BackgroundMatrix-assisted laser desorption ionisation time of flight mass spectrometry (MALDI TOF-MS) allows the identification of most bacteria and an increasing number of fungi. The potential for the highest clinical benefit of such methods would be in severe acute infections that require prompt treatment adapted to the infecting species. Our objective was to determine whether yeasts could be identified directly from a positive blood culture, avoiding the 1–3 days subculture step currently required before any therapeutic adjustments can be made.Methodology/Principal FindingsUsing human blood spiked with Candida albicans to simulate blood cultures, we optimized protocols to obtain MALDI TOF-MS fingerprints where signals from blood proteins are reduced. Simulated cultures elaborated using a set of 12 strains belonging to 6 different species were then tested. Quantifiable spectral differences in the 5000–7400 Da mass range allowed to discriminate between these species and to build a reference database. The validation of the method and the statistical approach to spectral analysis were conducted using individual simulated blood cultures of 36 additional strains (six for each species). Correct identification of the species of these strains was obtained.Conclusions/SignificanceDirect MALDI TOF-MS analysis of aliquots from positive blood cultures allowed rapid and accurate identification of the main Candida species, thus obviating the need for sub-culturing on specific media. Subsequent to this proof-of-principle demonstration, the method can be extended to other clinically relevant yeast species, and applied to an adequate number of clinical samples in order to establish its potential to improve antimicrobial management of patients with fungemia.

e Echinocandin drugs are widely used for the treatment of candidemia. Resistance is considered rare, and only a few cases of breakthrough candidiasis in patients receiving echinocandin have been reported worldwide. We report here for the first time a Candida kefyr isolate that acquired echinocandin resistance very rapidly after the initiation of caspofungin treatment for candidemia. We characterized the FKS gene mutation responsible for the resistance via the comparison of isolates sampled before and during treatment.A 61-year-old woman was admitted to the hematological unit at Pitié-Salpêtrière Hospital (Paris, France) for acute myeloid leukemia. The patient was not then receiving and had no history of receiving antifungal drugs. During chemotherapy-induced aplasia, a blood culture sampled at day 1 yielded positive for yeast at day 3. Considering the clinical condition of the patient, echinocandin antifungal therapy was chosen. Treatment by caspofungin was initiated on day 3 (70 mg the first day and then 50 mg/day; patient weight, 60 kg). Analysis of the strain with API 20C (bioMérieux, France) strips and mass spectrometry (Bruker Daltonics, Germany) clearly identified Candida kefyr (teleomorph, Kluyveromyces marxianus). Before the determination of the MICs, caspofungin treatment was left unchanged. The MICs for caspofungin, micafungin, and anidulafungin as determined by Etest (bioMérieux) were 0.06, 0.03, and 0.03 g/ml, respectively. For fluconazole, the MIC was 0.25 g/ml. As the patient's condition did not improve, treatment was switched to fluconazole at day 10. A blood culture sampled the same day was positive for yeast at day 11. Surprisingly, this culture gave C. kefyr again. For this isolate, the MICs for caspofungin, micafungin, and anidulafungin by Etest were 2, 4, and 8 g/ml, respectively. It should be noted that the determination of MIC by the CLSI reference method showed similar results (Table 1), but considering the recently proposed epidemiological cutoff value (ECV) for echinocandins against C. kefyr (1), for the initial isolate with low MIC echinocandin values, the MIC for caspofungin was above the ECV, whereas those for anidulafungin and micafungin were below. While receiving fluconazole, a blood culture sampled at day 19 was positive for yeast at day 21, and this time Candida krusei was identified. Treatment was then switched to voriconazole at day 23. The patient died at day 30. Of note, the two candidemias were surely of digestive origin; a stool sample collected a few days before the first candidemia was positive for both C. kefyr and C. krusei.Methods, results, and discussion. Faced with both microbiological resistance and clinical therapeutic failure, we decided to compare the hot spots (HS) of the FKS gene sequences of the two C. kefyr strains, the first identified in the blood culture sampled at day 1 (low echinocandin MIC) and the second at day 10 during caspofungin treatment (high echinocandin MIC). Resistance to echinocandin consistently involves these short FKS sequences that code f...