Direct inoculation of stored spore suspension for antifungal susceptibility testing of Aspergillus fumigatus

Jochem B Buil


Background: As azole resistance is increasingly reported for Aspergillus fumigatus,   antifungal susceptibility testing (AFST) of clinical  relevant A. fumigatusisolates is    important to guide antifungaltherapy in  geographic areas  were resistance is prevalent. Two reference methods of AFST  are recommended; broth microdilution based on either EUCAST or CLSI methodology, but these methods are not widely available. Alternatively, spore suspensions can be sent to mycology reference centers. However, this causes  delay in obtaining  the susceptibility results, as isolates need to be subcultered before performing broth microdilution. In order to accelerate AFST when isolates are sent to reference centers, we studied  whether  spore suspensions, that  are  sent in can  be  directly inoculated,  thereby eliminating the subculture-phase. 

Material/methods:Thirteen A. fumigatus isolates were used in    this study. Six azole resistant isolates; TR34/L98H (2  isolates) TR46/Y121F/T289A (2) and TR53(2)  and 7  azole susceptible isolates. Spore suspensions were prepared and inoculated after storage  of 0-1-2-3-4 days at  room temperature  to obtain MICs  of voriconazole, itraconazole,  posaconazole, amphothericin  B, and anidulafungin.  A second series of spore suspensions were stored for 0-4 days at 2ºC to 4ºC and at 28ºC. The effect of storage on optical density of spore suspensions was studied by measuring the transmission at 530nm with a spectrophotometer for four constitutive  days, for three isolates. All  measurements wereperformed in triplicate. 

Results: The MICs of azoles, amphothericin B, and anidulafungin did not differ more than one twofold dilution step when the inoculum had been stored in comparison with the MIC results obtained from non-stored spore suspensions. The susceptibility classification based on clinical breakpoints showed100% concordance for itraconazole, anidulafungin and amphothericin B. As the MICs of voriconazole and posaconazole were closer to the clinical breakpoint, variation in MICs of one twofold dilution step resulted in change in interpretation from intermediate to susceptible/resistant or visa-versa. This occurred for one of the replicates of voriconazole, 4 times for posaconazole at room temperature and once for posaconazole at 28ºC. In addition, one isolate intermediate/resistant for voriconazole at day0, tested susceptible at day 4 for 1 replicate. Storage of samples did not affect optical density.

Conclusions: We evaluated a strategy to accelerate susceptibility testing of A. fumigatus when isolates are sent to mycology reference laboratories. For isolates with MICs close to the breakpoints, small variations in measurements did result in a  change in interpretation for individual drugs. However, this is also the case for repeated measurements performed in accordance to EUCAST guidelines. Storage of spore suspensions at various temperatures did not result in changes of MIC of more thanone twofold dilution, indicating that the subcultering phase can be eliminated for spore suspensions up to 4 days old for mold active azoles, anidulafungin and amphotericin B.



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abstract No: 


Full conference title: 

European Congress of Clinical Microbiology and Infectious Diseases
    • ECCMID 26th (2016)