Rapid Species Diagnosis for Invasive Candidiasis Using Mass Spectrometry

Abstract

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.

Direct identification of yeasts from blood culture by MALDI-TOF mass spectrometry

“…Several studies have been carried out on direct identification of yeasts from positive blood culture bottles using various protocols with varying results (Buchan et al, 2012;Drancourt, 2010;Ferreira et al, 2011;Idelevich et al, 2014;Lavergne et al, 2013;Marinach-Patrice et al, 2010;Yan et al, 2011).…”

“…While some authors have had very low success rates with fungi (Buchan et al, 2012;Idelevich et al, 2014), others have reported excellent levels of identification with different sample processing methods (Ferroni et al, 2010(Ferroni et al, , 2011Idelevich et al, 2014;Lavergne et al, 2013;Marinach-Patrice et al, 2010;Yan et al, 2011).…”

“…Ferroni (2010), on the other hand, used saponin as the lysis buffer for 20 Candida strains and identified all species (100%), while Yan et al (2011) identified 100% of species using the Sepsityper kit preceded by two additional washing steps. In their study, Lavergne (2013) and Marinach-Patrice (2010) used the same protocol as ours and were able to rapidly and precisely identify the main Candida species, thus avoiding the need for culture on a specific medium. In a second study carried out by Pulcrano et al (2013), on Candida non-albicans with 0.5% SDS, all 82 strains were identified at the species level with a score greater than 2.…”

Molecular Identification of Closely Related Candida Species Using Two Ribosomal Intergenic Spacer Fingerprinting Methods

“…However, some studies from Chen and colleagues 54 described automated capillary electrophoresis means to distinguish species of Candida according to the amplicon size, but later abandoned it due to lack of uniformity Recently, mass spectrometry using matrix assisted laser desorption/ionization-time of flight technology has been developed for microbiological identification and showed very promising results for rapid and reliable determination of the most important yeast species isolated in the clinical setting. [55][56][57] However, to date, mass spectrometry spectra of closely related species forming the major clinical Candida complexes and of unusual species have not been reported and have not been introduced into the reference databases. 56,57 Thus, additional inputs into the current databases using strains with unambiguous molecular-based delineation are required to allow accurate identification of these species for which, in particular, phenotypical methods are unsatisfactory.…”

MALDI-TOF MS: Its Application in the Clinical Laboratory and a Paradigm Shift in Clinical Microbiology

“…In 2010, an evaluation of specieslevel identification of Candida isolates from positive blood culture broth was reported. In their report, Marinach-Patrice et al [38] noted that a direct identification from the positive broth, bypassing subculture and subsequent identification steps, allowed re- …”