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

Abstract

The antifungal susceptibility profiles of 77 clinical strains of Mucorales species, identified by internal transcribed spacer sequencing, were analyzed. MICs obtained at 24 and 48 h were compared. Amphotericin B was the most active agent against all isolates, except for Cunninghamella and Apophysomyces isolates. Posaconazole also showed good activity for all species but Cunninghamella bertholletiae. Voriconazole had no activity against any of the fungi tested. Terbinafine showed good activity, except for Rhizopus oryzae, Mucor circinelloides, and Rhizomucor variabilis isolates.

Mucormycosis (zygomycosis) is an aggressive and usually fatal human infection. The most common etiologic agents are Rhizopus spp., 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 mucormycosis has been observed (10, 11, 26). This increase has been particularly evident since the advent of voriconazole prophylaxis and treatment of aspergillosis infection in immunocompromised patients (20, 24, 27).

The gold standard therapy has not been well defined yet. It usually requires a combination of antifungal treatment, surgical intervention, and control of the underlying risk factors (19). The agent of choice for treating this infection is amphotericin B (5). However, mortality remains high, even with aggressive therapies. Posaconazole has been successfully used in salvage therapy for mucormycosis. In addition, other antifungals have potential utility in mucormycosis treatment.

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

The in vitro activity of posaconazole and those of five other antifungals against 77 clinical Mucorales isolates identified by sequencing the internal transcribed spacer (ITS) ribosomal DNA region are compared. For Mucorales species, CLSI and the European Committee for Antimicrobial Susceptibility Testing of the European Society of Clinical Microbiology and Infectious Diseases 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 ascertain whether there are differences in the results obtained at those two different times.

Identification of the strains.

Seventy-seven clinical isolates of Mucorales species were obtained between 1999 and 2008 in the Mycology Reference Laboratory of the National Centre for Microbiology of Spain. All isolates were identified to the genus level by means of morphology determinations by following the usual procedures (7). In addition, all strains were identified to the species level by analyzing the ITS sequences by parsimony analysis as described before (2). Rhizopus oryzae and Mucor circinelloides were the species most frequently encountered. Smaller numbers of isolates of species of other genera such as Rhizomucor, Cunninghamella, and Mycocladus were found. Two strains of Actinomucor elegans and one of Apophysomyces elegans were also found.

The identification of members of the Mucorales genus to the species level is a hard task, even for well-trained mycologists. Kontoyiannis et al. (11) have shown discordance of more than 20% between ITS sequencing results and morphological identification for this group of fungi. Sequencing of appropriate targets should be considered the gold standard for identification. In this study, the analysis of the ITS sequences allowed us to identify all species, indicating that the ITS region is an appropriate molecular target for identification of these fungi. The only clade not supported was that formed by Rhizomucor variabilis and M. circinelloides. R. variabilis has previously been found to be more closely phylogenetically related to Mucor species (28). Molecular data, together with susceptibility profiling, also support this finding.

Antifungal susceptibility testing.

Microdilution testing was performed by following the guidelines provided by the European Society of Clinical Microbiology and Infectious Diseases in European Committee for Antimicrobial Susceptibility Testing definitive document E.DEF 9.1 (1, 17, 22, 23). Aspergillus fumigatus ATCC 2004305 and Aspergillus flavus ATCC 2004304 were used as quality control strains (23). The antifungal agents used were amphotericin B (Sigma Aldrich Química S.A.), itraconazole (Janssen Pharmaceutica, Madrid, Spain), voriconazole (Pfizer S.A., Madrid, Spain), ravuconazole (Bristol-Myers Squibb, Princeton, NJ), posaconazole (Schering-Plough Research Institute, Kenilworth, NJ), and terbinafine (Novartis, Basel, Switzerland). Visual readings were performed with the help of a mirror at 24 and 48 h. The endpoint was the antifungal concentration that produced a complete inhibition of visual growth (Table 1). As the growth rate of most Mucorales species is 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 mandatory in order to detect resistant strains (21). In this work we have detected several strains showing more-than-twofold differences in dilutions between endpoint data obtained at 24 h and those obtained at 48 h (Table 2). Although the relevance of this change in MICs is not known, it must be taken into consideration that all strains analyzed in this work were fully resistant to voriconazole at 24 h, supporting an earlier reading at 24 h as appropriate. However, although we still recommend reporting endpoints for Mucorales species at 24 h, further research performed with strains susceptible at 24 h and resistant at 48 h is needed.

TABLE 1.

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

Organism (no. of isolates)	Antifungal agent	MIC (mg/liter) at 24 h		MIC (mg/liter) at 48 h
		GMa	Range	GM	Range
Rhizopus oryzae (26)	Amphotericin B	0.29	0.03-32	1	0.125-32
	Itraconazole	4	0.125-16	6.96	0.25-16
	Voriconazole	12.13	4-64	16	8-64
	Ravuconazole	1	0.25-16	1.32	0.25-16
	Posaconazole	1.15	0.06-16	2	0.06-16
	Terbinafine	24.25	16-32	32	16-32
Mucor circinelloides (20)	Amphotericin B	0.05	0.015-0.125	0.15	0.06-1
	Itraconazole	11.89	1-16	16	1-16
	Voriconazole	16	8-64	16	1-64
	Ravuconazole	11.89	1-16	13.13	2-32
	Posaconazole	1.49	0.25-16	2.69	0.25-16
	Terbinafine	21.53	2-32	32	1-32
Mycocladus corymbifer (7)	Amphotericin B	0.08	0.03-0.25	0.14	0.06-0.25
	Itraconazole	0.68	0.125-16	1.35	0.25-16
	Voriconazole	14.49	8-16	16	16
	Ravuconazole	1.22	0.5-4	2	1-8
	Posaconazole	0.41	0.125-16	0.67	0.25-16
	Terbinafine	0.5	0.25-16	1	0.5-32
Cunninghamella bertholletiae (6)	Amphotericin B	7.13	2-32	7.13	2-32
	Itraconazole	3.17	0.5-8	10.08	4-16
	Voriconazole	16	8-32	16	8-32
	Ravuconazole	8	4-16	12.13	8-16
	Posaconazole	2.30	0.5-8	4.59	1-16
	Terbinafine	0.40	0.03-16	0.49	0.03-32
Rhizopus microsporus (6)	Amphotericin B	0.45	0.25-1	0.89	0.5-1
	Itraconazole	1.59	0.5-16	3.56	0.5-16
	Voriconazole	8	4-16	8.98	4-16
	Ravuconazole	0.89	0.25-16	1.78	0.25-8
	Posaconazole	0.79	0.25-8	3.17	0.5-16
	Terbinafine	0.71	0.125-32	1.41	0.25-32
Rhizomucor pusillus (5)	Amphotericin B	0.05	0.02-0.125	0.10	0.06-0.125
	Itraconazole	0.29	0.125-0.5	0.44	0.125-1
	Voriconazole	5.28	0.5-16	6.06	0.5-16
	Ravuconazole	0.66	0.25-2	0.87	0.25-2
	Posaconazole	0.16	0.06-0.25	0.28	0.06-0.5
	Terbinafine	0.22	0.125-0.5	0.33	0.25-0.5
Rhizomucor variabilis (2)	Amphotericin B	0.03	0.02-0.06	0.09	0.06-0.125
	Itraconazole	16	16	16	16
	Voriconazole	16	16	16	16
	Ravuconazole	8	4-16	16	16
	Posaconazole	1.41	1-2	16	16
	Terbinafine	32	32	32	32
Cunninghamella spp. (2)	Amphotericin B	2.83	2-4	4	4
	Itraconazole	0.5	NAb	4	1-16
	Voriconazole	16	16	16	16-16
	Ravuconazole	1	NA	8	4-16
	Posaconazole	0.25	NA	4	1-16
	Terbinafine	0.13	NA	0.18	0.125-0.25
Actinomucor spp. (2)	Amphotericin B	1	1	1.41	1-2
	Itraconazole	2	0.5-8	2	0.5-8
	Voriconazole	16	8-32	22.63	16-32
	Ravuconazole	0.25	0.25	0.5	0.5
	Posaconazole	0.06	0.06	0.125	0.125
	Terbinafine	0.125	0.06-0.25	0.36	0.125-1
Apophysomyces spp. (1)	Amphotericin B	2	NA	8	NA
	Itraconazole	16	NA	16	NA
	Voriconazole	16	NA	16	NA
	Ravuconazole	2	NA	4	NA
	Posaconazole	0.5	NA	1	NA
	Terbinafine	2	NA	2	NA

^aGM, geometric mean.

^bNA, not applicable.

TABLE 2.

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

Drug and species	No. of isolates	MIC (mg/liter) at 24 h	MIC (mg/liter) at 48 h
Amphotericin B
Mucor circinelloides	2	0.015	0.125
	1	0.03	0.5
	1	0.03	0.25
Rhizopus oryzae	1	0.06	1
	1	0.06	0.5
	1	0.25	2
Itraconazole
Cunninghamella spp.	2	0.5	16
Mucor circinelloides	1	2	16
Rhizopus oryzae	2	1	8
	1	1	16
	1	0.25	16
Rhizopus microsporus	1	1	8
Ravuconazole
Cunninghamella spp.	1	1	16
Rhizopus oryzae	1	0.25	4
	1	0.5	4
Rhizopus microsporus	1	0.5	8
	1	1	8
Posaconazole
Cunninghamella spp.	1	0.25	16
Mucor circinelloides	1	2	16
Rhizomucor variabilis	2	2	16
	1	1	16
Rhizopus microsporus	1	1	8
	1	0.5	16
Terbinafine
Mucor circinelloides	2	2	32
	1	2	16

Amphotericin B, which is the antifungal of choice for this mycosis, was the most active agent against all isolates, with the exception of those belonging to the genera Cunninghamella and Apophysomyces. High drug MICs for Cunninghamella species have been reported before (8, 9, 15, 29, 30). Terbinafine was active against all species tested, except for R. oryzae, M. circinelloides, and R. variabilis. Azole drugs showed various levels of activity. Itraconazole showed activity against only R. pusillus and Mycocladus corymbifer. Similar results have been found in other studies (5, 25). Itraconazole has also shown good activity in animal models of infection with M. corymbifer (6, 12). Therefore, itraconazole could be useful for some cases of mucormycosis when susceptible strains are involved. Voriconazole has no in vitro activity against these fungi. In addition, it has been shown that patients with leukemia and bone marrow transplant recipients on voriconazole prophylaxis can develop breakthrough infections caused by Mucorales species (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 most of the species. Posaconazole was the azole drug which showed the 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 spectrum of activity against the Mucorales species and has proven to be useful in combination with amphotericin B to treat rhinocerebral mucormycosis or patients for whom treatment with amphotericin B alone has failed (13, 16).

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