582 0.1 25.23 0.042 0.379 38.055 0.348 0.307 0.082 0.343 0.087 0.065 >32 0.062 0.111 0.371 0.093 0.886 0.063
the spores were mature, followed by the collection of spores with sterile normal saline containing 0.5% Tween 20 (Sigma, Missouri, USA). The suspension was settled for about 15 minutes before transferring the supernatant into another tube to adjust the turbidity to 0.5 McF. RPMI 1640 agar (GIBCO, USA) containing MOPS (Xinjingke, Beijing, China) and 2% glucose (Guoyao, Beijing, China) was smeared with the supernatant in three directions with cotton swabs. Finally, the antifungal agent strips were put onto the plates after the surface of the plates was dry. Etest MIC was read after incubation for 48 hours at 35°C when the concentration at the border of the elliptical inhibition zone intercepted with the scale on the antifungal strip. Analysis of results The Whonet 5.4 software was employed to calculate parameters MIC50, MIC90, and MIC range. RESULTS The result for the quality control strain ATCC22019 was in the permissible range. The MIC of itraconazole was in the range of 0.064 μg/ml to 0.5 μg/ml while for amphoterincin B it was 0.25 μg/ml to 1 μg/ml. The Table summarizes the in vitro susceptibilities of 107 Aspergillus spp. to the 5 antifungal agents. The data demonstrates that the MIC90 of amphotericin B against the five Aspergillus spp. was the highest among the five antifungal drugs: 2 μg/ml against A. fumigatus and A. nidulans, >32 μg/ml against A. flavus, A. niger and A. terrus. For A. fumigatus, A. flavus, A. niger and A. nidulans, the MIC90 of caspofungin was the lowest: 0.094 μg/ml against A. fumigatus, A. flavus and A. nidulans,
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0.19 μg/ml against A. niger and A. terrus. For A. fumigatus and A. flavus, in order of increasing MIC90 were caspofungin, posaconazole, voriconazole, itraconazole and amphotericin B. For A. niger, the MIC90 of itraconazole corresponded to that of amphotericin B; however, the MIC50 of amphotericin B was remarkably lower than that of itraconazole. Noticeable, A. nidulans was sensitive to all the antifungal drugs tested with an MIC90 in the range of 0.047 μg/ml to 2 μg/ml. The MIC90 of posaconazole and voriconazole were at the same level of 0.125 μg/ml. A. terrus had a different antifungal susceptibility spectrum compared with the other four species. First, the MIC90 of posaconazole was the lowest for A. terrus among the drugs in contrast to the MIC90 of caspofungin which was the lowest for the other species. Second, the MIC of amphotericin B to A. terrus was higher than 32 μg/ml. DISCUSSION Recently, the research focus on the in vitro susceptibility to voriconazole, itraconazole, posaconazole, amphotericin B and caspofungin is rare in China and therefore, we gave a relatively comprehensive study to this field. In this study, we compared the activities of these five antifungal drugs against Aspergillus spp. using the Etest method. Research on the correlation between the broth microdilution method recommended by CLSI and Etest method has shown that they have good agreement and that Etest can be a substitute in antifungal susceptibility testing.4-6 The Etest antifungal susceptibility data showed that the MIC90 of caspofungin was the lowest against A. fumigatus, A. flavus, A. niger and A. nidulans, consistent with a report by Ingroff et al.7 Caspofungin, which is a kind of echinocandin, has been approved for the treatment of invasive aspergillosis, especially for patients intolerant or having a low response to other antifungal agents. Echinocandin can interfere with the synthesis of the cell wall of fungi by inhibiting sythesization of 1,3-β-D-glucan. It has few side effects because mammals have no cell walls. In general, we measured in vitro activities of azole antifungal agents were better than amphotericin B, consistent with other papers.4-6 Posaconazole had the most effective activity against A. fumigatus, A. flavus and A. terrus among the three azoles. Noticeable, it was more effective than caspofungin against A. terrus. Posaconazole and voriconazole had similar antifungal activity against A. nidulans whereas posaconazole was inferior to voriconazole against A. niger. It is reported abroad that posaconazole has an extended antifungal spectrum both in vitro and in vivo. Most importantly, it has good antifungal activity against Aspergillus and other opportunistic fungi resistant to amphotericin B and/or itraconazole.6 Therefore it is promising in the treatment of invasive fungal infections.
The antifungal activity of voriconazole was superior to that of itraconazole, in agreement with a report by Mallie et al.8 Voriconazole was more effective against A. fumigatus that was resistant to itraconazole. However, in our study the MIC of voriconazole was slightly higher than that of itraconazole against A. terrus (0.5 μg/ml and 0.125 μg/ml, respectively), which was different from Mallie’s results.8 Either by oral administration or parental administration, voricanazole has good biological availability in vivo.8 It can be an alternative to traditional liposome amphotericin B for empirical treatment. The present study demonstrated that amphotericin B was inferior to azoles, opposite from the results reported by Pfaller et al,9 which used the micro dilution method. A. terrus had natural drug resistance to amphotericin B with the MICs higher than 32 μg/ml. At present, Aspergillus strains resistant to itraconazole are emerging.5 Our study showed that for most strains of A. fumigatus, the MIC90 was in the range of 0.38 μg/ml–3 μg/ml with 4 percent of strains higher than 32 μg/ml. Whether these strains with high MIC resistant ones depends on the detection of resistant genes by molecular biology methods. Generally speaking, in vitro antifungal susceptibility in this study showed caspofungin had good antifungal activity against the five species of Aspergillus and azole agents had better activity than amphotericin B. Noticeable, this result is just from a small scale in vitro susceptibility study and we did not take other factors into consideration. Recently, there has been more research focused on in vivo antifungal susceptibility testing, which embodies in two aspects: one is the use of animal experiments and the other is patient-based clinical oberservations. In an early study of voriconazole versus itraconazole in endocarditis, voriconazole was superior to itraconazole in both the prevention and treatment of left-sided A. fumigatus endocarditis.10 Posaconazole (2.5 mg·kg-1·d-1 and 10 mg·kg-1·d-1 per oral (PO) was more effective than similar dosages of itraconazole and as effective as amphotericin B in the clearance of infected lung tissue in disseminated A. fumigatus aspergillosis in cyclophosphamide and corticosteroid immunosuppressed rabbits.11 Although caspofungin in our test showed good antifungal susceptibility with lower MICs, Arendrup et al12 reported that a clinical isolate of A. fumigatus obtained from a patient was resistant to caspofungin therapy, confirmed by Etest with an MIC>32 μg/ml. In neutropenic mice with cerebral A. fumigatus aspergillosis, caspofungin was inferior for prolonging survival and reducing brain infection when compared to posaconazole.13 In conclusion, in vitro antifungal activity may not be parallel with in vivo outcome. The outcome is relevant to immune status and basic diseases of the patient, the interactions of different drugs in vivo and the characteristics of the drugs. Investigations on a large sample of strains and pharmacodynamic and pharmacokinetic studies together with the participation of
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clinicians are needed to give further insight into the activity of these antifungal drugs.
8.
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(Received December 24, 2009) Edited by WANG De
