Susceptibility testing of Aspergillus spp. by means of an automated blood culture system

P.-M. Rath*, J. M. Freise and R. Ansorg

Institut für Medizinische Mikrobiologie, Universitätsklinikum Essen, Hufelandstrasse 55, D-45122 Essen, Germany

Received 4 July 2001; returned 28 November 2001; revised 14 January 2002; accepted 27 March 2002


    Abstract
 Top
 Abstract
 Introduction
 Materials and methods
 Results and discussion
 References
 
The BacT/Alert blood culture system was evaluated for the MIC determination of amphotericin B for 27 strains of Aspergillus from five different species. An inoculum of c. 108 cfu/mL and an incubation time of 48 h resulted in MICs for 26 of 27 strains that corresponded with published MICs obtained in a microdilution assay. Twelve of 13 strains with MIC48 values >=2 mg/L showed growth within 24 h in bottles containing 0.25 mg/L amphotericin B, indicating that results can be obtained the day following inoculation. It is concluded that the BacT/Alert blood culture system can be used for susceptibility testing of Aspergillus spp. with amphotericin B.


    Introduction
 Top
 Abstract
 Introduction
 Materials and methods
 Results and discussion
 References
 
Moulds of the genus Aspergillus are opportunistic pathogens. Transplant recipients in particular are at risk of acquiring invasive infections, and amphotericin B is currently the drug of choice.1 In the past, technical procedures for susceptibility testing of moulds were not standardized. Recently, the NCCLS proposed a reference method,2 but the procedure is time consuming and can be difficult to perform in laboratories where susceptibility testing of moulds is not performed routinely.

Automated blood culture systems are widely distributed in diagnostic laboratories and can be used for susceptibility testing of mycobacteria.3 To our knowledge, only a few studies have been published on the usefulness of such systems for susceptibility testing of fungi.46 Therefore, the BacT/Alert system was evaluated for susceptibility testing of Aspergillus species by testing 27 strains from international culture collections and comparing the results with MICs for the same strains obtained using a microdilution technique.7


    Materials and methods
 Top
 Abstract
 Introduction
 Materials and methods
 Results and discussion
 References
 
Twenty-seven Aspergillus strains obtained from international culture collections were investigated (Table 1). The results of susceptibility testing with amphotericin B and itraconazole in a microdilution assay have been published elsewhere.7 The strains were subcultured on Sabouraud dextrose agar (Oxoid, Basingstoke, UK) at 35°C for 3 days. Conidia were harvested by swabbing the surface of the agar and suspending the cells in sterile distilled water. The inoculum concentration was determined by measuring the transmission (T) at 530 nm and adjusted to produce aliquots of T c. 10, 50 and 90%.8 Aliquots were plated on Sabouraud dextrose agar to determine the number of cfu/mL.


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Table 1.  Results of susceptibility testing of 27 Aspergillus reference strains to amphotericin B in the microdilution assay and in the BacT/Alert system
 
A stock solution of amphotericin B/desoxycholate (Bristol-Myers Squibb, Munich, Germany) in sterile distilled water was injected into the aerobic blood culture bottles (40 mL broth) of the BacT/Alert system (Organon Teknika, Eppelheim, Germany) to produce a final concentration of 0.25, 0.5, 1 or 2 mg/L, respectively. One millilitre of conidial suspension was added. One bottle without amphotericin B was prepared as a growth control to be run in parallel. The system incubated the bottles at 36°C with agitation, and detected growth by monitoring the chromogenic change in a CO2-sensitive indicator located at the bottom of the bottle. Growth was indicated via transmission of a message to the appropriate software. The MIC24 and MIC48 were defined as the minimum amphotericin B concentrations that inhibited the growth (no chromogenic change) for 24 or 48 h, respectively. The maximum incubation period was 96 h (MIC96). All experiments were carried out 2–5 times using different subcultures to verify the reproducibility of the results.


    Results and discussion
 Top
 Abstract
 Introduction
 Materials and methods
 Results and discussion
 References
 
The time between inoculation and a positive signal depended on the concentration of the inoculum and the amphotericin B concentration. An inoculum with a transmission of c. 10% at T530 (geometric mean 5.6 x 107 cfu/mL, range 1.9–11 x 107 cfu/mL) in amphotericin B-free bottles produced positive results after 8–16 h, which was 5–12 h earlier than lower inoculum concentrations (c. 50% at T530 = 1.3 x 107 cfu/mL; c. 90% at T530 = 3.9 x 106 cfu/mL). When testing the amphotericin B susceptibility of one strain of each species with different inoculum concentrations, the bottles with an inoculum of c. 50% T and c. 90% T gave positive signals 1–13 and 3–35 h later, respectively, than the bottles with an inoculum of c. 10% T. Therefore, the highest inoculum concentration (c. 10% at T530) was chosen for subsequent susceptibility tests.

Table 1 shows the MICs obtained after different incubation periods and the corresponding data obtained by a standardized microdilution assay.7 When applying the criterion of a difference of <=1 dilution step, the results of the two methods agreed in 20 of 27 strains when the MIC24 was analysed. However, on examination of the MIC48 values an agreement was found in 26 of 27 strains. Only one Aspergillus flavus strain (ATCC 32612) showed a MIC48 in the BacT/Alert system that was three dilution steps higher (2 mg/L) than in the microdilution assay (0.25 mg/L).

These data show that the BacT/Alert system may be useful for the determination of the susceptibility to amphotericin B of Aspergillus species. In two earlier studies a radiometric method was used in a similar system to detect growth inhibition by amphotericin B of yeasts4 and different filamentous fungi including Aspergillus spp.5 In another study, yeasts were tested in the BacT/Alert system; however, yeast nitrogen base was used as growth medium.6 The present data show that the aerobic ready-for-use medium in the BacT/Alert bottles can also be used for susceptibility testing.

The system has some advantages compared with conventional techniques (e.g. micro- or macrodilution assays): (i) no preparation of medium is necessary; (ii) susceptibility testing can be performed in one system along with diagnostic blood cultures; (iii) the detection of growth is automated and does not depend on the experience of the investigator; and (iv) although a high inoculum (c. 10% at T530) gave the earliest results, the influence of innoculum concentration is negligible in the range down to c. 50% at T530.

The results of longer incubation periods (MIC72–96) revealed that six of eight A. flavus strains, four of five Aspergillus nidulans strains and the two Aspergillus terreus strains were not inhibited by 2 mg/L amphotericin B, whereas the Aspergillus fumigatus and Aspergillus niger strains were inhibited by this concentration. Retrospective clinical studies and results in animal models indicate that infections with filamentous fungi with MICs >= 2 mg/L show no response to amphotericin B.9,10 Therefore, a tentative breakpoint concentration of 1 mg/L amphotericin B seems to be suitable for in vitro susceptibility testing. With this concentration and an incubation period of 48 h, not only the A. terreus strains but also most of the strains of the species A. flavus and A. nidulans would be classified as resistant to amphotericin B.

In this study, five bottles were used for susceptibility testing. However, it would be possible to conduct the test with only two bottles, one without an antifungal agent as a growth control and a second bottle with 1 mg/L amphotericin B.

To obtain results within 24 h it would be possible to use an amphotericin B concentration of 0.25 mg/L as a screening concentration, because all strains except one (A. terreus CBS 469.81) with MIC48s >= 2 mg/L showed MIC24s > 0.25 mg/L. Further work is required to assess the suitability of this method for use in routine microbiology laboratories.


    Footnotes
 
* Corresponding author. Tel: +49-201-3538; Fax: +49-201-5729; E-mail: rath{at}uni-essen.de Back


    References
 Top
 Abstract
 Introduction
 Materials and methods
 Results and discussion
 References
 
1 . Stevens, D. A., Kan, V. L., Judson, M. A., Morrison, V. A., Dummer, S., Denning, D. W. et al. (2000). Practice guidelines for diseases caused by Aspergillus. Clinical Infectious Diseases 30, 696–709.[ISI][Medline]

2 . 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.

3 . Inderlied, C. B. & Salfinger, M. (1999). Antimycobacterial agents and susceptibility tests. In Manual of Clinical Microbiology, 7th edn (Murray, P. R., Baron, E. J., Pfaller, M. A., Tenover, F. C. & Yolken, R. H., Eds), pp. 1601–23. ASM Press, Washington, DC.

4 . Hopfer, R. L. & Gröschel, D. (1977). Amphotericin B susceptibility testing of yeasts with a Bactec radiometric system. Antimicrobial Agents and Chemotherapy 11, 277–80.[ISI][Medline]

5 . Merz, W. G., Fay, D., Thumar, B. & Dixon, D. (1984). Susceptibility testing of filamentous fungi to amphotericin B by a rapid radiometric method. Journal of Clinical Microbiology 19, 54–6.[ISI][Medline]

6 . Hazen, K. C., Chery, M. P. & Han, Y. (1994). Potential use of BacT/Alert automated blood culture system for antifungal susceptibility testing. Journal of Clinical Microbiology 32, 848–50.[Abstract]

7 . Rath, P.-M. (1998). Susceptibility of Aspergillus strains from culture collections to amphotericin B and itraconazole. Journal of Antimicrobial Chemotherapy 41, 567–70.[Abstract]

8 . Espinel-Ingroff, A., Bartlett, M., Bowden, R., Chin, N. X., Cooper, C., Jr, Fothergill, A. et al. (1997). Multicenter evaluation of proposed standardized procedure for antifungal susceptibility testing of filamentous fungi. Journal of Clinical Microbiology 35, 139–43.[Abstract]

9 . Odds, F. C., Van Gerven, F., Espinel-Ingroff, A., Bartlett, M. S., Ghannoum, M. A., Lancester, M. V. et al. (1998). Evaluation of possible correlations between antifungal susceptibilities of filamentous fungi in vitro and antifungal treatment outcomes in animal infection models. Antimicrobial Agents and Chemotherapy 42, 282–8.[Abstract/Free Full Text]

10 . Lass-Flörl, C., Kofler, G., Kropshofer, G., Hermans, J., Kreczy, A., Dierich, M. P. et al. (1998). In-vitro testing of susceptibility to amphotericin B is a reliable predictor of clinical outcome in invasive aspergillosis. Journal of Antimicrobial Chemotherapy 42, 497–502.[Abstract]





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