Skip to main page content

• HOME
• Current Issue
• Archive
• Alerts
• About ASM
• Contact us
• Tech Support
• Journals.ASM.org

This Article Abstract Full Text (PDF) Alert me when this article is cited Alert me if a correction is posted Services Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Reprints and Permissions Copyright Information Books from ASM Press MicrobeWorld Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Alvarado-Ramírez, E. Articles by Torres-Rodríguez, J. M. Search for Related Content PubMed PubMed Citation Articles by Alvarado-Ramírez, E. Articles by Torres-Rodríguez, J. M.

 Previous Article  |  Next Article 

Antimicrobial Agents and Chemotherapy, July 2007, p. 2420-2423, Vol. 51, No. 7
0066-4804/07/$08.00+0     doi:10.1128/AAC.01176-06
Copyright © 2007, American Society for Microbiology. All Rights Reserved.

In Vitro Susceptibility of Sporothrix schenckii to Six Antifungal Agents Determined Using Three Different Methods

Eidi Alvarado-Ramírez and Josep M. Torres-Rodríguez^*

Infectious Diseases and Mycology Research Unit (URMIM), Institut Municipal d'Investigació Mèdica (IMIM), Faculty of Medicine, Universitat Autònoma de Barcelona, Barcelona, Spain

Received 21 September 2006/ Returned for modification 28 November 2006/ Accepted 9 April 2007

Top ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

 
The in vitro susceptibility of Sporothrix schenckii to antifungal drugs has been determined with three different methods. Nineteen Peruvian clinical isolates of S. schenckii were tested against amphotericin B (AB), flucytosine (FC), fluconazole (FZ), itraconazole (IZ), voriconazole (VZ), and ketoconazole (KZ). Modified NCCLS M38-A, Sensititre YeastOne (SYO), and ATB Fungus 2 (ATBF2) methods were used to determine the MICs. ATCC isolates of Candida parapsilosis, Candida krusei, and Aspergillus flavus were used for quality control. Sporothrix inocula were prepared with the mycelial form growing on potato dextrose agar at 28 ± 2°C. MICs of AB, FC, FZ, and IZ were determined with all three methods, VZ with M38-A and SYO, and KZ with only SYO. The three methods showed high MICs of FZ and FC (MIC₉₀ of 0.5 µg/ml), being homogeneously lower than those of IZ and KZ. The M38-A method showed a variable MIC range of VZ (4.0 to 16 µg/ml); the geometric mean (GM) was 9.3 µg/ml. The MIC range of AB was wide (0.06 to 16 µg/ml), but the GM was 1.2 µg/ml, suggesting that the MIC is strain dependent. Agreement (two log₂ dilutions) between commercial techniques and the modified M38-A method was very high with FZ, IZ, and FC. In AB and VZ, the agreement was lower, being related to the antifungal concentrations of each method. The highest activity against S. schenckii was found with IZ and KZ. Lack of activity was observed with FZ, VZ, and FC. When AB is indicated for sporotrichosis, the susceptibility of the strain must be analyzed. Commercial quantitative antifungal methods have a limited usefulness in S. schenckii.

Top ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

 
Sporotrichosis is a subacute or chronic infection that affects humans and other mammals and is produced by the dimorphic fungus Sporothrix schenckii. The infection is acquired when the fungus penetrates through the skin due to trauma, at times minimal, or more rarely through the respiratory tract, causing pulmonary infection (20). The most frequent manifestation of sporotrichosis is the cutaneous-lymphatic form followed by fixed cutaneous infection. In patients with severe underlying disease and the immunodepressed, the disease can spread and cause death (5). The standard treatment of cutaneous-lymphatic sporotrichosis is potassium iodine, with which complete remission of lesions can be achieved in 2 to 3 months (11). Despite the development of new antifungal drugs, potassium iodine continues to be a commonly used therapy probably because of its wide availability. However, the use of potassium iodine may be associated with mild adverse effects, such as gastric intolerance, or more serious adverse events, such as vasculitis, periarthritis, and hypothyroidism, among others (23). Orally administered itraconazole (IZ) has been shown to be effective in the treatment of cutaneous-lymphatic and systemic sporotrichosis (1, 24), whereas visceral and disseminated forms have classically been treated with amphotericin B (AB), with various results (12, 22).

Studies on the susceptibility of S. schenckii to commonly used antifungals have rarely been reported. On the basis of MIC, McGinnis et al. (14) concluded that the susceptibility of S. schenckii to AB was dependent upon the strain being studied. They also found that the geometric mean (GM) was lower for IZ (1.56 µg/ml) than for voriconazole (VZ) (6.50 µg/ml). Aside from the scant information available on the susceptibility pattern of this species, particularly to the newer antifungals, no information exists on the possible utility of the methods currently offered by the industry to determine MICs. More recent publications base their data on the microdilution technique recommended by the Clinical and Laboratory Standards Institute (CLSI [formerly National Committee for Clinical Laboratory Standards]) in their document M38-A (17). Nevertheless, considering the reference pattern used by most authors, this method is relatively impractical for application in a routine microbiology laboratory.

The objectives of this study were to determine the in vitro susceptibilities of a considerable number of clinical S. schenckii strains to six antifungals (AB, flucytosine [FC], fluconazole [FZ], IZ, VZ, and ketoconazole [KZ]) and to compare the results obtained by using a modified M38-A method as a reference with the commercially available Sensititre YeastOne (SYO) and ATB Fungus 2 (ATBF2) kits; both of these methods have been initially standardized only for yeasts.

Top ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

 
A total of 19 S. schenckii isolates were used that had been obtained from patients with various forms of cutaneous-lymphatic sporotrichosis from different hospitals in Peru. These strains were identified in the Mycology Laboratory at the Von Humboldt Institute of Tropical Medicine and stored in sterile distilled water at 4°C within the URMIM collection up until their use.

ATCC 22019 Candida parapsilosis and ATCC 6258 Candida krusei strains were used as quality control (QC), and Aspergillus flavus ATCC 204304 was used as a reference mycelial isolate (M38-A). The M27-A2 method was used for the inoculation, incubation, and reading times of the yeasts.

Assays were carried out with the mycelial phase of S. schenckii, which was obtained by growing for 5 to 7 days at 28 ± 2°C in potato dextrose agar. QC yeasts were grown in Sabouraud dextrose agar with chloramphenicol for 24 h at 35°C. The reference strain A. flavus ATCC 204304 was grown in potato dextrose agar during 72 h at 35°C. Thereafter, the production of abundant conidia of S. schenckii was assessed by examination at x400 with optical microscopy.

To prepare the inoculum, conidia were removed from the colony surface with 3 to 4 ml of sterile saline solution by gently scraping the surface with a handle. The stem suspension was diluted in saline solution in order to visually adjust it to the values established by the manufacturer's instructions, corresponding to the scale 2 McFarland for the ATBF2 method (ATB Fungus2 2 14 201 C; bioMérieux SA, Marcy l'Etoile, France) and to 0.5 McFarland for the SYO method (Sensititre Technical Product Information; Yeast One MIC Susceptibility Test; Accu Med International Ltd., Sussex, United Kingdom). The counts in the Neubauer chamber corresponded to 6 x 10⁶ CFU/ml and 1.5 x 10⁶ CFU/ml, respectively. For the modified M38-A method, the inoculum used was 1 x 10⁵ CFU/ml, according to previous results (15). In all inocula, the viability of the isolate was confirmed by sowing in plates with Sabouraud dextrose agar with chloramphenicol and counting of CFU/ml.

For the test procedure, instructions as indicated in the M38-A document were followed as well as those indicated for each manufacturer (13, 21). After seeding, SYO and ATBF2 plates were incubated at 30°C for 3 to 5 days. For MIC readings, the control growths were always considered for each technique. MIC visual reading was performed at 48 to 72 h of incubation according to the growth control. With ATBF2, visual reading was done at 72 h, but with SYO, only after 72 to 96 h of incubation was clear color change observed. MIC endpoints for M38-A and ATBF2 were considered as no growth with AB, IZ, and VZ and >50% inhibition with respect to growth control for FZ and FC (17). Colorimetric reading was done with SYO, according to the manufacturer's instructions.

The SYO microplates contained six antifungals, FZ, IZ, VZ, KZ, AB, and FC, at increasing concentrations, plus an oxide reduction growth indicator (Alamar blue dye). The endpoint was assessed by a change in color, from blue (negative) to red (positive). The ATBF2 gallery presented four antifungals: AB, FC, FZ, and IZ. Endpoint reading was established after 96 h of incubation for the ATBF2 and 72 h of incubation for the other two methods. Candida QC strains were incubated for 24 to 48 h at 35°C, and the A. flavus reference strain was incubated for 72 h at 35°C. Readings were made visually by comparison with the growth control well.

The concentration ranges for each antifungal according to the technique used are shown in Table 1.

View this table: [in this window] [in a new window]

TABLE 1. Ranges of antifungal concentrations according to technique utilized

Five randomized (25%) isolates of S. schenckii as well the QC isolates were studied three times on different days. MICs were the same or changed in only one dilution concentration.

Differences in MICs of no more than two dilutions log₂ between the two methods were used to calculate the percent agreement (19).

Statistical analysis. Range values, GM, MIC₅₀, and MIC₉₀ were calculated. The values obtained were compared through the Wilcoxon test, with P of <0.05 considered to be statistically significant. Results were analyzed with SPSS- Windows 14.0 (SPSS Inc., Chicago, IL).

Top ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

 
Control plates of each inoculum showed the viability and purity; colony counts agreed with the microscopy counts performed in the Neubauer chamber.

The MICs obtained with the QC yeasts and A. flavus were within the ranges indicated in the CLSI reference documents (16, 17). The C. parapsilosis and C. krusei QC strains were also within the ranges described in the manufacturers’ instructions of both commercial methods. The values, not described until now, that were obtained with those methods for the reference A. flavus strain are presented in Table 2.

View this table: [in this window] [in a new window]

TABLE 2. MICs of 6 antifungals on 19 S. schenckii and 3 quality control strains determined by three methods: modified M38-A (M27-A2 for yeast reference strains), SYO, and ATBF2

MIC results for each one of the 19 S. schenckii strains are also depicted in Table 2.

Table 3 shows the ranges, MIC₅₀s, MIC₉₀s, and GMs for the modified M38-A method as well for the two commercial techniques.

View this table: [in this window] [in a new window]

TABLE 3. In vitro susceptibilities of 19 S. schenckii strains for six antifungals analyzed by three techniques and percentages of agreement with the modified M38-A reference method

With the reference method M38-A, all isolates showed highly elevated MICs for FC and FZ. A very wide range of MICs was found with AB (0.06 to >16 µg/ml) and VZ (4 to 16 µg/ml). The MICs for IZ were the most homogenous, presenting a narrower range (0.25 to 1 µg/ml), and the MIC₅₀ and MIC₉₀ were the same: 0.5 µg/ml being the lower ones. Only one isolate showed a maximum value of 1 µg/ml.

With the two commercial methods, high MICs were also found for FC and FZ; agreement with the reference method was very high in FZ (100%) and IZ (SYO, 95%; ATBF2, 89%), followed by FC (SYO, 68%; ATBF2, 84%) and AB (SYO, 58%; ATBF2, 68%). The lower agreement was found in VZ (SYO, 53%; this antifungal is not present in ATBF2).

KZ, which was only available in the SYO panel, showed a very low MIC (range, 0.06 to 0.5 µg/ml).

Top ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

 
Until now, for S. schenckii the breakpoint discriminating between a susceptible strain and a resistant one had not been determined for any of the currently available antifungals. Nevertheless, the results of this study demonstrate that IZ presents the lowest MIC score.

The values obtained for IZ are in agreement with those observed by Espinel-Ingroff (8). Meanwhile, the clinical correlation between MICs and therapeutic responses has not been established; the low MICs of IZ give certain support to consider this azole as the treatment of choice for mild sporotrichosis (10). KZ has also been administered at doses from 400 to 800 mg/day in patients infected by S. schenckii with favorable results (3).

VZ presented low activity in S. schenckii, coinciding with the results of several other authors (7, 9, 14, 18). In one of these publications (9), a GM of 8 µg/ml was found, similar to our data with the modified M38-A method. Nevertheless, with the SYO method, the GM value was lower (1.9 µg/ml), because six of the isolates demonstrated MICs of 0.5 µg/ml. Currently there are no publications available referring to the treatment with VZ in experimental models or in clinical cases of sporotrichosis.

AB also presents a wide susceptibility range. This antifungal has been used with various results in the treatment of the visceral and severe disseminated forms of sporotrichosis, including in patients infected with human immunodeficiency virus (12, 22). In 1972, Brandsberg and French (2) published the first data on the activity of AB on filamentous-phase S. schenckii; these authors, using an agar-microdilution method, found a range of 0.68 to 2.12 µg/ml (GM, 1.4 µg/ml). Despite the described variability, we have found nine strains with MICs of 0.5 µg/ml. These results would indicate that the susceptibility of this fungus to AB is dependent on the strain. Ellis (6), in a review, indicates that some isolates of this fungus show primary resistance to this polyene. For this reason, in severe sporotrichosis it is advisable to determine the MICs of AB to predict what the therapeutic response might be.

KZ, which was determined only by SYO, also showed lower MICs, being similar to those of IZ with the same method and very near those obtained for IZ with the reference method.

The global agreement among the two commercial methods and modified M38-A is very poor, except with IZ and FZ, in the last case because very high MICs have been shown, suggesting an intrinsic resistance of S. schenckii to this drug. Casali and Hamdan (4) showed that three azoles, IZ, KZ, and FZ, inhibited the synthesis of ergosterol and accumulated lanosterol in S. schenckii, with FZ being the least active. The low activity of FZ in S. schenckii has been shown in other studies (9).

Many routine laboratories find it difficult to carry out the reference assay because it must be prepared by hand and it is time-consuming. It is advisable to have accessible commercial methods on hand which are easy to carry out and interpret; both of the selected methods are commonly used in many laboratories.

The various ranges of antifungal concentrations observed for each method are a disadvantage for the assessment of agreement.

The present results suggest that both methods, ATBF2 and SYO, which were initially standardized for yeasts, are of limited interest for detecting MICs of filamentous-phase S. schenckii. Considering that IZ is currently the most useful antifungal agent for treating sporotrichosis, the colorimetric method most closely approximates the reference method. However, further studies should be carried out with S. schenckii strains recovered from other geographical regions to determine whether this pattern of in vitro susceptibility might be generalized.

 
We thank Beatriz Bustamante and Edgar Neyra from the Department of Microbiology, Von Humbolt Institute of Tropical Medicine, Lima, Peru, for providing the isolates used in this study.

 
* Corresponding author. Mailing address: Infectious Diseases and Mycology Research Unit (URMIM), Institut Municipal d'Investigació Mèdica (IMIM), Doctor Aiguader 80, E-08003 Barcelona, Spain. Phone: 34-93-2211009. Fax: 34-93-2213237. E-mail: jmtorres{at}imim.es

Published ahead of print on 16 April 2007.

Top ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

 

1
1. Baker, J. H., H. C. Goodpasture, H. R. Kuhns, and M. G. Rinaldi. 1989. Fungemia caused by an amphotericin B-resistant isolate of Sporotrhix schenckii. Successful treatment with itraconazole. Arch. Pathol. Lab. Med. 113:1279-1281.[Medline]
2
2. Brandsberg, J. W., and M. E. French. 1972. In vitro susceptibility of isolates of Aspergillus fumigatus and Sporothrix schenckii to amphotericin B. Antimicrob. Agents Chemother. 2:402-404.[Abstract/Free Full Text]
3
3. Calhoun, D. L., H. Waskin, M. P. White, J. R. Bonner, J. H. Mulholland, L. W. Rumans, D. A. Stevens, and J. N. Galgiani. 1991. Treatment of systemic sporotrichosis with ketoconazole. Rev. Infect. Dis. 13:47-51.[Medline]
4
4. Casali, A. K., and J. S. Hamdan. 1997. Effects of three azole derivatives on the lipids of different strains of Sporothrix schenckii. Can. J. Microbiol. 43:1197-1202.[Medline]
5
5. Castrejón, O. V., M. Robles, and O. E. Zubieta. 1995. Fatal fungemia due to Sporothrix schenckii. Mycoses 38:373-376.[Medline]
6
6. Ellis, D. 2002. Amphotericin B, spectrum and resistance. J. Antimicrob. Chemother. 49:7-10.[Free Full Text]
7
7. Espinel-Ingroff, A., K. Boyle, and D. J. Sheehan. 2001. In vitro antifungal activities of voriconazole and reference agents as determined by NCCLS methods: review of the literature. Mycopathologia 150:101-115.[CrossRef][Medline]
8
8. Espinel-Ingroff, A. 1998. In vitro activity of the new triazole voriconazole (UK-109,996) against opportunistic filamentous and dimorphic fungi and common and emerging yeast pathogens. J. Clin. Microbiol. 36:198-202.[Abstract/Free Full Text]
9
9. Espinel-Ingroff, A., K. Dawson, M. Pfaller, E. Anaissie, B. Breslin, D. Dixon, A. Fothergill, V. Paetznick, J. Peter, and M. Rinaldi. 1995. Comparative and collaborative evaluation of standardization of antifungal susceptibility testing for filamentous fungi. Antiomicrob. Agents Chemother. 39:314-319.
10
10. Kauffman, C. A., R. Hajjeh, S. W. Chapman, et al. 2000. Practice guidelines for the management of patients with sporotrichosis. Clin. Infect. Dis. 30:684-687.[CrossRef][Medline]
11
11. Kauffman, C. A. 1995. Old and new therapies for sporotrichosis. Clin. Infect. Dis. 21:981-985.[Medline]
12
12. Kurosawa, A., S. C. Pollock, M. P. Collins, C. R. Kraff, and M. O. Tso. 1988. Sporotrhix schenckii endophtalmitis in a patient with human immunodeficiency virus infection. Arch. Optalmol. 106:376-380.
13
13. Martin-Mazuelos, E., J. Peman, A. Valverde, M. Chaves, M. C. Serrano, and E. Canton. 2003. Comparison of the Sensititre YeastOne colorimetric antifungal panel and Etest with the NCCLS M38-A method to determine the activity of amphotericin B and itraconazole against clinical isolates of Aspergillus spp. J. Antimicrob. Chemother. 52:365-370.[Abstract/Free Full Text]
14
14. McGinnis, M. R., N. Nordoff, R. K. Li, L. Pasarell, and D. W. Warnock. 2001. Sporothrix schenckii sensitivity to voriconazole, itraconazole and amphotericin B. Med. Mycol. 39:369-371.[Medline]
15
15. Morera-Lopez, Y., J. M. Torres-Rodríguez, and T. Jiménez-Cabello. 2005. Estudio de la sensibilidad in vitro de aislamientos clínicos de mohos y levaduras a itraconazol y voriconazol. Rev. Iberoam. Micol. 22:103-107.
16
16. National Committee for Clinical Laboratory Standards. 2002. Reference method for broth dilution antifungal susceptibility testing for yeasts. Approved guideline M27-A2. National Committee for Clinical Laboratory Standards, Wayne, PA.
17
17. National Committee for Clinical Laboratory Standards. 2002. Reference methods for broth dilution antifungal susceptibility testing of filamentous fungi. Approved guideline M38-A. National Committee for Clinical Laboratory Standards, Wayne, PA.
18
18. Odabasi, Z., V. L. Paetznick, J. R. Rodríguez, E. Chen, and L. Ostrosky-Zeichner. 2004. In vitro activity of anidulafungin against selected clinically important mold isolates. Antimicrob. Agents Chemother. 48:1912-1915.[Abstract/Free Full Text]
19
19. Pujol, I., J. Capilla, B. Fernandez-Torres, M. Ortopeda, and J. Guarro. 2002. Use of Sensititre colorimetric microdilution panel for antifungal susceptibility testing of dermatophytes. J. Clin. Microbiol. 40:2618-2621.[Abstract/Free Full Text]
20
20. Schell, W. A. 1998. Agents of chromoblastomycosis and sporotrichosis, p. 323-336. In L. Ajello and R. J. Hay (ed.), Topley & Wilson's Medical Mycology, 9th ed. Arnold Press, London, United Kingdom.
21
21. Skrodeniene, E., A. Dambrauskiene, and A. Vitkausikene. 2006. Susceptibility of yeast to antifungal agents in Kaunas University of Medicine Hospital. Medicina (Kaunas) 42:294-299.[Medline]
22
22. Ware, A. J., C. J. Cockerell, D. J. Skiest, and H. M. Kussman. 2000. Disseminated sporotrichosis with extensive cutaneous involvement in a patient with AIDS. J. Am. Acad. Dermatol. 40:350-355.[CrossRef]
23
23. Weeke, J. 2000. Thyroid and antithyroid drugs, p. 1492-1493. In M. N. G. Dukes and J. K. Aronson (ed.), Meyler's side effects of drugs, 14th ed. Elsevier Press, Amsterdam, The Netherlands.
24
24. Winn, R. E., J. Anderson, J. Piper, N. E. Aronson, and J. Pluss. 1993. Systemic sporotrichosis treated with itraconazole. Clin. Infect. Dis. 17:210-217.[Medline]

---

Antimicrobial Agents and Chemotherapy, July 2007, p. 2420-2423, Vol. 51, No. 7
0066-4804/07/$08.00+0     doi:10.1128/AAC.01176-06
Copyright © 2007, American Society for Microbiology. All Rights Reserved.

This article has been cited by other articles:

• Pereira Silveira, C., Torres-Rodriguez, J. M., Alvarado-Ramirez, E., Murciano-Gonzalo, F., Dolande, M., Panizo, M., Reviakina, V. (2009). MICs and minimum fungicidal concentrations of amphotericin B, itraconazole, posaconazole and terbinafine in Sporothrix schenckii. J Med Microbiol 58: 1607-1610 [Abstract] [Full Text]  

This Article Abstract Full Text (PDF) Alert me when this article is cited Alert me if a correction is posted Services Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Reprints and Permissions Copyright Information Books from ASM Press MicrobeWorld Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Alvarado-Ramírez, E. Articles by Torres-Rodríguez, J. M. Search for Related Content PubMed PubMed Citation Articles by Alvarado-Ramírez, E. Articles by Torres-Rodríguez, J. M.