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Synergistic effects of terbinafine and itraconazole on clinical isolates of Fonsecaea monophora

European Journal of Dermatology. Volume 19, Numéro 5, 451-5, September-October 2009, Investigative report

DOI : 10.1684/ejd.2009.0728

Summary

Auteur(s) : Jun-min Zhang, Li-yan Xi, Hui Zhang, Zhi Xie, Jiu-feng Sun, Xi-qing Li, Sha Lu , Department of Dermatology, the Second Affiliated Hospital, Sun Yat-sen University, 107 West Yanjiang Road, Guangzhou 510120, China.

ARTICLE

Auteur(s) : Jun-min Zhang, Li-yan Xi, Hui Zhang, Zhi Xie, Jiu-feng Sun, Xi-qing Li, Sha Lu

Department of Dermatology, the Second Affiliated Hospital, Sun Yat-sen University, 107 West Yanjiang Road, Guangzhou 510120, China

accepté le 15 Avril 2009

Chromoblastomycosis is a cutaneous and subcutaneous fungal infection caused by dimorphic, filamentous fungi of the Dematiaceae family [1]. The disease, characterized by verrucose lesions and the appearance of sclerotic cells in pathological specimens, is found worldwide, with most reports coming from tropical and subtropical areas [2]. Chronic chromomycosis has a potential association with epidermoid carcinoma. Central nervous system invasion is possible and may be fatal [2, 3].

The most common chromoblastomycosis-causing filamentous fungus is Fonsecaea pedrosoi; others are Phialophora verrucosa, Cladophialophora carrionii, and Rhinocladiella cerophilum [4-6]. In 2004, De Hoog et al. isolated Fonsecaea monophora from F. pedrosoi and identified it as a new species by phylogenetic analysis based on confidently aligned the ITS rDNA sequence [2]. F. monophora has a more variable clinical spectrum than F. pedrosoi [7].

Testing the susceptibility of F. monophora to antifungal agents could become an important tool in selecting and monitoring appropriate antifungal drugs for the treatment and prophylaxis of chromoblastomycosis. There is no existing assay available for the in vitro susceptibility of F. monophora to antifungal agents. Since itraconazole and terbinafine are effective agents in the treatment of chromoblastomycosis [8, 9], we hypothesized that they can be used in combination to treat invasive F. monophora infection. To test the hypothesis, in the present study, we investigated the in vitro activity of itraconazole and terbinafine, used alone or in combination, against clinically relevant F. monophora isolates. Our results indicate that the two agents can be best used in combination to treat F. monophora infection, since a synergistic effect between the two agents was shown in the majority of these clinical isolates.

Materials and methods

Identification of fungal strains of the clinical isolates

Fifteen clinical isolates of F. monophora were provided by the Center of Fungal Research, the Second Affiliated Hospital, Sun Yat-sen University, Guangzhou, China. The patient information is listed in table 1. The study protocol was approved by the local ethics committee, and all patients gave written informed consent to their participation in the study. Skin scrapings were observed from each of the 15 patients and collected in 10% potassium hydroxide. The samples were cultured with Sabouraud’s Glucose Agar (SGA) at 26 °C. All the fungal strains grown had the typical colony and microscopic appearance of the genus Fonsecaea. DNA was extracted using 6% InStaGene^TMMatrix (BioRad, USA). Ribosomal DNA ITS domains were amplified in a Biometra T-Gradient Thermoblock (Germany) using primers ITS-4 (5′-TCCTCCGCTTATTGATATGC-3′) and ITS-5 (5′-GGAAGTAAAAGTCGTAACAAGG-3′). PCR conditions were 95 °C for 4 min, followed by 30 cycles of 94 °C for 60 s, 55 °C for 90 s, and 72 °C for 90 s. The DNA fragments were sequenced with an ABI PRISM 3100 sequencer after labeling with BigDye Terminator Cycle Sequencing Ready Reaction (Applied Biosystems, Foster City, Calif.); the sequences had 100% homology with type strains CBS 269.37 in GenBank and were confirmed to be F. monophora.

The isolates were subcultured on potato dextrose agar (PDA) for 7 to 10 days at 35 °C. Three strain types (CBS269.37, CBS102225 and CBS10222) were provided by Centraalbureau voor Schimmelcultures (CBS, the Netherlands). Candida parapsilosis ATCC22019, obtained from the American Type Culture Collection (Rockville, MD, USA), was used as a quality control. All isolates were tested in duplicate on two different days.
Table 1 Patient information of the clinical isolates of Fonsecaea monophora

Number of strains (SUMS)	Gender	Age (year)	Specimen site	Date (month/day/year)
0012	Male	45	Right buttock	11/07/1995
0013	Male	60	Right leg	01/18/1999
0014	Female	82	Left leg	08/17/1998
0034	Male	67	Right ankle	11/25/1998
0147	Male	54	Right dorsum of foot	02/20/2001
0158	Male	60	Left lower extremity	05/10/2001
0190	Male	72	Right leg	05/28/2002
0192	Male	76	Right ankle	11/29/2002
0200	Male	40	Right leg	05/07/2003
0228	Male	53	Face	02/23/2005
0246	Male	62	Left heel	03/06/2006
0247	Male	39	Left dorsum of foot	04/11/2006
0250	Male	55	Right leg	08/24/2006
0254	Male	82	Right back of hand	06/05/2006
0295	Male	55	Both legs	03/20/2007

Test drugs and reagents

Itraconazole was provided by Xian-Janssen Pharmaceutical Ltd. (Xi’an, China). Terbinafine was purchased from Beijing Novartis Pharmaceutical Ltd. (Beijing, China). Dimethyl sulfoxide (DMSO) was provided by Tianjin benchmark chemical reagent company Ltd. (Tianjin, China). RPMI 1640 medium (with L-glutamine and without bicarbonate) and morpholinepropanesulfonic acid (MOPS) were purchased from Sigma (Sigma Chemical Co., MO). Itraconazole and terbinafine were dissolved in 100% DMSO as stock solution (3200 μg/mL) and then diluted with culture medium to obtain the final concentrations from 0.002 to 16 μg/mL. The culture medium was RPMI 1640 with 0.165 M MOPS, pH7.0 at 25 °C.

Inoculum preparation

The isolates were subcultured twice on PDA slants at 35 °C for 7-10 days. Mature colonies were covered with approximately 1 mL of sterile saline (0.85% NaCl) and collected by scraping the surface with the tip of a Pasteur pipette. The resultant mixture of the conidia and hyphal fragments was withdrawn and transferred to a new sterile tube. The large particles in the mixture were allowed to settle for 3 to 5 min at room temperature; the supernatant was then transferred and vortexed for 15 s. The turbidity of the supernatant was counted by using a hemocytometer and the inoculum was adjusted with saline to achieve an inoculum concentration of 10⁶ conidia/mL. Each suspension was diluted 1:50-100 with RPMI 1640 to obtain the final test inoculum (0.5-5 × 10⁴ conidia/mL). Stock inoculum suspensions of the quality control fungi were prepared from 1- to 2-day culture; final test inoculum concentration was 0.5-2.5 × 10³ conidia/mL.

Susceptibility testing

The susceptibility of 15 clinical isolates of F. monophora was performed using the National Committee for Clinical Laboratory Standards (NCCLS) approved standard broth microdilution method [10]. Susceptibility testing was performed in 96-well flat-bottom microtitration plates. The minimal inhibitory concentration (MICs) of the drugs against all isolates were determined in duplicate and calculated as previously reported [11]. The final concentration of itraconazole or terbinafine was 16-0.01565 μg/mL. The concentration of combined use of itraconazole or terbinafine was 0.002-1 μg/mL, respectively.

A two-dimensional, two-agent broth microdilution checkerboard technique was used to study the interaction between the two drugs. Drug interaction was analyzed by the fractional inhibitory concentration index (FICI) [12]. The FICI values were calculated as follows: MIC of terbinafine - itraconazole/MIC of terbinafine+MIC of terbinafine - itraconazole/MIC of itraconazole. The interpretation of the FICI values in relation to the mode of drug interaction was made based on the following: ≤ 0.5, synergistic effect; > 0.5 but ≤ 1, additive effect; > 1 but ≤ 4, indifferent effect; and > 4, antagonistic effect [12, 13].

Data analysis

The mean MICs ± standard error of the mean (SEM) were calculated for each species, inoculum concentration, and time point from two separate experiments. The significance of differences between mean values for inocula was determined by using the Wilcoxon matched pairs test. P-values of equal or less than 0.05 were considered statistically significant.

Results

Inoculum quantification and growth

Candida parapsilosis (ATCC22019) grew well after 48 h of incubation at 35 °C and its MICs were within the reference range for itraconazole [14]. The MICs of itraconazole and terbinafine were 0.125-0.25 μg/mL and > 8 μg/mL, respectively. Synergism was found for itraconazole and terbinafine, according to the FICI (0.31-0.38).

MIC Values for clinical isolates

All the 18 clinical isolates (15 from our laboratory and 3 from others) were successfully cultured with satisfactory growth curves and morphology. The isolates began to grow in 72 h and showed significant growth in 5-7 days. Table 2 shows the results of the susceptibility testing of itraconazole and terbinafine against the F. monophora isolates. The MICs of terbinafine ranged from 0.0313 to 0.5000 μg/mL, with the MIC₅₀ and MIC₉₀ (at which 50% and 90% of the isolates were inhibited) being 0.125 and 0.25 μg/mL, respectively. The MICs of itraconazole ranged from 0.008-0.125 μg/mL, with the MIC₅₀ and MIC₉₀ being 0.0313 and 0.0625 μg/mL, respectively.

As shown in table 2, when the two drugs were combined, the geometric mean MICs were 0.018 μg/mL (range, 0.008 to 0.0313 μg/mL; MIC₅₀, 0.01565 μg/mL; MIC₉₀, 0.0313 μg/mL) and 0.006 μg/mL (range, 0.004 to 0.01565 μg/mL; MIC₅₀, 0.004 μg/mL; MIC₉₀, 0.008 μg/mL), respectively. The geometric mean MIC of the terbinafine in combination decreased from 0.116 to 0.018 (P = 0.00) while MIC of the Itraconazole in combination decreased from 0.034 to 0.006 (P = 0.00).
Table 2 Ranges and G-means of the minimal inhibitory concentrations of tebinafine and itraconazole

Antifungal		MIC range (μg/mL)	MIC₉₀	MIC₅₀	G-mean
Terbinafine	Single	0.5-0.0313	0.25	0.125	0.116*
Combination	0.0313-0.008	0.0313	0.01565	0.018*
Itraconazole	Single	0.125-0.008	0.0625	0.0313	0.034**
Combination	0.01565-0.004	0.008	0.004	0.006**

In vitro activities of terbinafine and itraconazole combinations

Synergism between the two drugs was found for 12 of 18 isolates (67%), according to the FICI (FICI < 0.5). 4 isolates (22%) showed an additive effect (0.5 < FICI < 1.0). According to the FICI, 2 isolates showed indifferent effect (FICI > 1.0 or FICI < 4.0). There was no antagonistic effect observed (table 3).

Clinical responses

Table 4 describes the antifungal dosages, therapy regimen, and the corresponding clinical responses. We studied 5 F. monophora isolates from 15 patients with F. monophora infection that occurred from July 1995 to March 2007. Three patients (synergism was found for their isolates, strain: SUMS0158; SUMS0200; SUMS0295) received terbinafine 0.25 g and itraconazole 0.2 g combination therapy after drug sensitivity testing. The combination treatment with terbinafine and itraconazole for 4 weeks resulted in a remarkable improvement of clinical symptoms. Following oral terbinafine 250 mg/day for 3 to 6 weeks, 2 of the 3 patients were cured, 1 patient was aggravated. The patient who failed terbinafine therapy recovered after receiving itraconazole therapy (200 mg/day p.o.) for another 6 weeks. 2 patients (strain: SUMS0228; SUMS0254) were cured after receiving itraconazole therapy (100-400 mg/day p.o.) for the complete 9- to 16-week course. No liver toxicity or other adverse effects were observed during the therapy.
Table 3 The MIC, FICI, and outcome of terbinafine and itraconazole against 18 isolates of Fonsecaea monophora after 5 days of incubation

Isolate	MIC (μg/mL)		MICs of the Combination (μg/mL)	FICI	Outcome
ITZ	TBF	ITZ/TBF
0012	0.0313	0.5	0.004/0.01565	0.16	Synergy
0013	0.0625	0.0625	0.008/0.01565	0.38	Synergy
0014	0.0313	0.0625	0.004/0.01565	0.38	Synergy
0034	0.0625	0.0625	0.004/0.0313	0.56	Additivity
0147	0.008	0.125	0.008/0.01565	1.13	Indifference
0158(A)	0.0625	0.0313	0.004/0.008	0.32	Synergy
0190	0.0313	0.0625	0.004/0.01565	0.38	Synergy
0192	0.125	0.0625	0.004/0.01565	0.28	Synergy
0200(B)	0.0313	0.0625	0.004/0.01565	0.38	Synergy
0228	0.01565	0.0625	0.008/0.01565	0.76	Additivity
0246	0.125	0.125	0.004/0.0313	0.28	Synergy
0247	0.01565	0.25	0.01565/0.01565	1.06	Indifference
0250	0.0625	0.125	0.008/0.01565	0.25	Synergy
0254	0.0625	0.5	0.008/0.0313	0.19	Synergy
0295(C)	0.0313	0.125	0.004/0.01565	0.25	Synergy
CBS269.37	0.01565	0.125	0.004/0.01565	0.38	Synergy
CBS102225	0.01565	0.25	0.008/0.0313	0.64	Additivity
CBS102229	0.01565	0.25	0.008/0.0313	0.64	Additivity

Table 4 The MICs of F. monophora isolates and clinical response to treatment

Isolate (SUMS)	MIC (μg/mL)		MICs of the combination	FICI	Therapy regimen (grams/day)	Clinical Follow up
ITZ	TBF	ITZ/TBF
0158	0.0625	0.0313	0.004/0.008	0.32	ITZ 0.2 and TBF 0.25 × 4 weeks, then TBF0.25 × 3 weeks	Cured
0200	0.0313	0.0625	0.004/0.01565	0.38	ITZ 0.2 and TBF 0.25 × 4 weeks, then TBF 0.25 × 6 weeks	Cured
0228	0.01565	0.0625	0.008/0.01565	0.76	ITZ 0.4 × 9 weeks, then ITZ 0.2 × 5 weeks, last ITZ 0.1 × 2 weeks	Cured
0254	0.0625	0.5	0.008/0.0313	0.19	ITZ 0.2 × 9 weeks,	Cured
0295	0.0313	0.125	0.004/0.01565	0.25	ITZ 0.2 and TBF 0.25 × 4 weeks, then TBF0.25 × 6 weeks last, ITZ 0.2 × 6 weeks	Improvement

Discussion

The fractional inhibitory concentration index (FICI) has been one of the most commonly used parameters for testing the interaction of antifungal drugs in vitro [15]. Using a checkerboard microdilution method based on FICI, we demonstrated the in vitro synergism of itraconazole and terbinafine in the present study. Gupta et al. reported [16] that they successfully treated four patients with chromoblastomycosis, caused by F. pedrosoi, using itraconazole and terbinafine on alternate weeks or in combination. Although the exact mechanisms of action for such a synergistic effect are not fully understood, it is possible that itraconazole and terbinafine block different steps of the same pathway of fungal ergosterol biosynthesis, resulting in an enhanced efficiency in antifungal activity. To our best knowledge, there is no report on the testing of in vitro susceptibility of F. monophora to antifungal agents.

Our results showed that the MIC₅₀ and MIC₉₀ of terbinafine were 0.125 and 0.25 μg/mL; the MIC₅₀ and MIC₉₀ of itraconazole were 0.0313 and 0.0625 μg/mL, respectively. The MICs of the terbinafine and itraconazole were within the range that can be achieved in a patient’s serum. The achievable maximum concentrations of terbinafine are approximately 0.9 or 1.7 mg/mL within 2 h of oral administration of a dose of 250 or 500 mg [17, 18], while 0.5 or 1.9 μg/mL within 4 h of oral administration of a dose of 200 or 400 mg can be achieved with itraconazole [19].

Treatment failure often occurs to monotherapy for invasive fungal infections. In a study conducted by Andrade et al., [20] fourteen F. pedrosoi isolates from six chromoblastomycosis patients were subjected to susceptibility testing. The results suggest the development of microbiological resistance to itraconazole in four instances, with two of them showing a lack of clinical response to itraconazole. Combination therapy may be effective in treating multi-resistant fungi [21-23]. In the present study, FICI analysis indicates that synergistic interaction exists with terbinafine and itraconazole in most F. monophora isolates from chromoblastomycosis patients, suggesting that terbinafine and itraconazole may be effective in vivo.

Thus far, it remains uncertain if there is a correlation between the results from fungal susceptibility testing and the clinical response. Andrade et al. observed a correlation between in vitro testing and clinical response [20]. In contrast, some investigators believe that the in vitro fungal susceptibility testing is not indicative for clinical response, but may be useful to identify resistant strains. If that is the case, susceptibility testing can be of help when clinical improvement is not satisfactory [24, 25]. In the present study, successful treatment with itraconazole and terbinafine combination was observed in three patients (table 3; patients A, B and C), who presented a synergistic effect for each isolate. Three isolates presented MICs against terbinafine of 0.0313 μg/mL, 0.062 μg/mL and 0.125 μg/mL, respectively. The three patients showed positive responses to the combination therapy. After combination therapy, patients were given mono-therapy with terbinafine. Patients C, who presented MIC of terbinafine as 0.125μg/mL, did not show clinical improvement, while patients B and A, who presented low MICs of terbinafine, showed clear clinical improvement. Then, patient C underwent itraconazole monotherapy (MIC of the terbinafine-itraconazole: 0.0313 μg/mL) and showed clear clinical improvement. We presume that the phenomena may be explained by the following: first, terbinafine has a high MIC (MIC: 0.125 μg/mL) and there is a correlation between terbinafine concentrations and therapeutic effect. Second, it is well known that severe chromoblastomycosis may correlate with the response to therapy, due to the difficult penetration of the drug in the infected tissues. Patient C presented severe lesions and 250 mg terbinafine (p.o.) might not have reached effective concentration in infected tissues. Finally, as Takemoto et al. reported, the peak level/MIC ratio of amphotericin is the best parameter that correlates with the in vivo activity of amphotericin [26]. The phenomena mentioned above may be partially ascribed to the higher peak level/MIC ratio of itraconazole than that of terbinafine.

In conclusion, this study represents our first effort to determine the activity of itraconazole and terbinafine against F. monophora, used alone or in combination. Our results indicate the combination may be an effective therapy for this disease, which should be tested in a clinical setting with patients with F. monophora infection.

Acknowledgements

There is no conflict of interest to disclose in the study. Financial support: none.

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