Activity of Posaconazole and Other Antifungal Agents against Mucorales Strains Identified by Sequencing of Internal Transcribed Spacers

The antifungal susceptibility profiles of 77 clinical strainsof Mucorales species, identified by internal transcribed spacersequencing, were analyzed. MICs obtained at 24 and 48 h werecompared. Amphotericin B was the most active agent against allisolates, except for Cunninghamella and Apophysomyces isolates.Posaconazole also showed good activity for all species but Cunninghamellabertholletiae. Voriconazole had no activity against any of thefungi tested. Terbinafine showed good activity, except for Rhizopusoryzae, Mucor circinelloides, and Rhizomucor variabilis isolates.

INTRODUCTION

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INTRODUCTION

Mucormycosis (zygomycosis) is an aggressive and usually fatalhuman infection. The most common etiologic agents are Rhizopusspp., although other species have also been associated (19).Most cases of disease occur among immunocompromised individuals(18). Recently, an increase in the number of cases of mucormycosishas been observed (10, 11, 26). This increase has been particularlyevident since the advent of voriconazole prophylaxis and treatmentof aspergillosis infection in immunocompromised patients (20,24, 27).

The gold standard therapy has not been well defined yet. Itusually requires a combination of antifungal treatment, surgicalintervention, and control of the underlying risk factors (19).The agent of choice for treating this infection is amphotericinB (5). However, mortality remains high, even with aggressivetherapies. Posaconazole has been successfully used in salvagetherapy for mucormycosis. In addition, other antifungals havepotential utility in mucormycosis treatment.

Species identification is epidemiologically and clinically important,because Mucorales species can exhibit differences in their antifungalin vitro susceptibilities (4, 5). Unfortunately, identificationby morphology examination requires a high level of expertise.

The in vitro activity of posaconazole and those of five otherantifungals against 77 clinical Mucorales isolates identifiedby sequencing the internal transcribed spacer (ITS) ribosomalDNA region are compared. For Mucorales species, CLSI and theEuropean Committee for Antimicrobial Susceptibility Testingof the European Society of Clinical Microbiology and InfectiousDiseases recommend obtaining endpoint data at 24 h. However,since growth rates across the Mucorales species are not uniform,MICs obtained at 24 and 48 h were compared in order to ascertainwhether there are differences in the results obtained at thosetwo different times.

Identification of the strains.

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Identification of the strains.

Seventy-seven clinical isolates of Mucorales species were obtainedbetween 1999 and 2008 in the Mycology Reference Laboratory ofthe National Centre for Microbiology of Spain. All isolateswere identified to the genus level by means of morphology determinationsby following the usual procedures (7). In addition, all strainswere identified to the species level by analyzing the ITS sequencesby parsimony analysis as described before (2). Rhizopus oryzaeand Mucor circinelloides were the species most frequently encountered.Smaller numbers of isolates of species of other genera suchas Rhizomucor, Cunninghamella, and Mycocladus were found. Twostrains of Actinomucor elegans and one of Apophysomyces eleganswere also found.

The identification of members of the Mucorales genus to thespecies level is a hard task, even for well-trained mycologists.Kontoyiannis et al. (11) have shown discordance of more than20% between ITS sequencing results and morphological identificationfor this group of fungi. Sequencing of appropriate targets shouldbe considered the gold standard for identification. In thisstudy, the analysis of the ITS sequences allowed us to identifyall species, indicating that the ITS region is an appropriatemolecular target for identification of these fungi. The onlyclade not supported was that formed by Rhizomucor variabilisand M. circinelloides. R. variabilis has previously been foundto be more closely phylogenetically related to Mucor species(28). Molecular data, together with susceptibility profiling,also support this finding.

Antifungal susceptibility testing.

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Antifungal susceptibility...

Microdilution testing was performed by following the guidelinesprovided by the European Society of Clinical Microbiology andInfectious Diseases in European Committee for AntimicrobialSusceptibility Testing definitive document E.DEF 9.1 (1, 17,22, 23). Aspergillus fumigatus ATCC 2004305 and Aspergillusflavus ATCC 2004304 were used as quality control strains (23).The antifungal agents used were amphotericin B (Sigma AldrichQuímica S.A.), itraconazole (Janssen Pharmaceutica, Madrid,Spain), voriconazole (Pfizer S.A., Madrid, Spain), ravuconazole(Bristol-Myers Squibb, Princeton, NJ), posaconazole (Schering-PloughResearch Institute, Kenilworth, NJ), and terbinafine (Novartis,Basel, Switzerland). Visual readings were performed with thehelp of a mirror at 24 and 48 h. The endpoint was the antifungalconcentration that produced a complete inhibition of visualgrowth (Table 1). As the growth rate of most Mucorales speciesis high, antifungal susceptibility reference methods (14, 23)recommend obtaining endpoint data at 24 h. For Aspergillus fumigatus,however, it has been proven that obtaining data at 48 h is mandatoryin order to detect resistant strains (21). In this work we havedetected several strains showing more-than-twofold differencesin dilutions between endpoint data obtained at 24 h and thoseobtained at 48 h (Table 2). Although the relevance of this changein MICs is not known, it must be taken into consideration thatall strains analyzed in this work were fully resistant to voriconazoleat 24 h, supporting an earlier reading at 24 h as appropriate.However, although we still recommend reporting endpoints forMucorales species at 24 h, further research performed with strainssusceptible at 24 h and resistant at 48 h is needed.

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TABLE 1. Geometric means and MIC ranges of the antifungal agents tested with the 77 Mucorales isolates

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TABLE 2. Isolates with >2-fold dilution differences in drug MICs between 24 and 48 h of incubation

Amphotericin B, which is the antifungal of choice for this mycosis,was the most active agent against all isolates, with the exceptionof those belonging to the genera Cunninghamella and Apophysomyces.High drug MICs for Cunninghamella species have been reportedbefore (8, 9, 15, 29, 30). Terbinafine was active against allspecies tested, except for R. oryzae, M. circinelloides, andR. variabilis. Azole drugs showed various levels of activity.Itraconazole showed activity against only R. pusillus and Mycocladuscorymbifer. Similar results have been found in other studies(5, 25). Itraconazole has also shown good activity in animalmodels of infection with M. corymbifer (6, 12). Therefore, itraconazolecould be useful for some cases of mucormycosis when susceptiblestrains are involved. Voriconazole has no in vitro activityagainst these fungi. In addition, it has been shown that patientswith leukemia and bone marrow transplant recipients on voriconazoleprophylaxis can develop breakthrough infections caused by Mucoralesspecies (11). Ravuconazole showed some activity against M. corymbifer,R. pusillus, R. oryzae, R. microsporus, and Actinomucor elegans,although isolates resistant to this drug were found in mostof the species. Posaconazole was the azole drug which showedthe best in vitro activity. The geometric mean of the MICs was $≤$ 2 mg/liter for all species but Cunninghamella bertholletiae.This is the first drug in the azole class to show a broad spectrumof activity against the Mucorales species and has proven tobe useful in combination with amphotericin B to treat rhinocerebralmucormycosis or patients for whom treatment with amphotericinB alone has failed (13, 16).

In summary, Mucorales species represent a heterogeneous groupof fungi with various levels of susceptibility to antifungals(3) that can be easily identified by sequencing the ITSs ofthe ribosomal DNA. These differences in susceptibility occuramong the same genera and species; therefore, a correct determinationof the susceptibility profile for an individual clinical isolateis essential for devising the best therapy for mucormycosis.

ACKNOWLEDGMENTS

We thank Grit Walther from the Fungal Biodiversity Centre, Centraalbureauvoor Schimmelcultures, for helping with the molecular identification.A.A.-I. and M.V.C. contributed equally to this work.

A.A.-I. has a predoctoral fellowship from the Fondo de InvestigacionesSanitarias (grant FI05/00856). M.V.C. has a research contractfrom the Agencia Española de Cooperación Internacional.I.C. has a contract from the Spanish Network for Research inInfectious Diseases (REIPI RD06/0008). This work was supportedin part by research projects PI05/32 from the Instituto de SaludCarlos III and by the Spanish Network for Research in InfectiousDiseases (REIPI RD06/0008).

In the past 5 years, J.L.R.-T. and M.C.-E. have received grantsupport from Astellas, Merck, Pfizer, Gilead, and Schering-Plough.They have been advisors or consultants to Astellas, Pfizer,Schering-Plough, Gilead, and Merck. They have been paid fortalks on behalf of Astellas, Pfizer, Schering-Plough, Gilead,and Merck. Other authors have no potential conflicts of interestto declare.

FOOTNOTES

* Corresponding author. Mailing address: Servicio de Micologia, Centro Nacional de Microbiologia, Instituto de Salud Carlos III, Ctra. Majadahonda Pozuelo km 2 28220, Majadahonda, Spain. Phone: 34918223919. Fax: 34915097966. E-mail: jlrtudela{at}isciii.es

Published ahead of print on 26 January 2009.

REFERENCES

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