In Vitro Triple Combination of Antifungal Drugs against Clinical Scopulariopsis and Microascus Species

Abstract

Broth microdilution checkerboard techniques based on the methodology of the Clinical and Laboratory Standards Institute (CLSI) were employed to study the triple antifungal combination of caspofungin, posaconazole, and terbinafine against 27 clinical isolates of Scopulariopsis and Microascus species. Synergy was observed for 26 isolates, whereas antagonism was observed for Scopulariopsis candida in this study.

TEXT

Scopulariopsis species and their teleomorphs, Microascus species, include both hyaline and dematiaceous mold forms. These species are usually saprophytic and are commonly isolated from the soil, air, plant debris, and moist indoor environments (1, 2). The most frequently occurring species in clinical cases is Scopulariopsis brevicaulis (3–7).

These filamentous fungi commonly act as opportunistic pathogens in humans and are mainly associated with superficial tissue infections, such as onychomycosis (3, 8) and corneal ulcers (4). However, during the last 2 decades, Scopulariopsis species have been found as pathogens involved in deep mycoses in immunocompromised and occasionally in immunocompetent hosts, causing pneumonia (9), sinusitis (5), prosthetic valve endocarditis (6), pulmonary or brain abscesses (7, 10), and other fatal infections.

The appropriate therapy for Scopulariopsis species infections has yet to be defined. S. brevicaulis has been reported to be resistant in vitro to almost all the currently used drugs, including amphotericin B (AMB), flucytosine, terbinafine (TRB), caspofungin (CAS), and azole compounds (11–15). In double-drug combinations, synergy was observed in only a few strains of S. brevicaulis with posaconazole (POS) plus TRB and AMB plus CAS (16). Regarding in vivo treatments, Yang et al. (17) published a report on an acute monocytic leukemia patient with an infection caused by S. brevicaulis for whom voriconazole-and-CAS combination treatment failed. To our knowledge, a triple combination of antifungal drugs has not yet been assessed with these fungi. The aim of our study was to identify a practical triple combination therapy against clinical isolates.

The drugs tested included TRB (Novartis, Basel, Switzerland), POS (Merck, Rahway, NJ, USA), and CAS (Merck), which show synergy in a double combination against some strains of S. brevicaulis (16). POS and TRB target lanosterol 14-α-demethylase or squalene epoxidase in the membrane to inhibit the synthesis of ergosterol in different stages, while CAS targets the cell wall to noncompetitively inhibit β-(1,3)-d-glucan synthase. The combination of these drugs may be able to disrupt the integrity of the fungi cell wall and membrane. AMB binds with the ergosterol in cell membranes to further disturb ion permeability. The decreased ergosterol in the cell membrane caused by posaconazole and terbinafine might negatively affect the antifungal ability of AMB. Therefore, we did not choose amphotericin as a candidate, although it has some synergy with CAS.

Twenty-seven clinical isolates of Scopulariopsis and Microascus spp. preserved by the Research Center for Medical Mycology at Peking University were used in this study. The strains were isolated from nails (25 strains), pulmonary tissue (1 strain), and bronchoalveolar lavage fluid (1 strain) (Table 1). All the strains were identified by morphological methods and sequences of the β-tubulin (TUB) and elongation factor 1-α (EF1-α) genes (18, 19). Candida parapsilosis strain ATCC 22019, Candida krusei strain ATCC 6258, Aspergillus flavus strain ATCC 204304, and Trichophyton mentagrophytes strain ATCC-MYA 4439 were used for quality control purposes.

TABLE 1.

Origin of isolates of Scopulariopsis and Microascus spp. from clinical samples

Strain name	Species	Source	City of isolation	Date isolated (yr.mo)
BMU 00594	Scopulariopsis brevicaulis	Nail	Daqing	2003.09
BMU 01837	Microascus sp. nov. I	Nail	Beijing	2000.11
BMU 01895	Microascus sp. nov. I	Nail	Dalian	2000.12
BMU 02276	S. brevicaulis	Nail	Beijing	2001.09
BMU 02787	Microascus gracilis	Nail	Beijing	2003.05
BMU 03030	S. brevicaulis	Nail	Beijing	2004.06
BMU 03031	S. brevicaulis	Nail	Beijing	2004.06
BMU 03130	S. brevicaulis	Nail	Beijing	2004.07
BMU 03909	Microascus sp. nov. II	Nail	Beijing	2006.02
BMU 03910	Microascus sp. nov. II	Nail	Beijing	2006.02
BMU 03911	Microascus sp. nov. II	Nail	Beijing	2006.02
BMU 03912	Microascus croci	Nail	Beijing	2006.02
BMU 03913	S. brevicaulis	Nail	Beijing	2006.02
BMU 03915	S. brevicaulis	Nail	Beijing	2006.02
BMU 03916	S. brevicaulis	Nail	Beijing	2006.02
BMU 03917	S. brevicaulis	Nail	Beijing	2006.02
BMU 03918	S. brevicaulis	Nail	Beijing	2006.02
BMU 03919	Microascus brunneosporus	Nail	Beijing	2006.02
BMU 03920	Scopulariopsis candida	Nail	Beijing	2006.02
BMU 03977	S. brevicaulis	Nail	Beijing	2006.07
BMU 04091	S. brevicaulis	Nail	Shijiazhuang	2007.01
BMU 04786	M. gracilis	BALFa	Beijing	2009.04
BMU 04809	Microascus cirrosus	Pulmonary tissue	Beijing	2009.08
BMU 04915	Microascus intricatus	Nail	Beijing	2009.12
BMU 06573	M. brunneosporus	Nail	Shanghai	2011.11
BMU 06938	S. brevicaulis	Nail	Nanjing	2012.01
BMU 07493	Microascus verrucosus	Nail	Beijing	2013.04

^aBALF, bronchoalveolar lavage fluid.

The drug combinations were tested according to the guidelines presented in document M38-A2 (20) of the Clinical and Laboratory Standards Institute (CLSI).

As previously published, we utilized various double combinations of these three drugs and obtained unsatisfactory results, with high fractional inhibitory concentration indices (FICIs) in most strains. The triple drug combination was conducted with a three-dimensional checkerboard technique (21). POS and TRB were set as the double combination, and then CAS was added at a single concentration in each plate, from which we were able to determine the MIC of each agent alone and the combined effects on the same plate. Different concentrations of CAS (five dilutions were tested, at 1, 2, 4, 8, and 16 μg/ml) were initially used to obtain the following final definitive concentrations of the antifungal agents: 2 μg/ml for CAS, 0.125 to 8 μg/ml for POS (for individual therapy, we used 0.25 to 16 μg/ml), and 0.0313 to 16 μg/ml for TRB. Quality controls were included in each set of experiments to ensure that the potency of the drugs was maintained during storage.

We used the MICs to evaluate the combined effects of the drugs (21). The plates were incubated at 35°C, and the MIC was defined as the lowest concentration exhibiting total visual inhibition of growth with a concave mirror after 48 h. The incubation period was prolonged when no growth was observed in the control wells after the incubation time. The combined effects were analyzed by the summation of the FICIs. The FICI was calculated as (MIC_POS in combination/MIC_POS alone) + (MIC_CAS in combination/MIC_CAS alone) + (MIC_TRB in combination/MIC_TRB alone). When the MIC exceeded the highest concentration in the plate, we used double the highest value for the calculation, as was previously done (i.e., when MIC was >16, we used 32 to calculate the FICI). The interactions were defined as synergistic when the FICI was ≤0.5, antagonistic when the FICI was >4, and indifferent (or no interaction) when the FICI was >0.5 but ≤4. Each assay was performed twice for every isolate.

Table 2 summarizes the MIC and FICI results for the individual antifungal agents and the three-dimensional checkerboard study of the triple combination. All fungi produced detectable growth in vitro and were highly resistant to TRB (2 to >16 μg/ml), CAS (8 to 16 μg/ml), and POS (8 to >16 μg/ml) alone.

TABLE 2.

Interactions of triple combination of caspofungin, posaconazole, and terbinafine

Isolate (n)	MIC (μg/ml) range of drug alone fora:			MIC (μg/ml) ranges of drugs in POS-TRB-CAS combination	FICI range for POS-TRB-CAS combinationb
	POS	TRB	CAS
S. brevicaulis (13)	>16	4 to >16	≥16	0.125 to 1/0.125 to 1/2	0.156 to 0.390
Microascus sp. nov. II (3)	>16	4	≥16	0.125 to 0.5/0.0313 to 1/2	0.148 to 0.316
M. brunneosporus (2)	>16	2	≥16	0.5 to 1/0.5/2	0.344 to 0.391
M. gracilis (2)	>16	≥16	≥16	0.25 to 1/4/2	0.321 to 0.320
Microascus sp. nov. I (2)	8 to 16	2	16	0.125 to 0.25/0.25/2	0.258 to 0.281
M. intricatus	>16	8	16	0.25/1/2	0.258
M. verrucosus	16	4	8	0.25/0.5/2	0.391
M. croci	>16	>16	16	0.125/4/2	0.254
M. cirrosus	>16	>16	>16	1/4/2	0.219
S. candida	>16	8	8	>16/>32/2	9.250

^aMIC was determined visually as the concentration that resulted in 100% inhibition. POS, posaconazole; TRB, terbinafine; CAS, caspofungin.

^bFICI < 0.5, synergy; FICI of 0.5 to 4, no interaction; FICI > 4, antagonism.

Notably, with the triple combination in vitro, synergistic interactions were observed for 26 of the isolates tested. In all of the combinations showing synergism, distinctly lower MICs were achieved with TRB (0.0313 to 4 μg/ml) and POS (0.125 to 4 μg/ml), and the FICI ranged from 0.102 to 0.391. Antagonism was observed for S. candida, with the MIC for TRB increasing from 8 μg/ml to >16 μg/ml (FICI, 4.750). However, there is only one doubling dilution for the combination, which is within the allowable interassay variation for broth microdilution. Consequently, we raised the concentration of POS to 16 μg/ml and TRB to 32 μg/ml in the combination; antagonism was also observed (POS, >16 μg/ml; TRB, >32 μg/ml), as the FICI increased to 9.250.

This study examined 27 clinical isolates, most of which were S. brevicaulis. The antifungal susceptibilities of Scopulariopsis and Microascus species had been primarily tested in S. brevicaulis (11, 13, 14, 22). Carrillo-Muñoz et al. (7, 22–24) reported that S. brevicaulis is susceptible or intermediately susceptible to voriconazole, sertaconazole, AMB, and TRB. In contrast, the reports of Steinbach et al. (25, 26) demonstrated that these species were resistant to TRB, AMB, and azole compounds. These contradictory data might be due to the limited number of strains or to additional therapy, especially in the clinical cases. Sandoval-Denis et al. (19) used almost all the available antifungal drugs, including the newer drugs, against 99 strains of Scopulariopsis species in vitro; however, the results indicated high resistance, as in the previous data. It can be concluded that these filamentous fungi are multiresistant to the broad-spectrum antifungal agents available today. A study by Cuenca-Estrella et al. (16) was performed on S. brevicaulis with double combinations of antifungal agents in vitro. However, an average indifferent effect was detected for all the combinations.

Triple antifungal therapy may be a feasible choice to treat Scopulariopsis infections. Triple antifungal therapy has been used to treat invasive aspergillosis with triazoles, echinocandins, and AMB (27, 28) and to treat cryptococcal meningitis with AMB, flucytosine, and azoles (29–31). In theory, the synergism among POS, TRB, and CAS may be because these agents disrupt the fungal membrane or wall integrity through different pathways or different enzymes. In our study, the three-agent combination appears to be promising, with a synergistic effect on Scopulariopsis and Microascus species. Synergistic interactions were detected for a high proportion (26/27) of the clinical strains with this combination of drugs. With a concentration of 2 μg/ml for CAS, we obtained satisfactorily lower MICs for POS and TRB. As the most frequently pathogenic species, all the S. brevicaulis isolates were observed to respond in vitro in a synergistic manner to POS at 0.125 to 1 μg/ml and TRB at 0.125 to 1 μg/ml, whereas S. candida exhibited an antagonistic interaction in which the concentration of TRB increased from 8 μg/ml to >16 μg/ml; in this case, TRB had an FICI of 4.250. Only a small subset of strains showed synergy when the CAS concentration was 1 μg/ml, and when CAS was ≥2 μg/ml, synergy was observed in all assays.

However, the antagonism result for S. candida has limitations. We raised the concentrations of POS and TRB in the plate, and the result still showed antagonism (POS, >16 μg/ml, and TRB, >32 μg/ml in combination). We concluded that the antagonism existed in this one S. candida strain only. More strains should be tested to verify the antagonism in S. candida.

These three drugs have been shown to be well tolerated and produce mild side effects, such as nausea, fever, and chills. However, they may also induce liver injury. TRB may cause hepatotoxicity, with an incidence of 0.5 to 3/100,000 exposed, while CAS may cause an increase in alanine aminotransferase (ALT), aspartate transaminase (AST), and alkaline phosphatase (AKP) levels (32–34). Patient liver function should be regularly assessed during treatment. However, the expensive cost of POS and CAS make them unsuitable for use with superficial tissue infections. The triple combination in invasive infections may become a salvage therapy in clinical practice.

In conclusion, the triple combination of CAS, POS, and TRB may be an alternative for treating deep tissue infections due to Scopulariopsis and Microascus species but not S. candida. More strains of S. candida are necessary to verify the antagonism with the triple combination. The in vitro results presented in this study must be confirmed in animal models and subjected to clinical validation in vivo before therapeutic recommendations can be made.