Fast and accurate identification of molds and yeast-like fungi directly from positive blood cultures by MALDI-TOF mass spectrometry

Abstract

Molds and yeast-like fungi are potential life-threatening agents of fungemia in immunocompromised patients. Fast and accurate identification (ID) of these pathogens helps to hasten the best-targeted antifungal therapy, improving the patients’ prognosis. We describe a new strategy that enabled the identification of molds or yeast-like fungi directly from positive blood culture (BC) by MALDI-TOF mass spectrometry (MS). BCs with Gram staining revealing arthroconidia and/or hyphae suggestive of fungemia by genera other than Candida were prospectively selected from January to December 2015. Blood aliquots were submitted to an in-house protein extraction protocol. Briefly, 4 ml aliquot was submitted to a lysis centrifugation step (step a) followed by protein extraction with ethanol and 70 % formic acid (step b). One microliter of the supernatant was spotted in quadruplicate in MALDI-TOF MS target and overlaid with 1 μl of α-cyano-4-hydroxycinnamic acid matrix. The pathogens that grew on Sabouraud dextrose agar (SDA) after 72 hours incubation at 30 °C (standard procedure) were also analyzed by MALDI-TOF MS. A loop full of conidia was suspended in 300 μl of deionized water and processed as in step b above. Protein mass fingerprints were obtained using the MALDI-TOF Vitek MS™ system (bioMérieux) and processed by the research use only (RUO) mode using an extended database [SARAMIS™ (v.4.12) plus in-house database]. ID was valid when a spectrum matched with a SuperSpectrum with a confidence level ≥ 75 %, as defined by the manufacturer. The final IDs were confirmed by PCR and sequence analyses of either the ITS/IGS regions of the ribosomal DNA, or the elongation factor-1 alpha gene. Until December 2015, seven potential non-Candida fungi were identified in blood cultures from six immunocompromised patients and an additional patient under intensive care treatment due to thoracic aortic dissection. In all cases, MALDI-TOF MS gave correct genus ID directly from positive BCs (confidence levels 80–99.9 %). Species ID was achieved in the cases of fungemia by Fusarium solani (n = 2), Trichosporon asahii (n = 2) and Geotrichum clavatum (Saprochaete clavata), being the identification of Exophiala dermatitidis and Fusarium verticillioides restricted to genus level. Identical ID results were obtained from the isolates grown on SDA, but with higher confidence levels (96–99.9 %). Five patients (#1–4 and #7) had targeted antifungal therapy guided by the MALDI-TOF MS species ID directly from positive BCs ; four of them survived (80 %). Of these patients, four had risk factors of poor prognosis such as severe neutropenia (neutrophils < 100 cells/mm3) and/or renal failure ; the remaining (#4) was under chemotherapy and systemic corticosteroids at the moment of the catheter related bloodstream infection. Patient #2 had persistent neutropenia and several positive BCs for Saprochaete clavata, and despite targeted dual antifungal therapy with amphotericin B deoxycholate plus voriconazole promptly introduced after fungal ID, deceased 18 days after the first positive BC. Patients #5 and #6 had Fusarium solani fungemia but MALDI-TOF guided targeted antifungal therapy could not be prescribed in one (#5) because he died before final ID of the fungemia and in the other (#6) because the patient was under palliative treatment ; this patient died subsequently due to a polymicrobial bloodstream infection.