mycoses 31, No. 3 (1988) Aktivity of Enilconazole Against AspergiNus 143

mycoses 31 (3) 143-147 accepted/angenommen: December 16,1987 . 0 Grosse Verlag Berlin 1988

In Vitro Activity of Edconazole* Against Aspergillus Spp. and its Fungicidal Eff~cacy in a Smoke Generator Against Aspergillus fumigatus Die In Vitro-Aktivitat von Emlconazol* gegen Aspergillus spp. und seine fungizide Aktivitat gegen Aspergillus fumigatus bei Verraucherung J. Van Cutsem, F. Van Gerven and P. A. J. Janssen Department of Bacteriology and Mycology, Janssen Research Foundation, Beerse, Belgium

Key words: Enilconazole - Aspergillus - fungicidal activity - smoke generator Scbliisselworter: Enilconazol - Aspergillus - Fungkidie - Rauchgenerator

Summary: Enilconazole is a broad-spectrum imidazole antifungal with potent anti- aspergillar activity. Complete inhibition of the growth of Aspergihs spp. was achieved in vitro in serial decimal dilution tests in Sabouraud broth at concentrations of 0.1 and 1 p.rn1-I. Enilconazole was also active in its vapour phase. The fungicidal activity of enilconazole (Clinafarma) incorporated in smoke genera- tors against Aspergillus fumgatus was evaluated and compared with that of thiabenda- zole (smoke generator Fungitec@). Two diffe- rent experiments were performed with conidia of A.fumgatus in hanging and in lying drops (5 x 106 spores per drop). Enilconazole at concentrations of 0.146g and 0.254g per m3 was highly fungicidal to the spores in hanging as well as in lying drops, while thiabendazole at 0.409 and 0.475g m3 was not fungicidal, reducing only the number of viable spores in lying drops and was poorly active in the hanging drops. Enilconazole in a smoke generator formulation appeared to be a highly aspergil- licidal compound. Its activity was largely su- perior to that of thiabendazole.

* Trademark: C1indfam-P

Zusammenfassung: Enilconazol ist ein Breit- band-Antimykotium aus der Imidazolreihe und besitzt eine starke Wukung gegen AspergiUus. Erne voUige Wachstumshemmung von Asper- giflus-species in vitro wird in Dezimalverdiin- nungsserien in Sabouraud-Bouillon bei Kon- zentrationen von 0,l bis 1 pg.ml-I erreicht. Enilconazol wirkt auch in der Dampfphase. Die fungiziden Eigenschaften von Enilconazol in Form eines Rauchgenerators (Clinafarm@') ge- gen Aspergihs fumgatus wurden im Vergleich zu Tiabendazol (Rauchgenerator Fungitec@) beurteilt. Mit den Konidien von A. fumgatus wurden mei verschiedene Experimente durch- gefiihrt: in hangenden und in liegenden Tropfen (5x 106 Sporen pro Tropfen). Enilconazol wirkte in Konzentrationen von 0,146g und 0,254g pro m3 stark fungizid gegen die Koni- dien in beiden Tropfenformen, wiihrend Tiaben- dazol weder bei 0,409 noch bei 0,475 g pro m3 fungizid wirkte, sondem nur die Zahl der lebensfagen Sporen in den liegenden Tropfen reduzierte, diejenigen in den hiingenden Tropfen aber kaum beeinflukite. Damit emies sich Enilconazol im Rauchgenerator als stark as- pergillozid. Die Wirksamkeit war deqenigen des Thiabendazols weitaus uberlegen.

144 J. Van Cutsem et al. mycoses 31, No. 3 (1988)

Introduction

Enilconazole is a broad-spectrum antifungal imidazole (5,6). In vitro it is fungicidal as well as fungistatic (9, 10). In vivo it is active after topical application in experimental dermato- phyte infections in guinea-pigs. Clinical studies confirmed its activity in various animal species in dermatophytosis (1,2, 3, 8) and in aspergillosis (4). The vapour phase activity of enilconazole was described (1 l), as well as its activity in a smoke generator in experimental aspergillosis in chickens (7). The aim of the present study was to evaluate the in vitro activity of enilconazole against a large number of Aspergillus strains and to compare its fungicidal activity in a smoke generator against Aspergillus fumigatus with that of the commercial smoke generator of thiabend- azole.

In-vitro evaluation of enilconazole against A spergiuus

Materials and Methods

The in-vitro sensitivity to enilconazole (Fi- gure 1) of 86 strains of Aspergillus spp. was determined in Sabouraud broth. Initial so- lutions of enilconazole were made in ethanol 50% to obtain a test medium with serial decimal dilutions ranging from 100 to 0.001 pgm- ' . Controls without and with solvent were added and all tests were per- formed in duplicate. The inoculum consisted of square blocks of 2mm side, containing hyphae and spores to be introduced in tubes of 16 mm diameter with 5 ml of the drug- supplemented medium. All cultures were in- cubated at 25°C for 14 d. Final results are given as partial (minimum of 50 to 75% growth inhibition) or complete antifungal activity (100% growth inhibition).

RSults

All controls developed abundantly. For the 62 tested strains of A. fumigatus, complete growth inhibition was achieved for

9 strains at 0.1 pg.ml-' and for the others at 1 pg.ml- ' (Table 1). Pronounced partial inhi- bition was obtained at lower concentrations. All tested strains of A. niger were sensitive; for some of them slightly higher concentrations were needed. The other Aspergillus spp. were as sensitive as the A . fumigatus isolates.

Evaluation of activity of the smoke generators

Materials and Methods

1. Smoke generators Smoke generators ClinafarmO (Janssen Phar- maceutica, B-2340 Beerse, Belgium) contain- ing 5 g enilconazole (lot. No. 85 I 25 OH-5 D 8801) were used, as well as smoke generators Fungitecm containing 7 g thiabendazole (Avitec F-75886 Paris Cedex 18, France, lot No. 863123). The chemical structures of enilconazole and of thiabendazole are given in Figure 1.

2. Test fungus Conidia of Aspergillus fumigatus, strain B 191 19 were harvested from cultures on Sabouraud agar in Roux bottles, incubated at 37" C for 4 days. A homogeneous suspension of spores containing 25 x 1 O7 colony forming units (CFU) was prepared in saline. A drop of tween 80 was added per 100 ml saline. Saline with the same dose of tween was also used afterwards.

3. Test rooms Two air-tight rooms were used alternately for two tests. Walls and ceilings were painted, the floor was paved. The temperature was 2&22 O C and relative humidity vaned between 65 and 75%. With each test, the smoke ge- nerators were placed in the middle of the room. After each test, the rooms were aired, cleaned and decontaminated. The details con- cerning the two rooms and the antifungals are given in Table 2.

4. Preparation of the supports A volume of 0.02ml of the suspension con- taining 5 x 106 CFU of spores was put in the

mycoses 31, No. 3 (1988) Aktivity of Enilconazole Against Aspergillus 145

C l

E ni I con azol e

Thiabendazole

Fig. 1: Chemical structure of enilconazole and of thiabendazole

Table 1: Activity of enilconazole against Aspergillus isolates

Aspergillus spp. No. of Number of strains with inhibition at stated decimal concentrations (bg ml-’) strains 10 1 0.1 0.01 tested complete partial’ complete partial complete partial partial

A. fumigatus 62 - - 62 - 9 2a 2 A. niger 9 9 - 6 3 0 9 0 A. nidulans a - a - 4 4 3 A. flavus 2 A. amstelodami 1 R candidus 1 R penicilloides 1 A. restrictus 1 A. versimlor 1

- - 1

1

1 1

- - - - 2 1 1 1 1 1

- - - - - - - - - - -

- - - - - - - - - -

- - - - - -

* partial: at least 50 to 75% of growth inhibition after 14 days of incubation at 25°C

middle of Petri dishes of 100 mm in diameter. Series of 8 dishes were prepared for each test. Half of the dishes were turned with the opening downwards, the suspension drop hanging in them, the other half with the opening upwards, with a lying drop of the suspension, both for the non-treated control dishes and the ones treated with enilconazole and thiabendazole. The dishes were at a mean distance of 1.45 m from the smoke generators, 1.10 m high.

5. Fumigation The duration of smoke emission, starting at the moment fire was set to the fuse, was about 30 sec. All the Petri dishes were exposed to the fumigation product for a period of 17 hours.

6. Control After the fumigation, 10ml of sterile saline was introduced in each Petri dish. The inoculum was removed, homogenized and, after washing, four successive decimal di-

146 J. Van Cutsem et al. mycoses 31, No. 3 (1988)

Table 2 Volume of test rooms and distribution of smoke generators I Room and volume

I. 59 m3 (I. 34.2 m3

Test 1 Test 2 Compound No. of Compound No. of

smoke smoke genera- genera- tors tors

Clinafarm@ 3 Fungitecs 4 Fungitecs 2 Clinafarm@ 1

Table 3 Weight of the smoke generators in g before and after smoke emission

Product Test 1 Test 2 before after before after

CIinafarm@ 1 74.39 63.04 1 73.22 62.80 2 74.99 64.16 3 75.23 63.47

Fungitecs 1 45.14 22.95 1 44.83 24.05 2 45.09 24.36 2 41.04 22.18

3 44.87 23.76 4 40.82 22.46

lutions were made. Four cultures on Sabouraud agar were made of each dilution in Petri dishes in view of the CFU count. After 3 d of incubation at 37" C, the colonies that had grown were counted.

RSults

The weight of the smoke generators before and after fumigation is given in Table 3, indicating that smoke production was normal for each generator.

In the non-treated controls, a mean of 95 x 106 CFU per ml was counted for hanging and lying drops (Extr. 22 x 106 ~ 163 x 106). With a concentration of 0.146 g per m3 of enilconazole, 4 negative and 4 positive hang- ing drops (mean 38 x 103 CFU per ml), and 7 negative and one positive (3 x 1@ CFU per ml) lying drops were obtained. A concen- tration of 0.254 g perm" of enilconazole used as smoke gave 6 negatives and 2 positives (102 - 4 x lo2 CFU per ml) in hanging as well as in lying drops. With thiabendazole smoke ge-

Table 4 Activity of smoke generators containing enilconazole and thiabendazole on spores of Aspergillus fumigaluy

Treatment' Antifungal active dose

in g per m3

A. Controls 0 enilconazole*' 0.254 enilconazole 0.254 . thiabendazole'" 0.475 thiabendazole 0.475

6. Controls 0 enilconazole 0.1 46 enilconazole 0.1 46

thiabendazole 0.409 thiabendazole 0.409

Inoculum" No. of samples

Mean No. of CFU per ml x to3

95,300 (Extr.: 22,400 - 163,OO) 6 negatives; 0.3; 0.4 6 negatives; 0.1: 0.4 2.594 (Extr.: 100 - 8,000) 12,706 (Extr.: 1.1 150 - 23,500)

95,156 (Extr.: 77,500 - 120,000) 7 negatives: 3.1 4 negatives; 4 positives 38 (Extr.: 22 - 71) 10,640 (Extr.: 4,500 - 20,750) 43,047 (Extr.: 30,000 - 55,000)

* treatment: 17 h contact time ** inoculum as lying or hanging drop

*** enilconazole smoke generator Clinafarm@ of 5 g, Janssen Pharmaceutica, Belgium lot NO. 85 I 2 5 OH-5 D 8801

**** thiabendazole: Fungitecm smoke generator of 7 g Avitec - 75886 Pans Cedex 18 France lot No. 863123

mycoses 31, No. 3 (1988)

nerators with concentrations of 0.409 g and 0.475 g per m3 of room volume, none of the drops were negative and the hanging drops contained a mean of 43 x 106 and 12 x 106 of CFU per ml respectively, while in lying drops themean was 1 x lo7 and 2 x 106 CFU per ml (Table 4).

Discussion The in-vitro broad-spectrum antifungal acti- vity of enilconazole has been described earlier (6). Enilconazole possessed high potency against all Aspergillus spp. strains by direct contact. The vapour phase activity of enilconazole also contributed to its potent anti-aspergillar efficacy, in preventing sporu- lation and in affecting the growth of conidio- phores, stipes, vesicles, phialides, and the production of conidia (llJ2). The smoke experiments confirmed the aspergillicidal ac- tivity of enilconazole, fumigated in smoke generators, on lying as well as on hanging spores, indicating the diffusion in the air of the active compound. The superiority of the enilconazole smoke generator (Clinafarm@) over the thiabendazole smoke generator (Fungitec@) was clearly demonstrated. In spite of higher concentrations of thabenda- zole, this compound was only capable of reducing the total number of Aspergillus spores, while enilconazole was largely fun- gicidal. The concentrations of enilconazole per m3 room volume used in this study correspond to the concentration used in previ- ous experiments to kill Aspergillus spores that came in direct contact with chicks. That concentration was able to protect these animals from aspergillosis (2). Clinafarm@ smoke generators can be used for fungal decontamination in animal facilities and in various other conditions.

Acknowledgements: This investigation was supported by the “Instituut tot Aanmoedi- ging voor het Wetenschappelijk Onderzoek in Nijverheid en Landbouw” (IWONL). The authors are grateful to H. Vanhove for re-

Aktivity of Enilconazole Against Aspergillus 147

viewing and to S . Sas for typing the manuscript.

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Address: Dr. J. Van Cutsem, Dept. of Bacteriology & Mycology, Janssen Research Foundation, B-2340 Beerse, Belgium.