ABSTRACT
Forty-four isolates belonging to human pathogenic species of Lichtheimia were tested against nine antifungal agents by using the EUCAST methodology. No remarkable differences were found between the clinical species, although L. ramosa showed slightly higher MICs for all drugs. Amphotericin B was the most active drug. Among azole drugs, posaconazole had the best activity in vitro and voriconazole was inactive. Echinocandins showed activity for some isolates, suggesting a potential role in combination therapy.
In recent years, the number of zygomycosis cases (mucormycosis) has grown, probably due to the increasing population at risk (14). The course of zygomycosis infection progresses rapidly and is potentially fatal, with mortality and morbidity rates remaining high. A gold-standard therapy has not yet been found, and amphotericin B remains the agent of choice (6). Treatment usually requires a combination of measures, including antifungal treatment, surgical intervention, and control of the underlying risk factors (13). Patient outcome has improved using posaconazole as salvage therapy (7, 18). Echinocandins have been used in combination therapies, highlighting the potential utility of other antifungals.
Lichtheimia is the third-most-frequent genus isolated in these infections and is responsible for approximately 5% of cases (4, 14). This genus has undergone several taxonomical changes in recent years. Thus, Lichtheimia species were originally classified in the genus Absidia. However, molecular phylogenetic analyses revealed that they belonged to a separate genus, named Mycocladus (8), that had to be renamed as Lichtheimia (9). Based on morphological, physiological, and molecular data, five species were proposed in this genus (3), L. corymbifera, L. ornata, L. ramosa, L. hyalospora, and L. sphaerocystis, of which only the first three are clinically relevant. In the last decades, L. corymbifera and L. ramosa have been treated as synonymous, and subsequently, previous publications on the in vitro susceptibility profile of “Absidia corymbifera” may refer either to L. corymbifera or to L. ramosa. In our setting, L. ramosa is more common than L. corymbifera. The aim of this study was to ascertain the antifungal susceptibility profile of clinical Lichtheimia species.
Strains.
Forty-three clinical isolates and one environmental isolate of Lichtheimia species were obtained between 1999 and 2009 in the Mycology Laboratory of the Spanish National Centre for Microbiology. Twenty-four strains were identified as L. ramosa, 19 strains as L. corymbifera, and one isolate as L. ornata by internal transcribed spacer (ITS) sequence comparison by Alastruey-Izquierdo et al. (3). Sixteen strains were isolated from respiratory sites, 15 from superficial sites, one from gastric juice, one from peritoneal drainage, and one from hospital air. The origin of 10 strains was unknown.
Antifungal susceptibility testing.
Microdilution testing was performed following the EUCAST standard methodology (16). Inoculum preparations were performed by means of counting spores in a hematocytometer (1, 12, 15). Aspergillus fumigatus ATCC 2004305 and A. flavus ATCC 2004304 were used as quality control strains (11).
The antifungal agents used in the study were amphotericin B (Sigma-Aldrich Quimica, Madrid, Spain), itraconazole (Janssen Pharmaceutica, Madrid), voriconazole (Pfizer S.A., Madrid), ravuconazole (Bristol-Myers Squibb, Princeton, NJ), posaconazole (Merck & Co., Inc., Rahway, NJ), terbinafine (Novartis, Basel, Switzerland), caspofungin (Merck & Co., Inc.), micafungin (Astellas Pharma, Inc., Tokyo, Japan), and anidulafungin (Pfizer S.A.). The final concentrations tested ranged from 16 to 0.03 mg/liter for amphotericin B, terbinafine, caspofungin, micafungin, and anidulafungin and from 8 to 0.015 mg/liter for itraconazole, voriconazole, ravuconazole, and posaconazole. The plates were incubated at 35°C for 48 h in a humid atmosphere. Visual readings were performed at 24 and 48 h with the help of a mirror. The endpoint for amphotericin B, itraconazole, voriconazole, ravuconazole, posaconazole, and terbinafine was the antifungal concentration that produced complete inhibition of visual growth at 24 and 48 h. For the echinocandins, the endpoint was the antifungal concentration that produced a visible change in the morphology of the hyphae compared with the morphology of the hyphae in the growth control well (minimum effective concentration [MEC]) (5, 10).
Table 1 shows the geometric mean (GM) and range of the MICs for the three Lichtheimia species. Since time plays a critical role in the management of these infections and most members of the Mucorales are fast-growing fungi, it has been recommended that MIC results be given for this fungal group at 24 h (2). However, we provide results at both 24 and 48 h because it is not yet clear whether all resistant strains can be detected after 24 h. Although no remarkable differences were found between L. corymbifera and L. ramosa, the latter species showed slightly higher MICs to most drugs. The greatest differences were found for itraconazole (the GM of the MIC of L. ramosa was 2.38 mg/liter and of L. corymbifera was 1.035 mg/liter at 48 h). The MICs of L. ornata were based on one isolate, and therefore, no conclusions could be obtained and more isolates are needed to evaluate the action of antifungals against this species. Amphotericin B was the most active drug, showing a GM of 0.07 mg/liter at 24 h. Among the azoles, voriconazole was inactive, whereas posaconazole had the highest activity (GM of MICs of 0.34 mg/liter at 24 h). Although itraconazole has shown poor activity against most zygomycetes (2), several isolates of Lichtheimia showed low MICs to this drug. These results concur with previous reports where L. corymbifera showed the lowest MICs to itraconazole among zygomycetes species (2, 6, 17). Terbinafine showed low MICs for most of the strains, although some isolates had MICs of >2 mg/liter. Regarding echinocandins, three out of 19 strains of L. corymbifera and three out of 24 of L. ramosa showed low MICs at 24 h. Anidulafungin was the echinocandin showing the best activity, especially against L. ramosa, where 10 out of 24 strains showed MICs of ≤2 mg/liter, pointing out the potential utility of this drug in combination therapies.
Antifungal susceptibility results for clinical isolates of Lichtheimia spp.
More data are needed in order to obtain a clear picture of the susceptibility profile and clinical importance of these species. Little is known about their prevalence, and there are no studies regarding epidemiology, pattern of disease, risk factors, etc. The treatment of systemic fungal infections has undergone changes in the last years, as several new antifungal agents are available. Because of the existence of these therapy alternatives, it is clear that not all fungal infections should be treated in the same manner. Consequently, the correct identification and susceptibility testing of fungal species are increasingly important. We strongly recommend sending all strains of Lichtheimia species involved in human infections to reference laboratories where those isolates can be properly identified to species level and where antifungal susceptibility testing can be performed. The importance of these species could thus be ascertained.
Nucleotide sequence accession numbers.
The GenBank nucleotide sequence accession numbers for ITS sequences from all the strains used in this work are as follows: CNM-CM4337:GQ342852, CNM-CM4427:GQ342853, CNM-CM4261:GQ342854, CNM-CM4849:GQ342855, CNM-CM4253:GQ342860, CNM-CM4228:GQ342861, CNM-CM4119:GQ342862, CNM-CM2166:GQ342863, CNM-CM5171:GQ342864, CNM-CM1638:GQ342866, CNM-CM4849:GQ342868, CNM-CM3590:GQ342869, CNM-CM5111:GQ342871, CNM-CM3148:GQ342872, CNM-CM4537:GQ342873, CNM-CM4978:GQ342892CNM-CM1503:HM104192, CNM-CM2071:HM104193, CNM-CM3013:HM104194, CNM-CM3346:HM104195, CNM-CM3415:HM104196, CNM-CM4374:HM104197, CNM-CM4671:HM104198, CNM-CM4738:HM104199, CNM-CM5039:HM104200, CNM-CM5166:HM104201, CNM-CM5175:HM104202, CNM-CM5222:HM104203, CNM-CM5223:HM104204, CNM-CM5224:HM104205, CNM-CM5256:HM104206, CNM-CM5358:HM104207, CNM-CM5396:HM104208, CNM-CM5397:HM104209, CNM-CM5398:HM104210, CNM-CM5399:HM104211, CNM-CM5400:HM104212, CNM-CM5538:HM104213, CNM-CM5565:HM104214, CNM-CM5581:HM104215, CNM-CM5637:HM104216, CNM-CM5677:HM104217, CNM-CM5738:HM104218, CNM-CM5848:HM104219, CNM-CM5861:HM104220, CNM-CM5984:HM104221.
ACKNOWLEDGMENTS
Ana Alastruey has a predoctoral fellowship from Fondo de Investigaciones Sanitarias (grant FI05/00856). Isabel Cuesta has a contract from the Spanish Network for Research into Infectious Diseases (REIPI RD06/0008). This work was supported in part by research projects PI05/32 from the Instituto de Salud Carlos III and by the Spanish Network for Research in Infectious Diseases (REIPI RD06/0008).
Over the past 5 years, M.C.-E. has received grant support from Astellas Pharma, bioMérieux, Gilead Sciences, Merck Sharp and Dohme, Pfizer, Schering Plough, Soria Melguizo S.A., the European Union, the ALBAN program, the Spanish Agency for International Cooperation, the Spanish Ministry of Culture and Education, the Spanish Health Research Fund, the Instituto de Salud Carlos III, the Ramon Areces Foundation, and the Mutua Madrileña Foundation. He has been an advisor/consultant to the Pan American Health Organization, Gilead Sciences, Merck Sharp and Dohme, Pfizer, and Schering Plough. He has been remunerated for talks on behalf of Gilead Sciences, Merck Sharp and Dohme, Pfizer, and Schering Plough.
Over the past 5 years, J.L.R.-T. has received grant support from Astellas Pharma, Gilead Sciences, Merck Sharp and Dohme, Pfizer, Schering Plough, Soria Melguizo S.A., the European Union, the Spanish Agency for International Cooperation, the Spanish Ministry of Culture and Education, the Spanish Health Research Fund, the Instituto de Salud Carlos III, the Ramon Areces Foundation, and the Mutua Madrileña Foundation. He has been an advisor/consultant to the Pan American Health Organization, Gilead Sciences, Merck Sharp and Dohme, Mycognostica, Pfizer, and Schering Plough. He has received payment for talks on behalf of Gilead Sciences, Merck Sharp and Dohme, Pfizer, and Schering Plough.
FOOTNOTES
- Received 7 September 2009.
- Returned for modification 19 September 2009.
- Accepted 18 April 2010.
- Copyright © 2010 American Society for Microbiology
