We described the impact of the capsule size for Cryptococcus neoformans and Cryptococcus gattii identification at the species level by Bruker matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS). After experimental capsule size modulation, we observed that reducing the capsule size resulted in improved identification by Bruker MALDI-TOF MS across all of the reference strains analyzed.
Cryptococcus neoformans and Cryptococcus gattii are relevant species among the pathogenic basidiomycetous yeasts responsible for infection in humans (1). These organisms usually produce a polysaccharide capsule that acts as an important virulence factor against the host's defenses (2). Besides its epidemiological importance, species differentiation in this genus has major clinical relevance since patients with central nervous system (CNS) infection by C. gattii have a higher risk of neurological complications, need a more prolonged course of induction antifungal therapy, and have poorer prognoses than those with C. neoformans infections (3, 4).Recently, matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS) has been shown to be a precise technology for Cryptococcus species identification (ID), replacing conventional and time-consuming phenotypic methods and providing an alternative to expensive and labor-intensive molecular techniques (5, 6). However, during routine practice in our clinical microbiology laboratory, we observed that some Cryptococcus isolates with prominent capsule sizes had low discriminatory ID when using Bruker MALDI-TOF MS analysis. These isolates required the application of old phenotypic methods, which delayed the release of the final result. This led us to investigate the impact of cryptococcal capsule size in correct species ID by Bruker MALDI-TOF MS analysis.For this purpose, reference strains of the eight genotypes of C. neoformans and C. gattii, WM148 (serotype A, VNI), WM626 (serotype A, VNII), WM 628 (serotype AD, VNIII), WM629 (serotype D, VNIV), WM179 (serotype B, VGI), WM178 (serotype B, VGII), WM161 (serotype B, VGIII), and WM779 (serotype C, VGIV), were subjected to capsule size modulation according to previously described methods (7,8). Briefly, 2 ml of capsule growth-inducing medium (CGIM) (Sabouraud dextrose broth [BD, Franklin Lakes, NJ, USA] diluted 10 times with sterile water, pH 7.3) containing 2 ϫ 10 6 yeast cells was incubated at 37°C with shaking. In an attempt to increase the variability in capsule size, all strains were subjected to a prolonged incubation in CGIM (up to 28 days) and were evaluated simultaneously on days 2, 3, 7, 14, 21, and 28, giving a total of 48 capsule size measurements (six replicates for each strain of the two Cryptococcus species). Next, yeast cells collected from the CGIM were submitted to a progressive capsule reduction protocol with four consecutive initial seedings in Sabouraud dextrose agar (SDA; BD) and two more seedings in the capsule-reducing medium (CRM) (SDA plus 2.9% NaCl). Dur...