Does the Capsule Interfere with Performance of Matrix-Assisted Laser Desorption Ionization–Time of Flight Mass Spectrometry for Identification of Cryptococcus neoformans and Cryptococcus gattii?

Abstract

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...

Comparison of the Bruker Biotyper and VITEK MS Matrix-Assisted Laser Desorption/Ionization Time-of-Flight Mass Spectrometry Systems Using a Formic Acid Extraction Method to Identify Common and Uncommon Yeast Isolates

“…Unreliable identification of C. neoformans by the Biotyper has been reported (identification rates of 50–66%) [ 7 17 18 ]. This may be due to the polysaccharide capsule of this pathogen, which renders the extraction and solubilization of proteins difficult, or due to insufficient database entries that would enable spectral matches [ 19 ]. We confirmed that the Biotyper system has some difficulties identifying C. neoformans when using direct FA extraction, and that an improved rate of the identification of C. neoformans isolates can be observed following the FA/ACN extraction, as reported previously [ 7 18 ].…”

A Moldy Application of MALDI: MALDI-ToF Mass Spectrometry for Fungal Identification

“…Growth on some media may yield low scores/percentages [75], and small or mucoid colonies may fail. Using experimental capsule size manipulation, it was demonstrated that capsule size of C. neoformans and C. gattii can compromise identification by the Bruker system [76]. Refined interpretive criteria may be needed to discriminate closely related species and distinguish them from the next best taxon match.…”

An analysis of the population of Cryptococcus neoformans strains isolated from animals in Poland, in the years 2015–2019

“…Correct identification percentage values obtained by authors studying the application of this identification method with regard to Cryptococcus ranged from 0 to 100% 64 – 68 . The differences in the observed values depended mainly on the strain tested (e.g.…”

“…The method enables rapid and reproducible identification and can be applied to a vast range of microorganisms. Though several authors reported the usefulness of this technique in the recognition of CNGSC at the species and even sub-species level, it was also observed that the presence of the capsule as well as the cell wall composition, make MALDI-TOF MS identification of these pathogens challenging in comparison to other clinically important fungal agents 63 , 64 . Additionally, it was noticed that in some of the manufacturer’s library versions, spectra of particular types belonging to the CNGSC were underrepresented, therefore construction of the in-house libraries were beneficial for the increase in identification quality 65 , 66 .…”