Abstract
Fungaemia is associated with high mortality rates and early appropriate antifungal therapy is essential for patient management. However, classical diagnostic workflow takes up to several days due to the slow growth of yeasts. Therefore, an approach for direct species identification and direct antifungal susceptibility testing (AFST) without prior time-consuming sub-culturing of yeasts from positive blood cultures (BCs) is urgently needed. Yeast cell pellets prepared using Sepsityper kit were used for direct identification by MALDI-TOF mass spectrometry (MS) and for direct inoculation of Vitek 2 AST-YS07 card for AFST. For comparison, MALDI-TOF MS and Vitek 2 testing were performed from yeast subculture. A total of twenty four positive BCs including twelve C. glabrata, nine C. albicans, two C. dubliniensis and one C. krusei isolate were processed. Applying modified thresholds for species identification (score ≥1.5 with two identical consecutive propositions), 62.5% of BCs were identified by direct MALDI-TOF MS. AFST results were generated for 72.7% of BCs directly tested by Vitek 2 and for 100% of standardized suspensions from 24 h cultures. Thus, AFST comparison was possible for 70 isolate-antifungal combinations. Essential agreement (minimum inhibitory concentration difference ≤1 double dilution step) was 88.6%. Very major errors (VMEs) (false-susceptibility), major errors (false-resistance) and minor errors (false categorization involving intermediate result) amounted to 33.3% (of resistant isolates), 1.9% (of susceptible isolates) and 1.4% providing 90.0% categorical agreement. All VMEs were due to fluconazole or voriconazole. This direct method saved on average 23.5 h for identification and 15.1 h for AFST, compared to routine procedures. However, performance for azole susceptibility testing was suboptimal and testing from subculture remains indispensable to validate the direct finding.
Highlights
Advanced treatment strategies in oncology, transplantation and other fields of medicine led to a considerable increase in the number of immunocompromised patients during the past decades [1]
Several novel technologies have been suggested to rapidly identify yeasts directly from positive blood cultures (BCs), including PNA-FISH [7] and procedures based on matrix-assisted laser desorption ionization–time of flight mass spectrometry (MALDI-TOF MS) [7, 12,13,14,15,16,17]
This approach can conveniently be combined with an early inoculation of an automated susceptibility testing device from the same biomass, contributing to an earlier identification and antimicrobial susceptibility testing result compared to standard methods [19]
Summary
Advanced treatment strategies in oncology, transplantation and other fields of medicine led to a considerable increase in the number of immunocompromised patients during the past decades [1]. Fungi can hardly be recognized on species level from Gram stain of positive blood culture (BC), as demonstrated in a recent study [7] In this setting, species prediction by Gram stain is further hampered by the current increase in non-albicans species among Candida causing BSIs [8, 9]. An approach of using very short-term cultures incubated only few hours after streaking of positive BC broth onto solid medium has recently been suggested for identification of BSI pathogens by MALDI-TOF [18] This approach can conveniently be combined with an early inoculation of an automated susceptibility testing device from the same biomass, contributing to an earlier identification and antimicrobial susceptibility testing result compared to standard methods [19]. Such method is very useful for bacteria, but poorly performable for yeasts due to their slow growth
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