In-vitro antifungal susceptibilities of Basidiobolus and Conidiobolus spp. strains

Josep Guarroa,*, Carme Aguilara and Isabel Pujolb

a Unitat de Microbiologia, Facultat de Medicina, Universitat Rovira i Virgili, Carrer Sant Llorenç, 21, 43201 Reus b Laboratori de Microbiologia, Hospital Universitari de Sant Joan de Reus, 43201 Reus, Tarragona, Spain


    Abstract
 Top
 Abstract
 Introduction
 Materials and methods
 Results and discussion
 References
 
The in-vitro antifungal susceptibilities of nine isolates belonging to Basidiobolus spp. and seven to Conidiobolus spp. against six antifungals (amphotericin B, ketoconazole, miconazole, itraconazole, fluconazole and flucytosine) were tested. A broth microdilution method, generally following the NCCLS guidelines, was used. Inoculum concentrations of the order of 100 cfu/mL were obtained by culturing fungi in a broth medium (Czapeck broth supplemented with 2% Tween 80 and 0.07% agar). MICs and MFCs were highly variable and isolate-dependent, with the exception of those of flucytosine which were constantly very high. In general, however, Basidiobolus spp. displayed low MICs of fluconazole, itraconazole, ketoconazole and miconazole, and Conidiobolus spp. were resistant to all antifungals tested.


    Introduction
 Top
 Abstract
 Introduction
 Materials and methods
 Results and discussion
 References
 
Some members of the order Entomophthorales are well recognized pathogens that can affect humans and animals, although some are saprophytes isolated from soil, plant detritus and faeces. The most common species from clinical sources are Basidiobolus ranarum and Conidiobolus coronatus (Entomophthora coronata, Delacroixia coronata). The former usually causes chronic subcutaneous infections, affecting predominantly buttocks or thighs, and the latter causes chronic granulomatous infections of the nasal submucosa, although many other body sites can also be affected and several cases of invasive infections have also been described.1 Both are related to Mucorales even though their clinical and histopathological patterns are very different. In the treatment of these infections, iodides and ketoconazole are usually employed. Amphotericin B is also used, although rarely effective in the management of Basidiobolus spp. infections. Itraconazole has also been described.2 We present data on the in-vitro susceptibility of 17 isolates of entomophthoralean fungi and review other published reports on the in-vitro susceptibility of these organisms.


    Materials and methods
 Top
 Abstract
 Introduction
 Materials and methods
 Results and discussion
 References
 
Seventeen isolates were tested, nine of them belonging to the genus Basidiobolus (three Basidiobolus haptosporus, three Basidiobolus meristosporus, one Basidiobolus heterosporus and two B. ranarum) and eight to Conidiobolus (six C. coronatus, one Conidiobolus incongruus and one Conidiobolus lamprauges). Paecilomyces variotiiATCC 36257 was used as a control. The following six antifungal agents were assayed: amphotericin B (Squibb & Sons, Barcelona, Spain), flucytosine (Hoffmann-La Roche, Basel, Switzerland), fluconazole (Pfizer, Madrid, Spain), ketoconazole (Roig-Farma, Barcelona, Spain), miconazole (Roig-Farma, Barcelona, Spain) and itraconazole (Janssen Pharmaceutica, Beerse, Belgium). The commercially available iv preparations of amphotericin B (Fungizone) and fluconazole (Diflucan) were used as stock solutions. Antifungal solutions were prepared as described previously.3 We used a broth microdilution method performed mainly according to NCCLS recommendations for filamentous fungi4 with a few modifications.

Sterile, 96-well microplates were used, each row comprising a decreasing concentration range of antifungal drug diluted with RPMI 1640 medium; 100 µL of the corresponding drug dilution was inoculated into each well. Microplates were stored at –70°C before use. The isolates were grown on plates of malt agar (MA) at 30°C for approximately 3 weeks before testing. The inoculum was prepared by scraping the superficial mycelium with a loop and directly suspending the fungal material in an Erlenmeyer flask (150 mL) which contained approximately 30 mL of LCTA (Czapek broth (Difco, Detroit, IL, USA) supplemented with 2% Tween 80 and 0.07% agar).5 The resulting suspension was maintained at 30°C in a shaking incubator for 3 days, and was then filtered through sterile gauze to obtain a homogeneous suspension of small hyphae. The suspension was adjusted spectrophotometrically to 70% transmission at 530 nm to produce the working suspension. Aliquots of 100 µL were inoculated into each well. Serial dilutions of the working suspension were spread on to MA plates to determine the cfu/mL. With this procedure we obtained inoculum concentrations of the order of 100 cfu/mL. Final antifungal concentration ranges were: 0.03–16 mg/L for amphotericin B, miconazole, itraconazole and ketoconazole, 0.125–64 mg/L for fluconazole, and 0.25–128 mg/L for flucytosine. The microplates were incubated without agitation at 30°C and readings were taken after 48 and 72 h. The MIC of amphotericin B was defined as the lowest drug concentration at which there was a complete absence of growth. The azoles and flucytosine MICs were defined as the lowest drug concentration that gave only a slight growth corresponding to approximately 25% of growth control. MFCs were obtained by placing 10 µL from each well showing total or partial inhibition on to MA plates. Fungal colonies were counted after incubation at 30°C for 48 h or until growth of the subcultures was visible in the growth control well. The MFC was defined as the lowest drug concentration from which one colony or less was visible on the agar plate. The high off-scale MICs and MFCs (e.g. >=16 mg/L) were converted to the next highest concentrations (32 mg/L), and the low off-scale MICs and MFCs were left unchanged.


    Results and discussion
 Top
 Abstract
 Introduction
 Materials and methods
 Results and discussion
 References
 
The MICs and MFCs of the 17 isolates tested are shown in Tables I and II. Except for those of flucytosine, which were always very high, they were very variable and isolate-dependent. In general, however, Basidiobolus spp. displayed low MICs of fluconazole, itraconazole, ketoconazole and miconazole, and Conidiobolus spp. were resistant to all antifungals tested.


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Table I. In-vitro susceptibilities of Entomophthorales isolates to six antifungals
 

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Table II. In-vitro activity of six antifungals against isolates of Conidiobolus spp. and Basidiobolusspp.
 
Over the last 10 years, approximately 20 cases of infection by Conidiobolus spp. have been published, the majority 1 ,6 ,7 caused by C. coronatus. One of the classical treatments used is the administration of potassium iodide (KI). Ketoconazole has been the most frequently used systemic agent, described in the literature on 10 occasions; five times alone, three times with KI, generally with success, and once unsuccessfully with itraconazole. Amphotericin B has been used in five cases; two as monotherapy and three in combination with other drugs with poor outcomes. Only in one case, where amphotericin B was administered together with terbinafine, was the infection cured. Fluconazole has been used in two cases with good results, one alone and the other with KI. Experience of treatment with itraconazole is still scarce and has yielded inconclusive results. Our results agree with those of Taylor et al.8 and Walsh et al.,1 and although they only tested one strain of C. coronatusand of C. incongruus, they found very high MICs of amphotericin B, ketoconazole, fluconazole, flucytosine and miconazole.

Over the same period, approximately 10 cases of human infection by Basidiobolus spp. have been reported, and all successfully treated. Three of these were with itraconazole, four with ketoconazole (one of them in combination with KI and one with amphotericin B), two with fluconazole and one with KI. This also agrees with our in-vitro results. Basidiobolus spp., in contrast to Conidiobolus spp., generally showed very low MICs, with the exceptions of flucytosine and fluconazole in two strains.

According to Taylor et al.,8 the high MIC for amphotericin B alone or in combination with other agents explains the failure of the patient involved in that case to respond. However, it is possible that variations in sensitivity exist between isolates.8 This is true in our experience, where of the six isolates of C. coronatus tested, four showed an MIC of 4 mg/L, one an MIC of 2 mg/L and the other an MIC of 0.5 mg/L.

In the only previous report where a considerable number of Entomorphthorales were tested, very different results were obtained, especially in the case of Conidiobolusspp.9 The three strains tested showed very low MIC and MFC values. A broth dilution method was used, although as inocula, the suspension created from scraping cultures on Sabouraud dextrose agar adjusted to a concentration of 104–105 cells/mL was used. We could not follow this procedure as the colonies formed by these fungi on solid media are not consistent: they are flat, very thin and folded, with sparse aerial mycelium and with diverse types of spores which makes it difficult to obtain homogeneous and reproducible working suspensions. For these reasons we used a broth medium assayed previously10 to prepare the inocula, which gave more reproducible results.


    Acknowledgments
 
This work was supported by the Fundació Ciència i Salut, Reus, Spain and by the CICYT grant PM98-0059.


    Notes
 
* Corresponding author. Tel: +34-977-759359; Fax: +34-977-759322; E-mail: umb{at}astor.urv.es Back


    References
 Top
 Abstract
 Introduction
 Materials and methods
 Results and discussion
 References
 
1 . Walsh, T. J., Renshaw, G., Andrews, J., Kwon-Chung, J., Cunnion, R. C., Pass, H. I. et al. (1994). Invasive zygomycosis due to Conidiobolus incongruus. Clinical Infectious Diseases 19, 423–30.[ISI][Medline]

2 . Richardson, M. D. & Warnock, D. W. (1997). Fungal Infection Diagnosis and Management. Blackwell Science, Oxford.

3 . Pujol, I., Guarro, J., Llop, C., Soler, L. & Fernández-Ballart, J. (1996). Comparison study of broth macrodilution and microdilution antifungal susceptibility tests for the filamentous fungi. Antimicrobial Agents and Chemotherapy 40, 2106–10.[Abstract]

4 . National Committee for Clinical Laboratory Standards. (1998). Reference Method for Broth Dilution Antifungal Susceptibility Testing of Conidium-Forming Filamentous Fungi: Proposed Standard M38-P. NCCLS, Wayne, PA.

5 . Bezjak, V. (1985). Standardization of a hyphal inoculum of aspergilli for amphotericin B susceptibility testing. Journal of Clinical Microbiology 21, 509–12.[ISI][Medline]

6 . Bittencourt, A. L., Arruda, S. M., de Andrade, J. A. F. & Carvalho, E. M. (1991). Basidiobolomycosis: a case report. Pediatric Dermatology 8, 325–8.[ISI][Medline]

7 . Jaffey, P. B., Haque, A. K., el-Zaatari, M., Pasarell, L. & McGinnis, M. R. (1990). Disseminated Conidiobolus infection with endocarditis in a cocaine abuser. Archives of Pathology and Laboratory Medicine 114, 1276–8.[Medline]

8 . Taylor, G. D., Sekhon, A. S., Tyrrell, D. L. J. & Goldsand, G. (1987). Rhinofacial zygomycosis caused by Conidiobolus coronatus: a case report including in vitro sensitivity to antimycotic agents. American Journal of Tropical Medicine and Hygiene 36, 398–401.

9 . Yangco, B. G., Okafor, J. I. & TeStrake, D. (1984). In vitro susceptibilities of human and wild-type isolates of Basidiobolus and Conidiobolus species. Antimicrobial Agents and Chemotherapy 25, 413–6.[ISI][Medline]

10 . Guarro, J., Llop, C., Aguilar, C. & Pujol, I. (1997). Comparison of in vitro antifungal susceptibilities of conidia and hyphae of filamentous fungi. Antimicrobial Agents and Chemotherapy 41, 2760–2.[Abstract]

Received 2 March 1999; returned 5 May 1999; revised 25 May 1999; accepted 8 June 1999