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
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Abstract |
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Introduction |
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Materials and methods |
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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: 0.0316 mg/L for amphotericin B, miconazole, itraconazole and
ketoconazole, 0.12564 mg/L for fluconazole, and 0.25128 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.
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Results and discussion |
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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 104105 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.
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Acknowledgments |
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Notes |
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References |
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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, 210610.[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, 50912.[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, 3258.[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, 12768.[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, 398401.
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, 4136.[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, 27602.[Abstract]
Received 2 March 1999; returned 5 May 1999; revised 25 May 1999; accepted 8 June 1999