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ORIGINAL CONTRIBUTION |
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| Year : 1995 | Volume
: 49
| Issue : 5 | Page : 109-113 |
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Antimycotic susceptibility testing of mould-fungi with allylamines by disk diffusion
Pankajalakshmi V Venugopal, Taralakshmi V Venugopal
Institute of Microbiology, Madurai Medical College, Madurai, India
| Date of Submission | 04-Aug-1994 |
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Correspondence Address:
Pankajalakshmi V Venugopal
Institute of Microbiology, Madurai Medical College, Madurai
India
PMID: 8772831
How to cite this article:
Venugopal PV, Venugopal TV. Antimycotic susceptibility testing of mould-fungi with allylamines by disk diffusion. Indian J Med Sci 1995;49:109-13 |
Considerable advances have been made in treating mycotic diseases with the introduction of several new antifungal drugs. Hence, routine susceptibility testing of pathogenic or opportunistic fungi involved in such infections would become a necessity in the near future. However, only macrobroth and micro-broth dilution and agar dilution methods are available till recently. Since reports on disk diffusion susceptibility testing of mould-fungi are very few [1],[2] , we decided to undertake a preliminary study for evaluating the antimycotic activity of the allylamines - naftifine and terbinafine against 17 clinical isolates of mould-fungi by agar dilution and disk diffusion methods.
| ¤ Materials and Methods | |  |
Naftifine and terbinafine were obtained from Sandoz Forchung-sinstitut. Preparation of the stock solution, further dilutions and drug containing media with the controls were made as previously described. [3] No. 1 What man filter paper was used for preapring the disks with 6 mm diameter. The disks were sterilized and each of the drug solution was loaded on to the disks at a concentration of 5 and 15 μg per disk. When dried, the disks were stored at 4°C. A total of 17 clinical isolates comprising of Aspergillus flavous 2, A. fumigates 1, A. niger 2, A. glaucus 1, A. terreus 2, Pencitlium spp 2, Paecilomyces spp., 1, Cfadosporium spp., 1 Rhizopus spp., 2 and Syncephalastrum spp., 1 from cases of otomycosis, P. romeroi 1 from a case of black grain eumycetoma and Mortierella spp., 1 from a case of rhinocerebral zygomycosis were tested. The isolates were maintained on potato dextrose agar without antibiotics and for the test, they were subcultured on Sabouraud's dextrose agar and incubated at 35°C for 3 to 10 days.
The preparation of the fungal inocula, spectrophotometrical standardization to an absorbance of 0.600 at 450 nm and susceptibility testing by agar dilution were done as previously described. [3] The tubes were incubated at 30°C until visible growth appeared in the control tubes (2 - 7 days) and the minimum inhibitory concentration (MIC) was recorded. Each test was performed in triplicate and the MICs represent the results of atleast two replications. Nutrient agar was poured to a depth of 5 mm in 150 mm Petri dish More Detailses and stored at 4 o C. The plates were dried and the standardized inoculum suspension was poured and after ensuring uniform spread, the excess was drained off. The inoculum was allowed to dry for 5 minutes and the disks were then applied. The 5 μg disks were used for all the isolates. For the zygomycetes, the 15 μg disks were used in addition. The plates were incubated at 30°C for 2 - 7 days. The readings were taken when visible growth appeared in the agar dilution control tubes (2 - 7 days). The diameters of the clear zones around the disks were measured with a ruler. Two readings were taken at right angles and two disks on separate plates were used for each drug and averaged to determine the zone diameter for a given isolate.
| ¤ Results | | |
Measurable zones of inhibition (mm) were obtained with the 5 μg disks for all the fungi other than zygomycetes. The diameters of the zones of inhibition ranged from 20-43 mm for terbinafine (mean 22.4 mm) and 14-45 m for naftifine (mean 19.9 mm). The 15μg disks were used in addition for zygomycetes and excepting a single isolate of Syncephalastrum spp., which was susceptible to terbinafine (MIC 1 μg/ml) with a inhibition zone of 20 mm, all the others were resistant with no measurable zone of inhibition and their MISs ranged from 100:->100 μg/ml for both the drugs. The average of each set of zones of inhibition for corresponding individual drug concentrations were calculated for each species [Figure 1] and the means of all the isolates were averaged. The values of the zones of inhibition for naftifine and terbinafine ranged from 45-0 mm and 33.8.0 mm for a corresponding MIC range 0.1 ->100 μg/ml of each of the drugs respectively.
The results were compared by regression analysis. Drug concentrations were expressed as loge values and each concentration was compared with its corresponding averaged zones of inhibition produced by the disks of terminafine and naftifine. The correlation coefficients for terbinafine naftifine were -0.9174 and -0.9597 (P'<0.001) respectively.
| ¤ Discussion | | |
The results show that terbinafine is more active against the mould-fungi tested other than zygomycetes, inhibiting 50 and 90% of the isolates at a concentration of 0.5 and 1 μg/ml (MIC range 0.1 - 5μg/ml) and the values for naftifine were 2.5 and 10 μg/ml (MIC range 0.1 -10μg/ml). The allylamines are a new class of potent antifungal agents with a broad spectrum of activity against dermatophytes, other moulds, dimorphic fungi and dematiaceous fungi. Preliminary pharmacokinetic studies in human have reported that peak drug concentation in plasma of 2 μg/ml were reached within 2 hours after a single oral dose of 500 mg of terbinafine and the elimination half life was 11.3 h. [4] The MIC range for the Aspergillus spp., obtained were 0.1 - 0.5μg/ml, well below 2 μg/ml of terbinafine. The 5 μg disks of both the drugs produced measurable zones of inhibition for all the isolates other than zygomycetes and good correlation has been seen between the MlCs and sizes of zones of inhibition. The allylamines does not have any appreciable activity on the zygomycetes. Shadomy et al [5] have reported an MIC range of 64 - 128 μg/ml for terbinafine and naftifine. Our values ranged from 100-> 100 μg/ml for all zygomycetes excepting a single isolate of Syncephalastrum spp., which had an MIC of 1 μg/ml of terbinafine. The 15 μg disks produced no zone of inhibition for the zygomycetes other than the isolate of Syncephalastrum spp., which had an inhibition zone of 20 mm for terbinafine. The results suggest that the allylamines naftifine and terbinafine could be used successfully for susceptibility testing of mouldfungi by disk difusion and the results could be obtained within 2 -7 days. Once the test conditions are properly standardized, susceptibility testing of mould-fungi by disk diffusion would gain the same prominence as the antibacterial susceptibility testing.
| ¤ Summary | | |
Susceptibility testing of 17 clinical isolates of mould-fungi, which included Aspergillus spp., (8) Penicillium spp., (2) Paec lomyces spp., (1) Cladosprium spp., (1) Pyrenochaeta romeroi (1) Rhizopus spp., 2. Syncephalastrum spp., (1) and Mortierella spp., (1) were carried out against allylamines - naftifine and terbinafine - (Sandoz Forchungsinstitut) by agar dilution and disk diffusion techniques. Terbinafine was more active than naftifine inhibiting 50 and 90%, of the fungi other than zygomycetes at a concentration of 0.5 and 1 μg/ml whereas the values for naftifine were 2.5 and 10 μg/ml The MiC range for zygomycetes for terbinafine and naftifine were 1 - > 100 and 100- > 100 respectively. The MICs and the sizes of zones of inhibition around the disks correlated well and the degree of correlation was measured by regression analysis. The correlation coefficients for naftifine and terbinafine were -0.9597 and --0.9174 (P<0.007) respectively.
| ¤ Acknowledgement | | |
We thank the Sandoz Forchungsinstitut (Austria) for kindly providing the test drugs. We are greatly indebted to Mr. R. E. Amalraj, Research Officer (Statistics) ACCERT (ICMR), Madras Medical College, Madras for the statistical evaluation.
| ¤ References | | |
| 1. | Drouhet E, Dupont B, Improvisi L, Biviani MA, Tortorano AM. Disc agar diffusion and microplate automatized technique in vitro evaluation of antifungal agents on yeasts and sporulated pathogenic fungi. In : Iwata K and Bossche HV, eds, In vitro and in vivo evaluation of antifungal agents, pp. 31, Amsterda: Elsevier, 1986.  |
| 2. | Petranyi G, Meingassner JG and Mieth H. Antifungal activity of the allylamine derivative terbinafine in vitro. Antimicrob Agents Chemother 1987;31: 1365-1368. |
| 3. | Pankajalakshmi V. Venugopal, Taralakshmi V. Venugopal. Antimycotic susceptibility testing of dematophytes, Indian J Med Microbiol 1993;11:61-65. |
| 4. | Stutz A, Petranyi G. Synthesis and antifungal activity of (E) - N - (6, 6 - dimethyl - 2 - hepten - 4 - ynyl) - N - methyl - 1 - naphthalene - methanamine (SF 86 - 327) and related allylamine derivatives with enhanced oral activity J Med Chem 1984:27:1539-1543. |
| 5. | Shadomy S. Espinal-Ingroff A, Gebhart J. In vitro activities with SF 86-327, a new orally active allylamine derivaties. J Med Vet Mycol 1985;23:125-132. |
[Figure 1]
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